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Home , Afterhyperpolarization, Himbacine, Isoguvacine, Linopirdine, Maurotoxin, SK channel

AHP, BK- and SK-channel references

This is an ongoing project (open-ended), so look at it only (ONLY!) as the first couple of hundred pages of a summary of what’s known about , the channels that gate them, and the that effects them. Where abstracts are not listed, I couldn’t get ‘em. Posted 6/14/02.

-Tres Thompson, Ph.D., Neuroscience

HIPPOCAMPAL PYRAMIDAL

•Alberi, S., Boeijinga, P.H., Raggenbass, M. & Boddeke, H.W. (2000). Involvement of -dependent protein kinase II in -induced rhythmic activity in the hippocampus of the rat. Brain Research 872(1-2): 11-19. The role of calcium and protein kinases in rhythmic activity induced by muscarinic receptor activation in the CA1 area in rat hippocampal slices was investigated. Extracellular recording showed that carbachol (20 µM) induced synchronized field potential activity with a dominant frequency of 7.39±0.68 Hz. Pretreatment with the membrane permeable Ca(2+) chelator BAPTA-AM (50 µM) or with thapsigargin (1 µM), a compound which depletes intracellular calcium stores, reduced the dominant power of carbachol-induced theta-like activity by 83% and 78%, respectively. Inhibition of calmodulin-dependent protein kinase II (CaMKII) by the cell permeable inhibitor KN-93 (10 µM) reduced the power of carbachol-induced theta-like activity by 80%. In contrast the protein kinase C (PKC) inhibitor calphostin C did not significantly (P>0.05) affect the effect of carbachol. Whole- cell recording indicated that KN-93 also blocked carbachol-induced suppression of slow I(AHP) and strongly inhibited the carbachol-induced plateau potential. Our data suggest that activation of CaMKII by carbachol is crucial for local theta-like activity in the CA1 area of the rat hippocampus in vitro. Furthermore, involvement of CaMKII in carbachol-induced suppression of the slow I(AHP) and the induction of plateau potentials could play a role in the induction of theta-like rhythmic activity by carbachol.

•Andreasen, M. (2002). Inhibition of slow Ca2+-activated K+ current by 4- aminopyridine in rat hippocampal CA1 neurons. British Journal of Pharmacology 135(4): 1013-1025. The effect of 4-aminopyridine (4-AP) on the slow (sAHP) seen after high frequency dendritic or somatic firing was investigated in rat

Page 1 AHP, BK- and SK-channel references hippocampal CA1 pyramidal neurones (PC). Intracellular recordings were obtained from the distal apical dendrites and somata and suprathreshold depolarizing current pulses were used to evoke a sAHP. The sAHP was blocked by low concentrations of carbacholine (Cch) but insensitive to high concentrations of . In the presence of extracellular 4-AP, the first dendritic sAHP evoked was reduced compared to a maximal sAHP evoked in the absence of 4-AP. The reduction was evident at submillimolar concentration and increased to about 80% with 4 mM 4-AP. The stability of the 4-AP-induced block was affected by the type of anion used in the electrode solution. With K(+) acetate (KAc) or K(+) methylsulphate (KMeSO(4)) containing electrodes, the block was progressively removed during the initial 300 - 400 s of recordings. With KCl containing electrodes, the block remained stable and was 10% larger than that obtained with acetate. Detailed investigations showed that intracellular acetate promotes the removal of the 4-AP-induced block in an activity-dependent manner. Intracellularly applied 4-AP also induced an acetate- sensitive block of the dendritic sAHP. 4-AP also blocked the somatic sAHP and the stability of the block showed the same sensitivity towards anions as the dendritic sAHP. Thus 4-AP appears to block the slow Ca2+-activated K(+) current underlying the sAHP in a complex manner which is sensitive to certain types of anions.

•Andreasen, M. & Lambert, J.D.C. (2001). 4-aminopyridine inhibits a slow calcium-activated K+ current in hippocampal CA1 neurons. Society for Neuroscience Abstracts 31(382.17): 196. 4-aminopyridine (4-AP) blocks several types of voltage-gated K+ currents, but is generally reported to have no effect on Ca2+-activated K+ currents. Here we report that 4-AP blocks the slow afterhyperpolarization (sAHP) observed in the apical dendrites following high frequency firing and/or Ca2+ spikes. Sharp microelectrodes were used to obtain intracellular recordings from the distal apical dendrites of hippocampal CA1 pyramidal neurones in slices from adult male Wistar rats. Suprathreshold current pulses (300 ms, 0.1 Hz), were used to evoke the sAHP. With K+ acetate (KAc) containing electrodes, the size of the first sAHP evoked in the presence of 4-AP was much smaller than expected considering the concurrent increase in Ca2+ spiking. Compared to maximal sAHPs evoked in the absence of 4-AP, the sAHPs in the presence of 4-AP was reduced by 20 % in 250 M and by 80 % in 4 mM 4-AP. The reduction was maximal on the first stimulation. Thereafter, there was a progressive increase in the sAHP until a plateau was reached after 300 - 400 s despite the continued presence of 4-AP. The rate of unblocking of 4-AP was partly

Page 2 AHP, BK- and SK-channel references dependent on the stimulation frequency. With KCl-containing electrodes, the reduction of the sAHP in the presence of 4-AP (4 mM) was 10 % larger than with KAc electrodes and, furthermore, remained stable for the whole of the recording period. Thus, 4-AP seems to block the slow Ca2+-activated K+ channels mediating the sAHP by binding to a site that is accessible when the channels are closed. Moreover, intracellular acetate somehow decreases the binding of 4-AP to the channel complex.

•Bacon, W.L. & Beck, S.G. (2000). 5-Hydroxytryptamine7 receptor activation decreases slow afterhyperpolarization amplitude in CA3 hippocampal pyramidal cells. Journal of Pharmacology and Experimental Therapeutics 294(2): 672-679. The 5-hydroxytryptamine(7) (5-HT(7)) receptor was originally defined by molecular biology techniques. The 5-HT(7) receptor protein and mRNA are found in brain areas, such as the CA3 subfield of the hippocampus, that are involved in various neuropsychiatric disease states. No functional response has previously been attributed to activation of the 5-HT(7) receptor in any of these brain areas. Calcium spike-induced slow afterhyperpolarizations (sAHP) were recorded from CA3 hippocampal pyramidal cells using intracellular recording techniques in a brain slice preparation maintained in vitro. A concentration-dependent inhibition of the sAHP amplitude was obtained when 5-HT was used as the agonist. To identify whether the 5-HT(7) receptor was one of the receptors mediating the inhibition of the sAHP amplitude, 5-HT agonists and antagonists were tested in the presence of WAY- 100635 and GR-113808 to block 5-HT(1A) and 5-HT(4) receptor activation, respectively. The rank order potency of the agonists was 5-carboxyamidotryptamine (5-CT) > 5-HT > 5-methoxytryptamine (5-MeOT). Other agonists with high affinity at 5-HT(2), 5-HT(3), 5-HT(1B), 5-HT(1D), or 5-HT(6) receptors did not produce any response when tested at 10 microM. Ritanserin, mesulergine, and SB-269770 were competitive antagonists of the 5-CT inhibition of sAHP amplitude, with affinity (pA(2)) values of 6.8, 7. 9, and 8.8, respectively. Methiothepin was also an effective antagonist but was insurmountable. Other antagonists with affinity for the 5- HT(2), 5-HT(3), or 5-HT(6) receptor had no effect. Based on the rank order potency of the agonists and antagonists, one of the receptors that mediates the decrease in sAHP amplitude in CA3 hippocampal pyramidal cells was concluded to be the 5-HT(7) receptor.

•Bekkers, J.M. (2000). Distribution of slow AHP channels on hippocampal CA1 pyramidal neurons. Journal of Neurophysiology 83(4): 2040-2046.

Page 3 AHP, BK- and SK-channel references

This work was designed to localize the Ca(2+)-activated K(+) channels underlying the slow afterhyperpolarization (sAHP) in hippocampal CA1 pyramidal cells. Cell-attached patches on the proximal 100 µm of the apical dendrite contained K(+) channels, but not sAHP channels, activated by backpropagating action potentials. Amputation of the apical dendrite approximately 30 µm from the soma, while simultaneously recording the sAHP whole cell current at the soma, depressed the sAHP amplitude by only approximately 30% compared with control. Somatic cell- attached and nucleated patches did not contain sAHP current. Amputation of the axon ≥20 microm from the soma had little effect on the amplitude of the sAHP recorded in cortical pyramidal cells. By this process of elimination, it is suggested that sAHP channels may be concentrated in the basal dendrites of CA1 pyramids.

•Bond, C.T., Maylie, J. & Adelman, J.P. (1999). Small-conductance calcium- activated channels. Annals of the NY Academy of Science 868: 370-8. SK channels play a fundamental role in all excitable cells. SK channels are potassium selective and are activated by an increase in the level of intracellular calcium, such as occurs during an . Their activation causes membrane hyperpolarization, which inhibits cell firing and limits the firing frequency of repetitive action potentials. The intracellular calcium increase evoked by action potential firing decays slowly, allowing SK channel activation to generate a long- lasting hyperpolarization termed the slow afterhyperpolarization (sAHP). This spike-frequency adaptation protects the cell from the deleterious effects of continuous tetanic activity and is essential for normal neurotransmission. Slow AHPs can be classified into two groups, based on sensitivity to the bee apamin. In general, apamin- sensitive sAHPs activate rapidly following a single action potential and decay with a time constant of approximately 150 ms. In contrast, apamin-insensitive sAHPs rise slowly and decay with a time constant of approximately 1.5 s. The basis for this kinetic difference is not yet understood. Apamin-sensitive and apamin-insensitive SK channels have recently been cloned. This chapter will compare with different classes of sAHPs, discuss the cloned SK channels and how they are gated by calcium ions, describe the molecular basis for their different pharmacologies, and review the possible role of SK channels in several pathological conditions.

•Borde, M., Bonansco, C., Ray, D.L. & Buño, W. (2000). Voltage-clamp analysis of the potentiation of the slow Ca2+-activated K+ current in hippocampal pyramidal

Page 4 AHP, BK- and SK-channel references neurons. Hippocampus 10(2): 198-206. Exploring the principles that govern activity-dependent changes in excitability is an essential step to understand the function of the nervous system, because they act as a general postsynaptic control mechanism that modulates the flow of synaptic signals. We show an activity-dependent potentiation of the slow Ca2+-activated K+ current (sIAHP) which induces sustained decreases in the excitability in CA1 pyramidal neurons. We analyzed the sIAHP using the slice technique and voltage-clamp recordings with sharp or patch-electrodes. Using sharp electrodes-repeated activation with depolarizing pulses evoked a prolonged (8-min) potentiation of the amplitude (171%) and duration (208%) of the sIAHP. Using patch electrodes, early after entering the whole-cell configuration (<20 min), responses were as those reported above. However, although the sIAHP remained unchanged, its potentiation was markedly reduced in later recordings, suggesting that the underlying mechanisms were rapidly eliminated by intracellular dialysis. Inhibition of L-type Ca2+ current by (20 M) markedly reduced the sIAHP (79%) and its potentiation (55%). Ryanodine (20 M) that blocks the release of intracellular Ca2+ also reduced sIAHP (29%) and its potentiation (25%). The potentiation of the sIAHP induced a marked and prolonged (>50%; 8 min) decrease in excitability. The results suggest that sIAHP is potentiated as a result of an increased intracellular Ca2+ concentration ([Ca2+]i) following activation of voltage-gated L-type Ca2+ channels, aided by the subsequent release of Ca2+ from intracellular stores. Another possibility is that repeated activation increases the Ca2+-binding capacity of the channels mediating the sIAHP. This potentiation of the sIAHP could be relevant in hippocampal physiology, because the changes in excitability it causes may regulate the induction threshold of the long-term potentiation of synaptic efficacy. Moreover, the potentiation would act as a protective mechanism by reducing excitability and preventing the accumulation of intracellular Ca2+ to toxic levels when intense synaptic activation occurs.

•Bowden, S.E., Fletcher, S., Loane, D.J. & Marrion, N.V. (2001). Somatic colocalization of rat SK1 and D class (Ca(v)1.2) L-type calcium channels in rat CA1 hippocampal pyramidal neurons. Journal of Neuroscience 21(RC175): 1-6. In hippocampal neurons, the firing of a train of action potentials is terminated by generation of the slow afterhyperpolarization (AHP). Recordings from hippocampal slices have shown that the slow AHP likely results from the activation of small-conductance calcium-activated potassium (SK) channels by calcium (Ca2+)

Page 5 AHP, BK- and SK-channel references entry through L-type Ca2+ channels. However, the relative localization of these two channel subtypes is not known. The cloning and characterization of three subtypes of SK channel has suggested that SK1 may underlie generation of the slow AHP. Using a novel antibody directed against rat SK1 (rSK1), it has been determined that the rSK1 channel is primarily in the soma of hippocampal CA1 neurons. In conjunction with antibodies directed against C (Ca(v)1.2) and D (Ca(v)1.3) class L-type Ca2+ channel alpha1 subunits, it was observed that rSK1 channels were selectively colocalized with D class L-type channels. This colocalization supports the functional coupling of L-type and SK channels previously observed in cell-attached patches from hippocampal neurons. However, it appears contrary to the slow rise and decay of the slow AHP. Induction of delayed facilitation of L-type Ca2+ channels in cell- attached patches from hippocampal neurons evoked delayed opening of coupled SK channels. Generation of ensemble currents produced waveforms identical to the ionic current underlying the slow AHP (I(sAHP)). Therefore, these data indicate that the slow AHP is somatic in origin, resulting from delayed facilitation of D class L-type Ca2+ channels colocalized with rSK1 channels.

•Brown, D.A. & Griffith, W.H. (1983). Calcium-activated outward current in voltage-clamped hippocampal neurones of the guinea-pig. Journal of Physiology (London) 337: 287-301. Slow clamp currents were recorded from CA1 and CA3 pyramidal neurones in slices of guinea-pig hippocampus maintained in vitro, using a single micro-electrode sample-and-hold technique. Depolarizing voltage commands evoked a time- and voltage-dependent outward current which was suppressed by removing external Ca or by adding Cd (0.5 mM) or Mn (5 mM). This Ca-dependent current (Ic) was not reduced by muscarinic agonists (unlike IM) but was greatly reduced by 5-20 mM- (TEA). Repolarizing IC tail currents reversed at -73 ± 5 mV in 3 mM-K solution. The reversal potential became about 30 mV more positive on raising [K]o to 15 mM. No clear change in current amplitude or tail-current reversal potential occurred on adding Cs (2 mM), reducing [Cl]o from 128 to 10 mM, or replacing external Na with Tris. The underlying conductance GC was activated at membrane potentials positive to -45 mV. At -32 mV GC showed an approximately exponential increase with time, with a time constant of approximately 0.6 sec at 26°C. Repolarizing tail currents declined exponentially with time, the time constant becoming shorter with increasing negative post-pulse potentials. When the clamp was switched off at the end of a depolarizing command of sufficient amplitude and

Page 6 AHP, BK- and SK-channel references duration to activate IC, a membrane hyperpolarization to -73 mV ensued, of similar amplitude and decay time to that following spontaneous action potentials. It is concluded that the clamp current observed in these experiments is probably the Ca- activated K current thought to contribute to the post-activation after- hyperpolarization in hippocampal neurones.

•Church, J. (1992). A change from HCO3--CO2- to HEPES-Buffered medium modifies membrane properties of rat CA1 pyramidal neurones in vitro. Journal of Physiology (London) 455: 51-71. 1. Intracellular recordings were obtained from CA1 pyramidal neurones in rat hippocampal slices. Perfusion with a HCO3(-)-CO2-free, HEPES-buffered medium at pH 7.4 produced a wide variety of reversible effects on neuronal excitability, compared to responses obtained under standard (21 mM-HCO3-, 5% CO2, pH 7.4) conditions. 2. Introduction of HCO3(-)-CO2-free medium most commonly elicited, within 5-20 min, a fall in resting (Vm), a rise in threshold for Na(+)-dependent action potential generation, and a reduction in input resistance. Anomalous inward rectification in the hyperpolarizing direction and subthreshold inward rectification were commonly reduced in HEPES-buffered medium. More prolonged exposure (> or = 25 min) to HCO3(-)-CO2-free medium produced, on occasion, Na+ spike inactivation. 3. The amplitudes of the fast and medium after- hyperpolarizations (AHPs) following a single depolarizing current-evoked action potential were attenuated during perfusion with HEPES-buffered medium at pH 7.4, as was the composite AHP following a train of action potentials. 4. Perfusion with HEPES-buffered medium at pH 7.4 reduced the degree of spike frequency adaptation and abolished depolarizing current-evoked burst-firing behaviour when this was present under standard conditions. 5. In (TTX)- and tetraethylammonium (TEA)-poisoned neurones, perfusion with HCO3(-)-CO2-free medium at pH 7.4 slightly raised the threshold for activation of Ca(2+)-dependent potentials and slightly reduced their duration, compared to responses obtained in HCO3(-)-CO2-buffered medium at the same pH. The AHP following the Ca2+ spike was, however, markedly attenuated. 6. Perfusion with a low-pH HCO3(-)-CO2- buffered medium (7 mM-HCO3-, 5% CO2, pH 6.9) produced changes qualitatively similar to those observed during perfusion with HEPES-buffered medium at pH 7.4. Raising the pH of the HEPES-buffered medium to 7.8 or 7.9 reversed inconsistently and then only in part the changes noted on the transition from a HCO3(-)-CO2- to a HEPES-buffered medium at the same pH (7.4). 7. The effects noted are unlikely to

Page 7 AHP, BK- and SK-channel references be due to a direct action of HEPES itself on neuronal membrane conductances. Rather, I suggest that they are likely to be caused by intracellular acidosis consequent upon the omission of HCO3- and CO2 from the extracellular medium.

•Church, J. (1999). Effects of pH changes on calcium-mediated potentials in rat hippocampal neurons in vitro. Neuroscience 89(3): 731-742. The effects of changes in extra- and intracellular pH (pHo and pHi, respectively) on potentials mediated by the influx of Ca2+ ions were investigated in intracellular "current-clamp" recordings from CA1 pyramidal neurons in rat hippocampal slices. In neurons which exhibited a "regular-spiking" discharge in response to depolarizing current injection at pH 7.3, perfusion with pH 7.7 medium led to the development of burst firing. Conversely, neurons which were "burst- firing" at pH 7.3 became regular spiking upon exposure to pH 6.9 medium. In addition, the rebound depolarization following a current-evoked hyperpolarization to >- 60 mV, which in part reflects activation of a low-voltage-activated Ca2+ conductance, was reduced at pHo 6.9 and enhanced at pHo 7.7. Neither the burst firing pattern of discharge nor the augmented rebound depolarization observed during perfusion with pH 7.7 medium was due to the reduction in [Cl-]o consequent upon the increase in [HCO3-]o at a constant PCO2. The magnitudes of the fast afterhyperpolarization which follows a single depolarizing current-evoked action potential and the slow afterhyperpolarization which follows a train of action potentials were attenuated and enhanced, respectively, during perfusion with pH 6.9 and pH 7.7 media, compared to responses obtained at pH 7.3. Reducing pHi at a constant pHo (by exposure to pH 7.3 HCO3-/CO2-free medium buffered with 30 mM HEPES) also attenuated fast and slow afterhyperpolarizations. In tetrodotoxin- and tetraethylammonium-poisoned slices, perfusion with pH 6.9 and pH 7.7 media reduced and increased, respectively, the magnitude of current-evoked Ca2+- dependent depolarizing potentials and their associated slow afterhyperpolarizations, compared with responses obtained at pH 7.3. In contrast, reducing pHi at a constant pHo elicited only a small reduction in the magnitude of Ca2+ spikes but markedly attenuated the subsequent slow afterhyperpolarization. The results suggest that, in rat CA1 hippocampal pyramidal neurons, Ca2+-dependent depolarizing potentials mediated by the influx of Ca2+ ions through voltage-activated Ca2+ channels are sensitive to changes in pHo. These effects of changes in pHo are not dependent upon changes in pHi consequent upon the changes in pHo. Changes in pHo also affect the magnitudes of fast and slow afterhyperpolarizations mediated by Ca2+-dependent K+

Page 8 AHP, BK- and SK-channel references conductances. In these cases, however, the effects of changes in pHo are mimicked by changes in pHi at a constant pHo, suggesting in turn that the effects of changes in pHo on fast and slow afterhyperpolarizations may be mediated both by changes in Ca2+ influx (reflecting mainly changes in pHo) and by direct effects of changes in pHi (consequent upon changes in pHo) on Ca2+-dependent K+ conductances.

•Church, J., Baxter, K.A. & McLarnon, J.G. (1998). pH modulation of Ca2+ responses and a Ca2+-dependent K+ channel in cultured rat hippocampal neurones. Journal of Physiology (London) 511(Pt. 1): 119-132. 1. The effects of changes in extra- and intracellular pH (pHo and pHi, respectively) on depolarization-evoked rises in intracellular free Ca2+ concentration ([Ca2+]i) and the activity of a Ca2+-dependent K+ channel were investigated in cultured fetal rat hippocampal neurones. 2. In neurones loaded with 2', 7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF), changes in pHo evoked changes in pHi. At room temperature, the ratio DeltapHi : DeltapHo (the slope of the regression line relating pHi to pHo) was 0.37 under HCO3-/CO2- buffered conditions and 0.45 under Hepes-buffered conditions; corresponding values at 37 C were 0.71 and 0.79, respectively. The measurements of changes in pHi evoked by changes in pHo were employed in subsequent experiments to correct for the effects of changes in pHi on the Kd of fura-2 for Ca2+. 3. In fura-2-loaded neurones, rises in [Ca2+]i evoked by transient exposure to 50 mM K+ were reduced and enhanced during perfusion with acidic and alkaline media, respectively, compared with control responses at pHo 7.3. Fifty percent inhibition of high-[K+]o-evoked rises in [Ca2+]i corresponded to pHo 7.23. In the presence of 10 microM nifedipine, 50 % inhibition of high-[K+]o-evoked responses corresponded to pHo 7.20, compared with a pHo of 7.31 for 50% inhibition of [Ca2+]i transients evoked by N-methyl-D- aspartate. 4. Changes in pHi at a constant pHo were evoked by exposing neurones to weak acids or bases and quantified in BCECF-loaded cells. Following pH-dependent corrections for the Kd of fura-2 for Ca2+, rises in [Ca2+]i evoked by high-[K+]o in fura-2-loaded cells were found to be affected only marginally by changes in pHi. When changes in pHi similar to those observed during the application of weak acids or bases were elicited by changing pHo, reductions in pH inhibited rises in [Ca2+]i evoked by 50 mM K+ whereas increases in pH enhanced them. 5. The effects of changes in pH on the kinetic properties of a BK-type Ca2+-dependent K+ channel were investigated. In inside-out patches excised from neurones in sister cultures to those used in the microspectrofluorimetric studies, with internal [Ca2+] at 20 µM,

Page 9 AHP, BK- and SK-channel references channel openings at an internal pH of 6.7 were generally absent whereas at pH 7.3 (or 7.8) the open probability was high. In contrast, channel activity in outside-out patches was not affected by reducing the pH of the bath (external) solution from 7.3 to 6.7. In inside-out patches with internal [Ca2+] at 0.7 microM, a separate protocol was applied to generate transient activation of the channel at a potential of 0 mV following a step from a holding level of -80 mV. In this case open probabilities were 0.81 (at pH 7.8), 0.57 (pH 7.3), 0.19 (pH 7.0) and 0.04 (pH 6.7). Channel conductance was not affected by changes in internal pH. 6. The results indicate that, in fetal rat hippocampal neurones, depolarization-evoked rises in [Ca2+]i mediated by the influx of Ca2+ ions through dihydropyridine-sensitive and - resistant voltage-activated Ca2+ channels are modulated by changes in pHo. The effects of pHo cannot be accounted for by changes in pHi consequent upon changes in pHo. However, changes in pHi affect the unitary properties of a Ca2+-dependent K+ channel. The results support the notion that pHo and/or pHi transients may serve a modulatory role in neuronal function.

•Cingolani, L.A., Gymnopoulos, M., Stocker, M. & Pedarzani, P. (2001). Developmental regulation of AHP currents and SK channel subunit expression in rat hippocampal pyramidal neurons. Society for Neuroscience Abstracts 31(382.14): 196. In CA1 pyramidal neurons two calcium-activated currents mediate the medium and slow afterhyperpolarizations following action potentials. I(AHP) presents faster kinetics and sensitivity to the toxin apamin; sI(AHP) presents slow kinetics and is modulated by many neurotransmitters. Cloned SK channels are likely candidates to mediate AHP currents. During development, action potentials and firing patterns undergo changes related to neuronal differentiation. Calcium- activated currents may link calcium influx to changes in excitability during neuronal differentiation. We have studied AHP currents and expression of SK channel subunits SK(1-3) in the rat hippocampus during postnatal development. All SK subunits were highly expressed in the CA1 layer starting from P1. Whole-cell and perforated patch recordings of AHP currents were performed. The neurons showed no significant changes in the resting membrane potential, a decrease in input resistance, and an increase in membrane capacitance with age. The apamin-sensitive I(AHP) was first observed at P6, and reached amplitude similar to the adult at P12. sI(AHP) presented instead a delayed developmental pattern. Corresponding changes in the early and late phases of adaptation of the firing pattern were observed. Developmental changes of the high voltage activated calcium currents have also been

Page 10 AHP, BK- and SK-channel references examined. The mechanisms regulating the different developmental patterns of the AHP currents are currently under investigation.

•Cloues, R.K., Tavalin, S.J. & Marrion, N.V. (1997). Beta-adrenergic stimulation selectively inhibits long-lasting L-type facilitation in hippocampal pyramidal neurons. Journal of Neuroscience 17(17): 6493-6503. L-type calcium channels are abundant in hippocampal pyramidal neurons and are highly clustered at the base of the major dendrites. However, little is known of their function in these neurons. Single-channel recording using a low concentration of permeant ion reveals a long-lasting facilitation of L-type channel activity that is induced by a depolarizing prepulse or a train of action potential waveforms. This facilitation exhibits a slow rise, peaking 0.5-1 sec after the train and decaying over several seconds. We have termed this behavior "delayed facilitation," because of the slow onset. Delayed facilitation results from an increase in opening frequency and the recruitment of longer duration openings. This behavior is observed at all membrane potentials between -20 and -60 mV, with the induction and magnitude of facilitation being insensitive to voltage. beta-Adrenergic receptor activation blocks induction of delayed facilitation but does not significantly affect normal L-type channel activity. Delayed facilitation of L-type calcium channels provides a prolonged source of calcium entry at negative membrane potentials. This behavior may underlie calcium-dependent events that are inhibited by beta-adrenergic receptor activation, such as the slow afterhyperpolarization in hippocampal neurons.

•Cohen, A.S., Coussens, C.M., Raymond, C.R. & Abraham, W.C. (1999). Long- lasting increase in cellular excitability associated with the priming of LTP induction in rat hippocampus. J Neurophysiol 82(6): 3139-3148. The mechanisms underlying the facilitation (priming) of long-term potentiation (LTP) by prior activation of metabotropic glutamate receptors (mGluRs) were investigated in area CA1 of rat hippocampal slices. In particular, we focused on whether a long-lasting increase in postsynaptic excitability could account for the facilitated LTP. Administration of the mGluR agonist 1S, 3R- aminocyclopentanedicarboxylic acid (ACPD) produced rapid decreases in the amplitude of both the slow spike afterhyperpolarization (AHP(slow)) and spike frequency adaptation recorded intracellularly from CA1 pyramidal cells. These changes persisted after drug washout, showing only a slow decay over 20 min. ACPD also caused a leftward shift of the field EPSP-population spike relation and an

Page 11 AHP, BK- and SK-channel references overall increase in population spike amplitude, but this effect was not as persistent as the intracellularly measured alterations in cell excitability. ACPD-treated cells showed increased spike discharges during LTP-inducing tetanic stimulation, and the amplitude of the AHP(slow) was negatively correlated with the degree of initial LTP induction. The beta-adrenergic agonist isoproterenol also caused excitability changes as recorded intracellularly, whereas in extracellular experiments it weakly primed the induction but not the persistence of LTP. ACPD primed both LTP measures. Isoproterenol administration during the tetanus occluded the priming effect of ACPD on initial LTP induction but not its effect on LTP persistence. We conclude that the persistent excitability changes elicited by ACPD contributes to the priming of LTP induction but that other ACPD-triggered mechanisms must account for the facilitated persistence of LTP in the priming paradigm.

•D'Hoedt, D., Doorty, K.B., Jeyaseelen, K., Wadsworth, J.D.F., Stocker, M., Pedarzani, P. & Strong, P.N. (2001). Tamapin: A new toxin from the Indian red targeting SK channels and AHP currents in central neurons. Society for Neuroscience Abstracts 31(382.12): 196. Tamapin, isolated from the venom of the Indian red scorpion, Mesobuthus tamulus, is a 31 peptide (mass 3458). The toxin inhibits 125I-apamin binding to rat synaptosomal plasma membranes (Ki = 12pM). Tamapin has 6 residues and its amino acid sequence is homologous to and other scorpion venom which block apamin-sensitive, low conductance Ca2+-activated K+ channels (SK channels). When applied to CA1 pyramidal neurons in brain slices, tamapin (10 nM) selectively blocked the apamin-sensitive, medium duration afterhyperpolarizing current (I(AHP); 98.21.2% block; n=5), but had no effect on the apamin-insensitive slow AHP current (sI(AHP); 2.04.4% block; n=5). In current clamp, tamapin reduced the medium AHP and caused an attenuation of spike frequency adaptation in CA1 neurons. To test the effect of tamapin on cloned SK channels with defined subunit compositions, SK2 and SK3 channels were stably expressed in HEK293 cells. Tamapin blocked SK2 currents (IC(50) = 38.9pM) in a partially reversible manner. Tamapin is 1.5-fold more potent than apamin and 10-fold more potent than scyllatoxin in blocking SK2 channels. Tamapin should prove a useful and potent new tool to study SK channel structure and function.

•Diewald, L., Heimrich, B., Busselberg, D., Watanabe, T. & Haas, H.L.

Page 12 AHP, BK- and SK-channel references

(1997). Histaminergic system in co-cultures of hippocampus and posterior hypothalamus: a morphological and electrophysiological study in the rat. European Journal of Neuroscience 9(11): 2406-2413. Neurons of the tuberomammillary nucleus in the posterior hypothalamus diffusely project to most parts of the , where their main transmitter, histamine, modulates the excitability of the target neurons. The development of a histaminergic hypothalamo-hippocampal pathway and its function were studied in organotypic co-cultures. Immunocytochemistry for histidine decarboxylase, the specific synthesizing enzyme, stained clusters of neurons in the hypothalamic tuberomammillary area. Immunolabelled varicose processes innervated the co-cultured hippocampus and established a few synaptic contacts on dendrites. Cultured tuberomammillary neurons displayed their typical membrane properties and were spontaneously active. In hippocampal pyramidal cells of the CA3 region the long-lasting afterhyperpolarization was reduced by histamine or impromidine and increased by the H2 antagonist cimetidine, but not by the mepyramine. The membrane potential was depolarized in presence of an H2 agonist and hyperpolarized by an H2 antagonist. In single hippocampal cultures histamine antagonists did not affect afterhyperpolarization and membrane potential. Histaminergic neurons retain their main morphological and physiological characteristics in slice cultures and establish a functional connection with co- cultured target cells.

•Egorov, A.V., Gloveli, T. & Muller, W. (1999). Muscarinic control of dendritic excitability and Ca2+ signaling in CA1 pyramidal neurons in rat hippocampal slice. Journal of Neurophysiology 82(4): 1909-1915. The system is critically involved in synaptic models of and memory by enhancing dendritic [Ca(2+)](i) signals. Diffuse cholinergic innervation suggests subcellular modulation of membrane currents and Ca(2+) signals. Here we use ion-selective microelectrodes to study spread of carbachol (CCh) after focal application into brain slice and subcellular muscarinic modulation of synaptic responses in CA1 pyramidal neurons. Proximal application of CCh rapidly blocked the somatic slow afterhyperpolarization (sAHP) following repetitive stimulation. In contrast, the time course of potentiation of the slow tetanic depolarization (STD) during synaptic input was slower and followed the time course of spread of CCh to the dendritic tree. With distal application, augmentation of the somatic STD and of dendritic Ca(2+) responses followed spread of CCh to the entire

Page 13 AHP, BK- and SK-channel references apical dendritic tree, whereas the sAHP was blocked only after spread of CCh to the proximal dendritic segment. In dendritic recordings, CCh blocked a small sAHP, augmented the STD, and rather reduced dendritic action potentials. Augmentation of dendritic Ca(2+) signals was highly correlated to augmentation of the STD. The NMDA DL-2-amino-5-phosphonovaleric acid (APV) blocked approximately 55% of the STD in control and during CCh application. In conclusion, muscarinic suppression of the proximal sAHP can augment firing and thereby Ca(2+) responses. Dendritic augmentation of the STD by blockade of the sAHP and direct enhancement of N-methyl-D-aspartate (NMDA) receptor-mediated currents potentiates Ca(2+) signals even when firing is not affected due to suprathreshold input. In this way, subcellular muscarinic modulation may contribute to parallel information processing and storage by dendritic synapses of CA1 pyramidal neurons.

•Empson, R.M. & Jefferys, J.G. (2001). Ca2+ entry through L-type Ca2+ channels helps terminate epileptiform activity by activation of a Ca2+ dependent afterhyperpolarisation in hippocampal CA3. Neuroscience 102(2): 297-306. In CA3 neurons of disinhibited hippocampal slice cultures the slow afterhyperpolarisation, following spontaneous epileptiform burst events, was confirmed to be Ca(2+) dependent and mediated by K(+) ions. Apamin, a selective blocker of the SK channels responsible for part of the slow afterhyperpolarisation reduced, but did not abolish, the amplitude of the post-burst afterhyperpolarisation. The result was an increased excitability of individual CA3 cells and the whole CA3 network, as measured by burst duration and burst frequency. Increases in excitability could also be achieved by strongly buffering intracellular Ca(2+) or by minimising Ca(2+) influx into the cell, specifically through L-type (but not N-type) voltage operated Ca(2+) channels. Notably the L-type Ca(2+) channel antagonist, nifedipine, was more effective than apamin at reducing the post-burst afterhyperpolarisation. Nifedipine also caused a greater increase in network excitability as determined from measurements of burst duration and frequency from whole cell and extracellular recordings. N-methyl D-aspartate receptor activation contributed to the depolarisations associated with the epileptiform activity but Ca(2+) entry via this route did not contribute to the activation of the post-burst afterhyperpolarisation.We suggest that Ca(2+) entry through L-type channels during an epileptiform event is selectively coupled to both apamin-sensitive and - insensitive Ca(2+) activated K(+) channels. Our findings have implications for how the route of Ca(2+) entry and subsequent Ca(2+) dynamics can influence network

Page 14 AHP, BK- and SK-channel references excitability during epileptiform discharges.

•Engel, J., Schultens, H.A. & Schild, D. (1999). Small conductance potassium channels cause an activity-dependent spike frequency adaptation and make the transfer function of neurons logarithmic. Biophysical Journal 76(3): 1310-9. We made a computational model of a single to study the effect of the small conductance (SK) Ca2+-dependent K+ channel on spike frequency adaptation. The model neuron comprised a Na+ conductance, a Ca2+ conductance, and two Ca2+- independent K+ conductances, as well as a small and a large (BK) Ca2+-activated K+ conductance, a Ca2+ pump, and mechanisms for Ca2+ buffering and diffusion. Sustained current injection that simulated synaptic input resulted in a train of action potentials (APs) which in the absence of the SK conductance showed very little adaptation with time. The transfer function of the neuron was nearly linear, i.e., both asymptotic spike rate as well as the intracellular free Ca2+ concentration ([Ca2+]i) were approximately linear functions of the input current. Adding an SK conductance with a steep nonlinear dependence on [Ca2+]i (. Pflugers Arch. 422:223- 232; Kohler, Hirschberg, Bond, Kinzie, Marrion, Maylie, and Adelman. 1996. Science. 273:1709-1714) caused a marked time-dependent spike frequency adaptation and changed the transfer function of the neuron from linear to logarithmic. Moreover, the input range the neuron responded to with regular spiking increased by a factor of 2.2. These results can be explained by a shunt of the cell resistance caused by the activation of the SK conductance. It might turn out that the logarithmic relationships between the stimuli of some modalities (e.g., sound or light) and the perception of the stimulus intensity (Fechner's law) have a cellular basis in the involvement of SK conductances in the processing of these stimuli.

•Erdemli, G., Xu, Y.Z. & Krnjevic, K. (1999). Potassium conductance causing hyperpolarization of CA1 hippocampal neurons during hypoxia. J Neurophysiol 80(5): 2378-2390. In experiments on slices (from 100- to 150-g Sprague-Dawley rats) kept at 33°C, we studied the effects of brief hypoxia (2-3 min) on CA1 neurons. In whole cell recordings from submerged slices, with electrodes containing only KMeSO4 and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and in the presence of kynurenate and (to minimize transmitter actions), hypoxia produced the following changes: under current clamp, 36 cells were hyperpolarized by 2.7 ± 0.5 (SE) mV and their input resistance (Rin) fell by 23 ± 2.7%; in 30 cells under voltage

Page 15 AHP, BK- and SK-channel references clamp, membrane current increased by 114 ± 22.3 pA and input conductance (Gin) by 4.9 ± 0.9 nS. These effects are much greater than those seen previously with K gluconate whole cell electrodes, but only half those seen with "sharp" electrodes. The hypoxic hyperpolarizations (or outward currents) were not reduced by intracellular ATP (1-5 mM) or bath-applied glyburide (10 µM): therefore they are unlikely to be mediated by conventional ATP-sensitive K channels. On the other hand, their depression by internally applied ethylene glycol-bis-(beta-aminoethyl ether)- N,N, N',N'-tetraacetic acid (1.1 and 11 mM) and especially 1, 2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (11-33 mM) indicated a significant involvement of Ca-dependent K (KCa) channels. The beta-adrenergic agonist isoprenaline (10 microM) reduced hypoxic hyperpolarizations and decreases in Rin (n = 4) (and in another 11 cells corresponding changes in Gin); and comparable but more variable effects were produced by internally applied 3':5'-adenosine cyclic monophosphate (cAMP, 1 mM, n = 6) and bath-applied 8-bromo-cAMP (n = 8). Thus afterhyperpolarization-type KCa channels probably take part in the hypoxic response. A major involvement of G proteins is indicated by the near total suppression of the hypoxic response by guanosine 5'-O-(3-thiotriphosphate) (0. 1-0.3 mM, n = 23) and especially guanosine 5'-O-(2-thiodiphosphate) (0.3 mM, n = 26), both applied internally. The adenosine antagonist 8-(p-sulfophenyl)theophylline (10-50 µM) significantly reduced hypoxic hyperpolarizations and outward currents in whole cell recordings (with KMeSO4 electrodes) from submerged slices but not in intracellular recordings (with KCl electrodes) from slices kept at gas/saline interface. In further intracellular recordings, antagonists of gamma-aminobutyric acid-B or serotonin receptors also had no clear effect. In conclusion, these G-protein- dependent hyperpolarizing changes produced in CA1 neurons by hypoxia are probably initiated by Ca2+ release from internal stores stimulated by enhanced glycolysis and a variable synergistic action of adenosine.

•Figenschou, A., Hu, G.Y. & Storm, J.F. (1996). Cholinergic modulation of the action potential in rat hippocampal neurons. European Journal of Neuroscience 8(1): 211-219. The cholinergic input to the hippocampus from the medial septum is important for modulating hippocampal activity and functions, including theta rhythm and spatial learning. Neuromodulation by transmitters in central nervous system neurons usually affects cell excitability by modifying the membrane potential, discharge pattern and spike frequency. Here we describe another type of

Page 16 AHP, BK- and SK-channel references neuromodulation: changing the action potential waveform. During intracellular recordings from CA1 pyramidal cells in hippocampal slices from rats, the cholinergic agonist carbachol caused several reversible changes in the action potential: low doses (2 µM) caused an increase in spike duration; high doses (10-40µM) or long- lasting applications also reduced the spike amplitude and rate of rise, and raised the spike threshold. These effects are similar to those of metabotropic glutamate receptor agonists or phorbol esters, both of which activate protein kinase C. The effects were blocked by the , and were prevented by Ca2+-free medium and by Ca2+-channel blockers. However, the cholinergic spike modulation was not occluded or mimicked by blocking the Ca2+-dependent K+ currents IC or IAHP, suggesting that these K+ currents are not involved in the modulation. We conclude that muscarinic receptor activation modulates the action potential in CA1 pyramidal cells via a Ca2+-dependent mechanism, possibly involving protein kinase C. This modulation and the similar effects mediated by metabotropic glutamate receptors to our knowledge provide the only examples of neuromodulation of the action potential in the central nervous system-a form of modulation known to regulate Ca2+ influx and transmitter release, and to mediate and learning in invertebrates.

•Gant, J.C. & Thompson, L.T. (2000). Calmodulin mediated plasticity in rat CA1 neurons following spatial learning. Society for Neuroscience Abstracts 30(73.11). Transient increases in pyramidal neuron excitability in the hippocampus following acquisition in multiple learning paradigms demonstrates a conserved role for K+ channel-mediated plasticity in memory consolidation. Following consolidation, long after the transient plasticity has decayed to a basal state, a strong relationship persists between neuronal excitability and learning rate. Rats with the highest basal hippocampal pyramidal neuron excitability exhibit the most rapid learning capacity. Both spike-frequency accommodation and the post-burst afterhyperpolarization (AHP) are involved, and an examination of intracellular signaling pathways should reveal mechanisms determining the excitability state under different conditions and at different times. The Ca2+-dependence of the K+ conductances responsible for the AHP indicate that cytosolic Ca2+-mediated pathways regulate long lasting changes in neuronal function associated with both learning and neural plasticity. Specifically, the multiple roles of calmodulin mediated pathways were examined in the present studies. Intracellular and whole-cell methods were utilized in a submerged ventral slice preparation to examine the influence of

Page 17 AHP, BK- and SK-channel references calmodulin inhibitors and calmodulin-linked enzyme systems on excitability measures. Ca2+ signaling was manipulated both directly and indirectly, and the results examined in the context of intrinsic and learning-dependent plasticity. Calmodulin has effects on excitability not only as the calcium sensor for the calcium-dependent IAHP, but also indirect effects via calmodulin-dependent effector pathways.

•Gant, J.C. & Thompson, L.T. (2001). Calmodulin influences neural excitability through interaction with SK channels in hippocampal CA1 neurons. Society for Neuroscience Abstracts 31(924.5): 476. Following learning there is a transient increase in excitability of CA1 and CA3 pyramidal neurons in the hippocampus. Changes in neuronal excitability are mediated by Ca2+-dependent K+ currents, which underlie the afterhyperpolarization (AHP). Calmodulin influences excitability in hippocampal neurons through interaction with SK channels, serving as the constitutive Ca2+ sensor for channel activation. Application of peptide CAM inhibitors results in a dose-dependent reduction of AHP amplitude and duration. In order to demonstrate that the influence of calmodulin inhibitors on excitability is mediated directly through SK channel interaction, SK channel specific antagonists were used in conjunction with calmodulin inhibitors. In the present study, we demonstrate that application of W-13 reduces AHP amplitude and decreases spike frequency accommodation. Dequalinium chloride also influences neural excitability in a similar fashion. Following saturating applications of W-13, a saturating concentration of dequalinium chloride fails to reduce AHP amplitude and accommodation further. The results of this experiment are consistent with CAM mediating CA1 neural excitability via its constitutive interaction with SK channels.

•Gant, J.C. & Thompson, L.T. (2002). Calmodulin protein expression, calmodulin- mediated neuronal excitability, and learning in the aging hippocampus. Society for Neuroscience Abstracts 32. Plasticity in the excitability of hippocampal pyramidal cell neurons has been linked to learning in multiple species and tasks. In young animals, changes in postsynaptic properties of CA1 neurons, including the post-burst AHP and spike frequency accommodation, reflect learning abilities transiently and predictively. This plasticity can be preserved in aging, although fewer aging individuals exhibit increased excitability. Age-dependent declines in modulation of post-synaptic plasticity are linked to age-dependent declines in learning ability. Although multiple calcium-dependent K+ currents may contribute to the AHP in CA1 neurons, those

Page 18 AHP, BK- and SK-channel references gated by SK channels are sensitive to calmodulin (CAM) and CAM inhibitors. We continue to study the role of CAM in learning-related plasticity and the impact of aging on this plasticity. Rat spatial learning has served as a useful model for studying age-dependent hippocampal plasticity, and aging Fisher, Long Evans, and Sprague Dawley rats all exhibit age-dependent deficits in acquisition of a long-delay radial-arm maze working memory task. Immunostaining of fixed rat brains using monoclonal antibodies demonstrated age-dependent changes in expression of CAM and CAM-dependent membrane-associated and intracellular proteins. In intracellular recordings from basal temporal lobe slices, CAM inhibitors dose- and age-dependently reduced both the AHP and accommodation. The effect of CAM inhibitors on the AHP was also compared to that of apamin and other SK channel inhibitors, again revealing age- and dose-dependent effects. The relation between these and earlier findings will be discussed.

•Gerlach, A.C., Khawaled, R., Tzounopoulos, T., Maylie, J. & Adelman, J.P. (2001). Calcium may activate the medium and slow afterhyperpolarization of hippocampal CA1 neurons by different mechanisms. Society for Neuroscience Abstracts 31(41.14): 20. Currents underlying the afterhyperpolarization (IAHP) in hippocampal CA1 pyramidal neurons were studied in freshly prepared brain slices using whole cell voltage clamp. Activation of the ImAHP and IsAHP, recorded upon , depended on the duration and amplitude of the activating pulse. The apamin-sensitive ImAHP activated during the depolarizing pulse and decayed immediately upon repolarization. The amplitude of the ImAHP increased exponentially as a function of pulse duration with a time constant of ~225 ms. In contrast, the IsAHP had a distinct rising phase, activating with a latency of approximately 150 ms from the beginning of the depolarizing pulse. The rising phase of the IsAHP reached half- maximal amplitude approximately 250 ms from the beginning of the depolarizing pulse. The amplitude of the IsAHP increased exponentially as a function of pulse duration with a time constant of ~50 ms. The latency to onset, the half-rise time, and the time constant of the envelope of tails of the IsAHP were independent of pulse duration or amplitude and were not altered when the current was reduced by isoproterenol. The results are consistent with the ImAHP channels corresponding to the cloned SK channels and direct gating by Ca2+ influx. The IsAHP demonstrates latency to onset and half-rise time suggesting that the IsAHP channels are not

Page 19 AHP, BK- and SK-channel references gated directly by Ca2+ influx through voltage-gated Ca2+ channels. A Ca2+- dependent intracellular process with voltage- and time-invariant kinetics may mediate activation of the IsAHP. Supported by: NIH

•Giese, K.P., Peters, M. & Vernon, J. (2001). Modulation of excitability as a learning and memory mechanism: A molecular genetic perspective. Physiology & Behavior 73(5): 803-810. Gene targeting has contributed substantially to the investigation of the neurobiological basis of mammalian learning and memory (L&M). These experiments start with an hypothesis as to a mechanism underlying L&M, then genes of interest are manipulated, and the impact on neuronal physiology and L&M is studied. Previous gene targeting studies have focussed mainly on the role of synaptic plasticity in L&M. Some of those reports provide evidence that processes other than, or additional to, long-term potentiation (LTP) are required for L&M. Accordingly, it is possible that altered neuronal excitability is an essential mechanism. The properties of ion channels determine neuronal excitability and so genetic alteration of properties is an appropriate method for testing whether the modulation of excitability affects L&M. K(v)beta 1.1-deficient mice were the first mutants used to study the role of altered excitability in mammalian L&M. K(v)beta 1.1 is a regulatory subunit with a restricted expression pattern in the brain, and it confers fast inactivation on otherwise noninactivating K(+) channel subunits. In hippocampal pyramidal neurones Kv beta 1.1-deficiency results in a reduced slow after- hyperpolarisation (sAHP), modulation of which is thought to contribute to L&M. The L&M phenotype of the mutants supports this sAHP hypothesis. It is expected that further gene targeting studies on excitability will lead to valuable insights into the processes of L&M.

•Giese, K.P., Storm, J.F., Reuter, D., Fedorov, N.B., Shao, L.R., Leicher, T., Pongs, O. & Silva, A.J. (1998). Reduced K+ channel inactivation, spike broadening, and after-hyperpolarization in Kvbeta1.1-deficient mice with impaired learning. Learning and Memory 5(4-5): 257-273. A-type K+ channels are known to regulate neuronal firing, but their role in repetitive firing and learning in mammals is not well characterized. To determine the contribution of the auxiliary K+ channel subunit Kvbeta1.1 to A-type K+ currents and to study the physiological role of A-type K+ channels in repetitive firing and

Page 20 AHP, BK- and SK-channel references learning, we deleted the Kvbeta1.1 gene in mice. The loss of Kvbeta1.1 resulted in a reduced K+ current inactivation in hippocampal CA1 pyramidal neurons. Furthermore, in the mutant neurons, frequency-dependent spike broadening and the slow afterhyperpolarization (sAHP) were reduced. This suggests that Kvbeta1.1-dependent A-type K+ channels contribute to frequency-dependent spike broadening and may regulate the sAHP by controlling Ca2+ influx during action potentials. The Kvbeta1.1-deficient mice showed normal synaptic plasticity but were impaired in the learning of a water maze test and in the social transmission of food preference task, indicating that the Kvbeta1.1 subunit contributes to certain types of learning and memory.

•Golding, N.L., Jung, H.Y., Mickus, T. & Spruston, N. (1999). Dendritic calcium spike initiation and repolarization are controlled by distinct subtypes in CA1 pyramidal neurons. Journal of Neuroscience 19(20): 8789-8798. In CA1 pyramidal neurons of the hippocampus, calcium-dependent spikes occur in vivo during specific behavioral states and may be enhanced during epileptiform activity. However, the mechanisms that control calcium spike initiation and repolarization are poorly understood. Using dendritic and somatic patch-pipette recordings, we show that calcium spikes are initiated in the apical dendrites of CA1 pyramidal neurons and drive bursts of sodium-dependent action potentials at the soma. Initiation of calcium spikes at the soma was suppressed in part by potassium channels activated by sodium-dependent action potentials. Low-threshold, putative D-type potassium channels [blocked by 100 µM 4-aminopyridine (4-AP) and 0.5-1 µM alpha- (alpha-DTX)] played a prominent role in setting a high threshold for somatic calcium spikes, thus restricting initiation to the dendrites. DTX- and 4-AP-sensitive channels were activated during sodium-dependent action potentials and mediated a large component of their afterhyperpolarization. Once initiated, repetitive firing of calcium spikes was limited by activation of putative BK-type calcium-activated potassium channels (blocked by 250 µM tetraethylammonium chloride, 70 nM , or 100 nM iberiotoxin). Thus, the concerted action of calcium- and voltage-activated potassium channels serves to focus spatially and temporally the membrane depolarization and calcium influx generated by calcium spikes during strong, synchronous network excitation.

•Gong, L., Gao, T.M., Li, X., Huang, H. & Tong, Z. (2000). Enhancement in activities of large conductance calcium-activated potassium channels in CA1

Page 21 AHP, BK- and SK-channel references pyramidal neurons of rat hippocampus after transient forebrain ischemia. Brain Research 884(1-2): 147-154. It has been reported previously that the neuronal excitability persistently suppresses and the amplitude of fast afterhyperpolarization (fAHP) increases in CA1 pyramidal cells of rat hippocampus following transient forebrain ischemia. To understand the conductance mechanisms underlying these post-ischemic electrophysiological alterations, we compared differences in activities of large conductance Ca(2+)-activated potassium (BK(Ca)) channels in CA1 pyramidal cells acutely dissociated from hippocampus before and after ischemia by using inside-out configuration of patch clamp techniques. (1) The unitary conductance of BK(Ca) channels in post-ischemic neurons (295 pS) was higher than that in control neurons (245 pS) in symmetrical 140/140 mM K(+) in inside-out patch; (2) the membrane depolarization for an e-fold increase in open probability (P(o)) showed no significant differences between two groups while the membrane potential required to produce one-half of the maximum P(o) was more negative after ischemia, indicating no obvious changes in channel voltage dependence; (3) the [Ca(2+)](i) required to half activate BK(Ca) channels was only 1 microM in post-ischemic whereas 2 microM in control neurons, indicating an increase in [Ca(2+)](i) sensitivity after ischemia; and (4) BK(Ca) channels had a longer open time and a shorter closed time after ischemia without significant differences in open frequency as compared to control. The present results indicate that enhanced activity of BK(Ca) channels in CA1 pyramidal neurons after ischemia may partially contribute to the post-ischemic decrease in neuronal excitability and increase in fAHP.

•Haug, T. & Storm, J.F. (2000). Protein kinase A mediates the modulation of the slow Ca2+-dependent K+ current, IsAHP, by the neuropeptides CRF, VIP, and CGRP in hippocampal pyramidal neurons. Journal of Neurophysiology 83(4): 2071-2079. We have studied modulation of the slow Ca2+-activated K(+) current (I(sAHP)) in CA1 hippocampal pyramidal neurons by three peptide transmitters: corticotropin releasing factor (CRF, also called corticotropin releasing hormone, CRH), vasoactive intestinal peptide (VIP), and calcitonin gene-related peptide (CGRP). These are known to be expressed in interneurons. Using whole cell voltage clamp in hippocampal slices from young rats, in the presence of tetrodotoxin (TTX, 0.5 microM) and tetraethylammonium (TEA, 5 mM), I(sAHP) was measured after a brief depolarizing voltage step eliciting inward Ca2+ current. Each of the peptides CRF (100-250 nM), VIP (400 nM), and CGRP (1 microM) significantly reduced the

Page 22 AHP, BK- and SK-channel references amplitude of I(sAHP). Thus the I(sAHP) amplitude was reduced to 22% by 100 nM CRF, to 17% by 250 nM CRF, to 22% by 400 nM VIP, and to 40% by 1 microM CGRP. We found no consistent concomitant changes in the Ca2+ current or in the time course of I(sAHP) for any of the three peptides, suggesting that the suppression of I(sAHP) was not secondary to a general suppression of Ca2+ channel activity. Because each of these peptides is known to activate the cyclic AMP (cAMP) cascade in various cell types, and I(sAHP) is known to be suppressed by cAMP via the cAMP- dependent protein kinase (PKA), we tested whether the effects on I(sAHP) by CRF, VIP, and CGRP are mediated by PKA. Intracellular application of the PKA-inhibitor Rp-cAMPS significantly reduced the suppression of I(sAHP) by CRF, VIP, and CGRP. Thus with 1 mM Rp-cAMPS in the recording pipette, the average suppression of I(sAHP) was reduced from 78 to 26% for 100 nM CRF, from 83 to 32% for 250 nM CRF, from 78 to 30% for 400 nM VIP, and from 60 to 7% for 1"µM CGRP. We conclude that CRF, VIP, and CGRP suppress the slow Ca2+-activated K(+) current, I(sAHP), in CA1 hippocampal pyramidal neurons by activating the cAMP-dependent protein kinase, PKA. Together with the monoamine transmitters norepinephrine, serotonin, histamine, and , these peptide transmitters all converge on the cAMP cascade modulating I(sAHP).

•Heuss, C., Scanziani, M., Gähwiler, B.H. & Gerber, U. (1999). G-protein- independent signaling mediated by metabotropic glutamate receptors. Nature Neuroscience 2(12): 1070-1077. Synaptically released glutamate activates ionotropic and metabotropic receptors at central synapses. Metabotropic glutamate receptors (mGluRs) are thought to modulate membrane conductances through transduction cascades involving G proteins. Here we show, in CA3 pyramidal cells from rat hippocampus, that synaptic activation of type 1 mGluRs by mossy fiber stimulation evokes an excitatory postsynaptic response independent of G-protein function, while inhibiting an afterhyperpolarization current through a G-protein-coupled mechanism. Experiments using peptide activators and specific inhibitors identified a Src-family protein kinase as a component of the G-protein-independent transduction pathway. These results represent the first functional evidence for a dual signaling mechanism associated with a heptahelical receptor such as mGluR1, in which intracellular transduction involves activation of either G proteins or tyrosine kinases.

Page 23 AHP, BK- and SK-channel references

•Hirschberg, B., Maylie, J., Adelman, J.P. & Marrion, N.V. (1999). Gating properties of single SK channels in hippocampal CA1 pyramidal neurons. Biophysical Journal 77(4): 1905-13. The activation of small-conductance calcium-activated potassium channels (SK) has a profound effect on membrane excitability. In hippocampal pyramidal neurons, SK channel activation by Ca2+ entry from a preceding burst of action potentials generates the slow afterhyperpolarization (AHP). Stimulation of a number of receptor types suppresses the slow AHP, inhibiting spike frequency adaptation and causing these neurons to fire tonically. Little is known of the gating properties of native SK channels in CNS neurons. By using excised inside-out patches, a small- amplitude channel has been resolved that was half-activated by approximately 0.6 µM Ca2+ in a voltage-independent manner. The channel possessed a slope conductance of 10 pS and exhibited nonstationary gating. These properties are in accord with those of cloned SK channels. The measured Ca2+ sensitivity of hippocampal SK channels suggests that the slow AHP is generated by activation of SK channels from a local rise of intracellular Ca2+.

•Hu, H., Sailer, C.A., Trieb, M., Schwarzer, C., Knaus, H.G. & Storm, J.F. (2000). Differential distribution and functional expression of apamin-sensitive small-conductance Ca-activated K channels in rat hippocampus and dentate gyrus. Society for Neuroscience Abstracts 30. Small-conductance Ca-activated K (SK) channels are important for excitability control and afterhyperpolarizations (AHPs) in vertebrate neurons. Using whole-cell recording in hippocampal slices from young rats, we have compared Ca-activated K currents (SK or AHP currents) in CA1 and CA3 pyramidal cells and dentate gyrus (DG) granule cells. To isolate the SK currents, recordings were done in presence of Na, M and BK channel blockers. In all 3 cell types, voltage steps eliciting Ca influx were followed by biphasic outward tail currents consisting of a medium I(mAHP) lasting < 1 s and a slow I(sAHP) current lasting 4-8 s. I(mAHP) was far smaller in the DG granule cells than in the CA1 and CA3 pyramidal cells. Furthermore, although apamin (100 nM) blocked I(mAHP) virtually completely in CA1/CA3, it had little or no effect on DG tail currents. The apamin-sensitive current was about 100 pA in the CA1 and CA3, but < 5 pA in DG cells. Immunohistochemistry revealed a restricted distribution of SK2 protein with high densities in the hippocampal CA1-CA3 and substantially lower level in the dentate gyrus (DG). In contrast, SK3 protein was distributed more diffusely throughout the

Page 24 AHP, BK- and SK-channel references brain. Immunoprecipitation detected solely homotetrameric SK channels in association with calmodulin, in native tissue. The close agreement between the SK2 distribution and expression of apamin-sensitive currents in CA1-CA3, strongly suggests that SK2 homotetramers underlie the apamin-sensitive medium afterhyperpolarizations in rat hippocampus.

•Hu, H., Shao, L.R., Chavoshy, S., Gu, N., Trieb, M., Behrens, R., Laake, P., Pongs, O., Knaus, H.G., Ottersen, O.P. & Storm, J.F. (2001). Presynaptic Ca2+-activated K+ channels in glutamatergic hippocampal terminals and their role in spike repolarization and regulation of transmitter release. Journal of Neuroscience 21(24): 9585-9597. Large-conductance Ca2+-activated K(+) channels (BK, also called Maxi-K or Slo channels) are widespread in the vertebrate nervous system, but their functional roles in synaptic transmission in the mammalian brain are largely unknown. By combining electrophysiology and immunogold cytochemistry, we demonstrate the existence of functional BK channels in presynaptic terminals in the hippocampus and compare their functional roles in somata and terminals of CA3 pyramidal cells. Double-labeling immunogold analysis with BK channel and glutamate receptor antibodies indicated that BK channels are targeted to the presynaptic membrane facing the synaptic cleft in terminals of Schaffer collaterals in stratum radiatum. Whole-cell, intracellular, and field-potential recordings from CA1 pyramidal cells showed that the presynaptic BK channels are activated by calcium influx and can contribute to repolarization of the presynaptic action potential (AP) and negative feedback control of Ca2+ influx and transmitter release. This was observed in the presence of 4-aminopyridine (4-AP, 40-100 microm), which broadened the presynaptic compound action potential. In contrast, the presynaptic BK channels did not contribute significantly to regulation of action potentials or transmitter release under basal experimental conditions, i.e., without 4-AP, even at high stimulation frequencies. This is unlike the situation in the parent cell bodies (CA3 pyramidal cells), where BK channels contribute strongly to action potential repolarization. These results indicate that the functional role of BK channels depends on their subcellular localization.

•Huang, C.C., Tsai, J.J. & Hsu, K.S. (1997). L-deprenyl (selegiline) limits the repetitive firing of action potentials in rat hippocampal CA1 neurons via a mechanism. Brain Research 753(1): 27-35.

Page 25 AHP, BK- and SK-channel references

The effects of L-deprenyl (selegiline), a highly selective monoamine oxidase type B (MAO-B) inhibitor, on cell excitability of rat hippocampal CA1 neurons were examined in slice preparations using intracellular recording techniques. Superfusion of L-deprenyl (10 and 20 µM) reversibly limited the repetitive firing (RF) of action potentials elicited by injection of depolarizing current pulses (100 ms) into the pyramidal cells. At a concentration of 1-50 µM, L-deprenyl did not alter resting membrane potential or input resistance of the hippocampal CA1 neurons. The limitation of RF by L-deprenyl (20 µM) was accompanied by the reduction of the maximal rate of rise (Vmax) of the action potentials in a non-voltage-dependent manner. In 80% of recorded cells, application of L-deprenyl (20 µM) produced an increase in the amplitude and duration of afterhyperpolarization (AHP). The limitation of L-deprenyl on RF was mimicked by other MAO-B inhibitors, pargyline and 4-phenylpyridine. In addition, the ability of L-deprenyl to limit RF was not observed in the hippocampal CA1 neurons taken from dopamine (DA)-depleted rats. Moreover, we also observed that the L-deprenyl-induced limitation of RF was specifically antagonized by (±)-7-bromo-8-hydroxy-3-methyl-1-phenyl-2,3,4,5- tetrahydro-1H-3-benzaz epine (SKF-83566, 5 µM), a selective D1 dopaminergic receptor antagonist. However, the D2 dopaminergic receptor antagonist, sulpiride (5 µM), had no effect on L-deprenyl's action. These results indicate that the MAO-B inhibitory ability leading to an increase of the dopaminergic tone in the hippocampus is involved, at least in part, in the L-deprenyl-induced reduction of neuronal excitability in the CA1 region of rat hippocampus and that the D1 dopaminergic receptor is involved in L-deprenyl's action.

•Ireland, D.R. & Abraham, W.C. (2001). Multiple metabotropic glutamate receptor subtypes mediate persistent excitability changes in hippocampal CA1 pyramidal neurons. Society for Neuroscience Abstracts 31(913.8). Previous studies have implicated Group I metabotropic glutamate receptors (mGluRs) in the mGluR-mediated increase in excitability in hippocampal CA1 pyramidal neurons. In this study we used mGluR antagonists to investigate in more detail the mGluR receptor types underlying this effect. Acute hippocampal slices from rats were maintained in vitro at 32.5°C and intracellular current-clamp recordings made from CA1 pyramidal neurons manually held at -65 mV. Application of the Group I mGluR agonist, (RS)-3,5-dihydroxyphenylglycine (DHPG), abolished the slow afterhyperpolarization (sAHP) and medium AHP (mAHP) and caused a consequent increase in cell excitability. Input resistance was increased and membrane

Page 26 AHP, BK- and SK-channel references depolarization was inferred by a change in holding current. With the exception of the suppression of the mAHP, these effects were persistent, with the suppression of the sAHP lasting for more than 1 hour after agonist washout. Preincubation of the specific mGluR5 antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), reduced but did not prevent the effects of DHPG. Preincubation of MPEP together with the mGluR1 antagonist, 1-aminoindan-1,5-dicarboxylic acid (AIDA) had no greater effect on the DHPG-induced changes than did MPEP alone. However, preincubation in the broad-spectrum mGluR antagonist, LY341495, completely prevented all DHPG-induced changes. These results demonstrate that the DHPG-induced increase in excitability in CA1 pyramidal neurons is partly mediated by mGluR5, consistent with previous pharmacological and localization studies, and partly by a different, possibly novel, type of mGluR.

•Jahromi, B.S., Zhang, L., Carlen, P.L. & Pennefather, P. (1999). Differential time-course of slow afterhyperpolarizations and associated Ca2+ transients in rat CA1 pyramidal neurons: Further dissociation by Ca2+ buffer. Neuroscience 88(3): 719-726. Hippocampal neurons exhibit a slow afterhyperpolarization following membrane depolarization; this is thought to reflect an underlying Ca2+-dependent K+ current. This current is potentiated by intermediate concentrations (0.1-1.0 mM) of exogenous Ca2+ buffer [Schwindt P. C. et al. (1992) Neuroscience 47, 571-578; Zhang L. et al. (1995) J. Neurophysiol. 74, 2225-2241]. The relationship between the slow afterhyperpolarization and associated Ca2+ transients was investigated in the presence and absence of added exogenous Ca2+ buffer. Slow afterhyperpolarizations and underlying K+ currents were measured using whole-cell patch-clamp recordings from hippocampal CA1 neurons in acute rat brain slices. Inclusion of fluorescent Ca2+ indicators in the patch pipette solution allowed simultaneous measurement of the evoked subcellular Ca2+ transients using a confocal microscope. The peak Ca2+ signal exhibited an incremental increase with each action potential. This increase eventually reached a plateau with increasing numbers of action potentials, suggesting dye saturation with peak Ca2+ concentrations. As the K(D) for Ca2+ of the indicator dyes used was between 200 and 300 nM, it is predicted that saturation will occur when the peak Ca2+ signal exceeds 1 microM. This occurred with fewer action potentials in dendritic vs somatic compartments. Neither compartment exhibited averaged Ca2+ transients matching the slow afterhyperpolarization time-course, dendritic Ca2+ transients being most divergent.

Page 27 AHP, BK- and SK-channel references

Intracellular accumulation of exogenous Ca2+ buffer, either by inclusion in the patch pipette or by incubation of the brain slice with its membrane-permeable form, caused a prolongation of the slow afterhyperpolarization but not of the somatic Ca2+ transient. The initial rate of decline of the dendritic Ca2+ transient was diminished, but remained faster than that of the slow afterhyperpolarization. We conclude that neither dendritic nor somatic Ca2+ signals match the slow afterhyperpolarization time-course, with this dissociation being further magnified by addition of exogenous Ca2+ buffer. The implications of this result are discussed.

•Jouvenceau, A., Dutar, P. & Billard, J.M. (1997). Is activation of the metabotropic glutamate receptors impaired in the hippocampal CA1 area of the aged rat? Hippocampus 7(5): 455-459. The effects of aging on activation of metabotropic glutamate (mGlu) receptors were studied in the CA1 field of hippocampal slices from young (3- to 4- month-old) and aged (24- to 27-month-old) Sprague-Dawley rats with the use of ex vivo electrophysiological recording techniques. The depolarization of membrane potential, the increase in input resistance, and the blockade of the afterhyperpolarization induced in pyramidal cells of young rats by bath application of the mGlu receptor agonist (±)-trans-1-aminocyclopentate-1,3-dicarboxylic acid were not altered in aged animals. No age-related changes of the depressive effects of the mGlu receptor agonist were found on either the excitatory glutamatergic postsynaptic potential or the GABA-mediated inhibitory postsynaptic potentials induced by the stimulation of the stratum radiatum. The magnitude of synaptic plasticity involving mGlu receptor activation, although weaker, was not significantly altered in aged rats. This absence of age-related effects on activation of mGlu receptors may be important in understanding the possible origins of the alterations in neuronal plasticity which occur in brain aging.

•Kamal, A., Ramakers, G.M.J., Artola, A., Biessels, G.-J. & Gispen, W.H. (2001). Increased sAHP in CA1 pyramidal cells of streptozotocin-induced diabetic rats. Society for Neuroscience Abstracts 31(382.19): 196. Spatial memory and hippocampal synaptic plasticity is impaired in streptozotocin (STZ)-induced diabetes mellitus (DM) rats. However, little is known about the effect of DM on the physiology of neurones in the CNS. Here, we compare basic electrophysiological properties of CA1 hippocampal pyramidal cells between control (22 cells), STZ-induced DM rats (24 cells) and DM rats treated with insulin

Page 28 AHP, BK- and SK-channel references

(7 cells). Hippocampal slices (450 µm) were prepared and CA1 pyramidal cells were impaled with sharp electrode (78120 M) filled with K-acetate. The basic passive (resting membrane potential, input resistance, membrane time constant) and active properties (action potential [AP] amplitude and duration) were not significantly different between the groups. However, CA1 pyramidal cells from STZ-induced DM rats showed significantly more spike broadening during a train of APs than cells from control animals (in controls the duration of the 3rd AP was 1.50±0.14 relative to the first AP, compared to 1.89±0.07 in DM rats). The amplitude of the slow afterhyperpolarization (sAHP) following a train of 5 APs was increased in the DM rats (7.87±0.88 mV in DM versus 4.25±0.61 mV in control rats). Insulin treatment prevented this increase (5.83±1.22 mV). The amplitude of the sAHP elicited by a Ca2+ -spike (in the presence of 1 µM TTX) was the same in DM and control rats. We conclude that the increased duration of AP in a train in CA1 pyramidal cells from STZ-induced DM rats most likely results in increased Ca2+ influx causing an increased sAHP. Increased amplitude of the sAHP is also observed in aged animals, however the cause of the increased sAHP seems to differ.

•Kelly, T. & Church, J. (2001). pH modulation of the slow Ca2+-dependent K+ current in rat hippocampal CA1 pyramidal neurons in vitro. Society for Neuroscience Abstracts 31(382.8): 196. In CA1 pyramidal neurons, the Ca2+-dependent slow afterhyperpolarization (sAHP) that modulates neuronal excitability, is itself modulated by changes in pH (Church, 1999, Neuroscience 89, 731). In the present study, the influence of pH changes on sIAHP, the current underlying the sAHP, was examined using blind patch- clamp recordings from CA1 neurons in 400 m hippocampal slices. Under control pH 7.4 HCO3-/CO2-buffered conditions in the presence of TTX and TEA, an unclamped Ca2+ current (ICa2+), elicited by a depolarizing step from 50 to 0 mV, evoked a sIAHP. Both the ICa2+ and the sIAHP increased in magnitude during perfusion with pH 7.7 medium and decreased during perfusion with pH 6.7 medium. In CA1 neurons, changes in extracellular pH (pHo) evoke changes in intracellular pH (pHi) in the same direction. To delineate between their effects, pHi was decreased at a constant pHo by replacing control medium with a pH 7.4 HEPES-buffered medium; this maneuver attenuated the sIAHP without a change in ICa2+. In addition, to prevent changes in pHi from occurring upon changing pHo, cytosolic H+ buffering power was increased by increasing [HEPES] in the patch pipette from 10 to 100 mM. However, even during perfusion with control pH 7.4 HCO3-/CO2-buffered medium,

Page 29 AHP, BK- and SK-channel references this maneuver consistently (n = 9) attenuated the sIAHP to <10 pA, compared to 110 19 pA (n = 12) recorded with patch-pipettes containing 10 mM HEPES. The results indicate that increases and decreases in pHi augment and attenuate sIAHP, respectively, suggesting a modulatory role for H+ on SK channel activity. Additionally, high concentrations of HEPES in the patch pipette appear to inhibit sIAHP through an unknown mechanism.

•Kimura, T., Tamura, R., Kurimoto, H. & Ono, T. (1999). Effects of T-588, a newly synthesized cognitive enhancer, on hippocampal CA1 neurons in rat brain tissue slices. Brain Research 831(1-2): 175-183. The effects of a newly synthesized cognitive enhancer, (-)-R-alpha-[[2- (diethylamino) ethoxy] methyl] benzo [b] thiophene-5-methanol hydrochloride (T- 588), on the membrane properties of hippocampal CA1 neurons were investigated in a rat brain slice preparation. T-588 produced a slow and long-lasting depolarization of CA1 neurons with an increase in membrane resistance; this action showed close similarity to that of (ACh). However, the action of T-588 was not affected by atropine, tetrodotoxin or DL-2-amino-5-phosphonovalerate, while the action of ACh was blocked by atropine. The estimated reversal potential of this T- 588 effect was near -90 mV which is the reversal potential of potassium ions in CA1 neurons. In the whole-cell voltage-clamp study, T-588 produced a reversible block of the outward potassium current in CA1 neurons. T-588 did not block the afterhyperpolarization evoked by an intracellular current injection, while ACh suppressed it. These results suggest that T-588 has a direct effect on CA1 neurons independent of its cholinergic activity, resulting from blockade of a conductance carried predominantly by potassium ions.

•Knöpfel, T., Vranesic, I., Gähwiler, B.H. & Brown, D.A. (1990). Muscarinic and beta-adrenergic depression of the slow Ca2+-activated potassium conductance in hippocampal CA3 pyramidal cells is not mediated by a reduction of the depolarization-induced cytosolic Ca2+ transients. Proceedings of the National Academy Sciences USA 87: 4083-4087. Combined intracellular and microfluorometric recording techniques were used to evaluate whether the inhibition by cholinergic or adrenergic transmitters of the Ca2(+)-activated potassium current (IAHP) in hippocampal CA3 pyramidal cells was mediated by an alteration of depolarization-induced change in cytosolic free Ca2+ concentration [(Ca2+]i). Low concentrations of isoproterenol (1-10 µM) and

Page 30 AHP, BK- and SK-channel references (0.25-1 µM) reversibly abolished IAHP without affecting concomitant Ca2+ transients or the steady-state [Ca2+]i. Only after application of higher concentrations of muscarine, [Ca2+]i increased; in the presence of potassium channel blockers, muscarine depressed Ca2+ currents and concomitant Ca2+ transients. These observations provide direct evidence that the inhibition of IAHP by isoproterenol and muscarine are not mediated by an alteration of Ca2+ dynamics.

•Krause, M., Offermanns, S., Stocker, M. & Pedarzani, P. (2002). Functional specificity of G-alpha-Q and G-alpha-11 in the cholinergic and glutamatergic modulation of potassium currents and excitability in CA1 pyramidal neurons. Journal of Neuroscience 22(3): 666-673. In hippocampal and other cortical neurons, action potentials are followed by a slow afterhyperpolarization (sAHP) generated by the activation of small- conductance Ca2+-activated K(+) channels and controlling spike frequency adaptation. The corresponding current, the apamin-insensitive sI(AHP), is a well known target of modulation by different neurotransmitters, including acetylcholine (via M(3) receptors) and glutamate (via metabotropic glutamate receptor 5, mGluR(5)), in CA1 pyramidal neurons. The actions of muscarinic and mGluR agonists on sI(AHP) involve the activation of -insensitive G-proteins. However, the pharmacological tools available so far did not permit the identification of the specific G-protein subtypes transducing the effects of M(3) and mGluR(5) on sI(AHP). In the present study, we used mice deficient in the Galpha(q) and Galpha(11) genes to investigate the specific role of these G-protein alpha subunits in the cholinergic and glutamatergic modulation of sI(AHP) in CA1 neurons. In mice lacking Galpha(q), the effects of muscarinic and glutamatergic agonists on sI(AHP) were nearly abolished, whereas beta-adrenergic agonists acting via Galpha(s) were still fully effective. Modulation of sI(AHP) by any of these agonists was instead unchanged in mice lacking Galpha(11). The additional depolarizing effects of muscarinic and glutamatergic agonists on CA1 neurons were preserved in mice lacking Galpha(q) or Galpha(11). Thus, Galpha(q), but not Galpha(11), mediates specifically the action of cholinergic and glutamatergic agonists on sI(AHP), without affecting the modulation of other currents. These results provide to our knowledge one of the first examples of the functional specificity of Galpha(q) and Galpha(11) in central neurons.

•Krause, M. & Pedarzani, P. (2000). A protein phosphatase is involved in the

Page 31 AHP, BK- and SK-channel references cholinergic suppression of the Ca2+-activated K+ current sIAHP in hippocampal pyramidal neurons. Neuropharmacology 39(7): 1274-1283. The slow calcium-activated potassium current sI(AHP) underlies spike- frequency adaptation and has a substantial impact on the excitability of hippocampal CA1 pyramidal neurons. Among other neuromodulatory substances, sI(AHP) is modulated by acetylcholine acting via muscarinic receptors. The second-messenger systems mediating the suppression of sI(AHP) by muscarinic agonists are largely unknown. Both protein kinase C and A do not seem to be involved, whereas calcium calmodulin kinase II has been shown to take part in the muscarinic action on sI(AHP). We re-examined the mechanism of action of muscarinic agonists on sI(AHP) combining whole-cell recordings with the use of specific inhibitors or activators of putative constituents of the muscarinic pathway. Our results suggest that activation of muscarinic receptors reduces sI(AHP) in a G-protein-mediated and phospholipase C-independent manner. Furthermore, we obtained evidence for the involvement of the cGMP-cGK pathway and of a protein phosphatase in the cholinergic suppression of sI(AHP), whereas release of Ca2+ from IP(3)-sensitive stores seems to be relevant neither for maintenance nor for modulation of sI(AHP).

•Krause, M., Stocker, M., Offermanns, S. & Pedarzani, P. (2000). G-alpha-Q mediates inhibition of sIAHP by acetylcholine and glutamate in CA1 pyramidal neurons. Society for Neuroscience Abstracts 30. In hippocampal and other cortical neurons action potentials are followed by a slow afterhyperpolarization (sAHP) generated by the activation of small- conductance Ca2+-activated K+ channels, and underlying spike frequency adaptation. The corresponding current, the apamin-insensitive sIAHP, is a well known target of modulation by different neurotransmitters, including acetylcholine (via M3 receptors) and glutamate (via mGluR5). The actions of muscarinic and mGluR agonists on sIAHP have been shown to involve activation of pertussis-toxin insensitive G- proteins. However, the pharmacological tools used in previous studies did not permit the identification of the specific G-proteins transducing the effects of M3 and mGluR5 on sIAHP. We used mice deficient in the Gaq gene to investigate the specific role of this G-protein a subunit in the cholinergic and glutamatergic modulation of sIAHP. The gross morphology of the hippocampus of Gaq-deficient mice was indistinguishable from wild-type (wt) animals. Additionally, characteristic features of AHP currents as well as neuronal passive properties were not significantly different in Gaq-deficient mice and wt littermates or other mice

Page 32 AHP, BK- and SK-channel references

(NMRI). In wt mice, the cholinergic agonist carbachol (CCh; 5 mM), and the mGluR agonists t-ACPD (10-20 mM) and DHPG (2-3 mM) strongly suppressed sIAHP, without affecting the apamin-sensitive IAHP. In mice lacking Gaq, the effects of CCh and DHPG were largely and significantly inhibited, arguing in favour of an involvement of Gaq in transducing their actions on sIAHP. Beside suppressing sIAHP, activation of M3 receptors and mGluR5 led to the generation of depolarizing inward currents, as previously observed in rat. This effect was preserved in wt as well as Gaq-deficient mice. Finally, the b-adrenergic agonist isoproterenol suppressed sIAHP both in wt and in Gaq-deficient mice. We conclude that the absence of Gaq impairs specifically the action of cholinergic and glutamatergic agonists on sIAHP, without affecting the modulation of other currents, or of sIAHP by other neurotransmitters coupled to different second-messenger pathways.

•Lancaster, B. & Adams, P.R. (1986). Calcium-dependent current generating the afterhyperpolarization of hippocampal neurons. Journal of Neurophysiology 55: 1268-1282. A single-electrode voltage-clamp technique was employed on in vitro hippocampal slices to examine the membrane current responsible for the slow afterhyperpolarization (AHP) in CA1 pyramidal cells. This was achieved by using conventional procedures to evoke an AHP in current clamp, followed rapidly by a switch into voltage clamp (hybrid clamp). The AHP current showed a dependence on extracellular K+, which was close to that predicted for a K+ current by the Nernst equation. The AHP current could be blocked by Cd2+ or norepinephrine. Although the AHP current showed a requirement for voltage-dependent Ca2+ entry, the current did not show any clear intrinsic voltage dependence. Once activated, AHP current is not turned off by hyperpolarizing the membrane potential. The effects of norepinephrine, Cd2+, and tetraethylammonium (TEA) were used to identify an AHP current component to the outward current evoked by depolarizing voltage commands from holding potentials that approximate to the for these cells. The AHP current can contribute significantly to the outward current during the depolarizing command. Upon repolarization it is evident as a slow outward tail current. This slow tail current had the same time constant as AHP currents evoked by hybrid clamp. Fast components to the tail currents were also observed. These were sensitive to Cd2+ and TEA. They probably represent a voltage-sensitive gKCa, sometimes termed C-current. The strong sensitivity to voltage and TEA displayed by the conventionally described gKCa (IC) are properties inconsistent with the AHP. It

Page 33 AHP, BK- and SK-channel references seems likely that the AHP current (IAHP) represents a Ca2+-activated K+ current separate from IC and that these two currents coexist in the same cell.

•Lancaster, B., Hu, H. & Storm, J.F. (2001). Rate of K+ current inhibition following photo-release of cAMP in CA1 pyramidal cells. Society for Neuroscience Abstracts 31(714.14): 366. Beta-adrenergic receptors activate a mechanism, involving cAMP and protein kinase A, to inhibit Ca2+-dependent K+ current (IsAHP) thereby enhancing CA1 cell excitability. Since the rate of this inhibition following noradrenaline release has been difficult to determine, we used photolysis of caged cAMP to assess the time course of IsAHP inhibition. Whole cell recordings were made from CA1 pyramidal cells with a KMeSO4-based internal solution plus 200 M DMNB-cAMP. IsAHP was evoked by depolarizing voltage steps or internal perfusion of dibromo-BAPTA (DBB 1 mM). At 30°C, release of cAMP following a single UV flash caused 80-90% inhibition of depolarization evoked IsAHP (n = 13). IsAHP recovery was initially rapid ( = 31 s), but typically incomplete up to 5 minutes postflash. Inhibition of IsAHP was less complete at 21 oC (65 10 %, n = 4) and contained no rapid component of recovery ( = 129 s). The onset kinetics were determined using DBB to generate persistent IsAHP. Inhibition of this persistent K+ current following photorelease of cAMP (61±8 %) occurred with = 1.89±0.24 s (n = 10). This value was significantly slower at 21 oC; = 6.35±0.66 s (P<0.001, n = 7). Intracellular application of Rp-cAMPS reduced the action of photoreleased cAMP on IsAHP in a dose-dependent manner. With 0.5-1 mM Rp-cAMPS (n=5) the UV flash caused 52 6% inhibition of IsAHP; with 4-5 mM Rp-cAMPS (n=4), it caused 11±7% inhibition of IsAHP, compared to 80±5% inhibition without Rp-cAMPS (n=5). Thus, 4-5 mM Rp-cAMPS inhibited the UV flash effect on IsAHP by 87%, consistent with a requirement for protein kinase A.

•Lancaster, B., Hu, H., Ramakers, G.M.J. & Storm, J.F. (2001). Interaction between synaptic excitation and slow afterhyperpolarization current in rat hippocampal pyramidal cells. Journal of Physiology (London) 536(Pt. 3): 809-823. 1. Whole cell recordings from CA1 pyramidal cells were performed to investigate the interaction between excitatory postsynaptic potentials (EPSPs) or currents (EPSCs), and the slow Ca2+-dependent K+ current, IsAHP. Blockers of the slow afterhyperpolarization (sAHP) such as isoprenaline (ISO) or noradrenaline (NA) reduced the hyperpolarization that followed a short train of EPSPs, and slowed the decay of summated EPSPs or EPSCs.

Page 34 AHP, BK- and SK-channel references

2. ISO/NA action on synaptic responses was observed in the absence of action potentials, but was curtailed by Ca2+ chelation (10 mM EGTA in the electrode) and was not observed with a caesium-based recording solution. This suggests the involvement of an ISO/NA-sensitive Ca2+-dependent K+ current without a requirement for regenerative spiking. 3. An ISO/NA-sensitive sAHP was observed following both NMDA and non- NMDA receptor-mediated EPSP trains in nominally zero Mg2+ medium. Isoprenaline sensitivity was blocked by hyperpolarization during EPSPs or by , suggesting a requirement for voltage-dependent Ca2+ influx during EPSPs. The data indicate that bursts of EPSPs can activate voltage-gated Ca2+ channels, which trigger IsAHP during synaptic responses. 4. A decrease in EPSP temporal summation occurred during both spike-evoked sAHPs and persistent activation of sAHP conductance following internal dialysis with diazo-2 (2 mM). At constant membrane potential, diazo-2 caused a decrease in membrane time constant and input resistance and accelerated the rate of EPSP decay. Photolysis of diazo-2 or application of NA reduced the resting sAHP conductance, causing an increased membrane time constant and input resistance in association with an increase in EPSP half-width. 5. These results indicate that short bursts of EPSPs can activate a Ca2+- dependent K+ current resembling IsAHP, and that activation of this current reduces the postsynaptic response to high-frequency synaptic input. The findings imply that modulation of IsAHP can regulate synaptic efficacy and may influence the threshold for tetanus-induced synaptic plasticity.

•Lancaster, B., Nicoll, R.A. & Perkel, D.J. (1991). Calcium activates two types of potassium channels in rat hippocampal neurons in culture. Journal of Neuroscience 11: 23-30. Several calcium-dependent potassium currents can contribute to the electrophysiological properties of neurons. In hippocampal pyramidal cells, 2 afterhyperpolarizations (AHPs) are mediated by different calcium-activated potassium currents. First, a rapidly activated current contributes to action- potential repolarization and the fast AHP following individual action potentials. In addition, a slowly developing current underlies the slow AHP, which occurs after a burst of action potentials and contributes substantially to the spike-frequency accommodation observed in these cells during a prolonged depolarizing current pulse. In order to investigate the single Ca2(+)-dependent channels that might underlie

Page 35 AHP, BK- and SK-channel references these currents, we performed patch-clamp experiments on hippocampal neurons in primary culture. When excised inside-out patches were exposed to 1 µM Ca2+, 2 types of channel activity were observed. In symmetrical bathing solutions containing 140 mM K+, the channels had conductances of 19 pS and 220 pS, and both were permeable mainly to potassium ions. The properties of these 2 channels differed in a number of ways. At negative membrane potentials, the small-conductance channels were more sensitive to Ca2+ than the large channels. At positive potentials, the small-conductance channels displayed a flickery block by Mg2+ ions on the cytoplasmic face of the membrane. Low concentrations of tetraethylammonium (TEA) on the extracellular face of the membrane specifically caused an apparent reduction of the large-channel conductance. The properties of the large- and small- conductance channels are in accord with those of the fast and slow AHP, respectively.

•Lancaster, B., Ramakers, G.M.J., Hu, H. & Storm, J.F. (2000). Interaction between excitatory synaptic input and slow AHP current (ISAHP) in CA1 pyramidal cells. Society for Neuroscience Abstracts 30. The slow AHP is a noradrenaline-sensitive, Ca2+-dependent K+ current typically linked to the Ca2+ influx which accompanies action potentials. We have now examined activation of IsAHP by the Ca2+ influx associated with excitatory synaptic responses, and have tested how this K conductance influences synaptic responses detected at the soma. Whole cell recordings from area CA1 were made with KMeSO4-based solutions containing QX-314 (0.5-2 mM) to suppress INa and IH. Trains of 5 stimuli (50 Hz, 0 Mg2+ medium) produced postsynaptic responses which included a noradrenaline (NA)-sensitive component (10 mM, n=7) which was an outward current of 500 ms duration. This current was not observed with Cs gluconate electrodes (n=4) and was inhibited by inclusion of 10 mM EGTA (n=7). In current clamp, this NA-sensitive outward current induced a hyperpolarization which was suppressed when synaptic activation was superimposed on hyperpolarization (n=10). These data suggest that IsAHP is activated during repetitive synaptic activity as a result of voltage dependent Ca2+ influx. Intracellular diazo-2 (2 mM) activates a steady state outward current which shares the properties of IsAHP (Lancaster & Batchelor 2000, J. Physiol. 522:231). We used this method to examine the influence of the K conductance on EPSPs. At constant membrane potential, activation of this conductance speeds the decay of EPSPs in tandem with decreases in input resistance and membrane time constant (n=5). These actions were reversed

Page 36 AHP, BK- and SK-channel references either by diazo-2 photolysis (n=5) or NA (n=6). The K+ current had no consistent effect on the amplitude or initial slope of EPSPs, and field responses were constant during the experimental manipulations. Temporal summation during high frequency (10 stimuli @ 100 Hz) synaptic input was enhanced (n=6) during block of the K+ conductance.

•Lancaster, B. & Wheal, H.V. (1987). Properties of two calcium-activated hyperpolarizations in rat hippocampal neurones. Journal of Physiology 389: 187-203. Intracellular recording from hippocampal CA1 pyramidal cells in the slice preparation was used to analyse the pharmacological sensitivity of action potential repolarization and the hyperpolarizations that follow the action potential. The Ca2+-activated after-hyperpolarizations (AHPs) could be divided into a fast AHP with a time course of milliseconds, and a slow AHP which lasted for a few seconds at a temperature of 30°C. 2. The repolarization of the action potential is sensitive to the Ca2+ Cd2+. This effect is simultaneous with a block of the fast AHP which follows immediately upon the repolarization of the action potential. The slow AHP was also blocked by Cd2+. 3. Low concentrations of the K+ channel blocker, tetraethylammonium (TEA; 200-500 µM), block the fast AHP and slow down action potential repolarization. The slow AHP was not affected by low concentrations of TEA. 4. The action potential repolarization and the fast AHP are also reversibly sensitive to charybdotoxin. This agent had no effect on the slow AHP. 5. When EGTA or BAPTA were added to the normal recording electrolyte (KMeSO4), the generation of slow AHPs was prevented. In addition, cells impaled with BAPTA- containing electrodes displayed broader action potentials and much reduced fast AHPs compared to recordings made with electrodes containing KMeSO4 alone or with EGTA. 6. The slow AHP can be eliminated by noradrenaline, 8-bromocyclic AMP or carbachol. Under these conditions there are no effects on the fast AHP or on action potential duration. 7. Block of the fast AHP with TEA or CTX (charybdotoxin) is associated with an increased frequency of the first few action potentials during a depolarization. This is a quite distinct effect from the greatly increased number of action potentials which results from block of the slow AHP 8. The results support a conclusion that the fast a.h.p. is generated by the TEA- and voltage-sensitive Ca2+-activated K+ current, IC. This current is involved in spike repolarization and turns off upon the return to resting potential. Thus block of IC has no effect on the slow AHP which is caused by a separate membrane current.

Page 37 AHP, BK- and SK-channel references

•Lancaster, B. & Zucker, R.S. (1994). Photolytic manipulation of Ca2+ and the time course of slow, Ca2+-activated K+ current in rat hippocampal neurones. Journal of Physiology (London) 475: 229-239. 1. Experiments were performed on hippocampal CA1 pyramidal cells to investigate the time course of a slow, Ca2+-activated K+ current that follows a burst of action potentials. At a temperature of 27-30°C, this current rises to a peak 200-400 ms following the cessation of Ca2+ entry before decaying to baseline in 4-8s. 2. Intracellular recordings were made using electrodes containing the photolabile calcium buffers nitr-5 or DM-nitrophen loaded appropriately with Ca2+. Under these conditions, photolysis of the compound using an ultraviolet flashlamp caused an instantanous increase in cytoplasmic Ca2+ throughout the cell. The response to flash photolysis was a membrane hyperpolarization with an onset limited by the membrane time constant. Multiple (up to twenty) flash responses could be generated. 3. The postspike slow after-hyperpolarization (AHP) and flash-induced hyperpolarizations showed a common sensitivity to the beta-adrenergic receptor agonist isoprenaline. 4. Following a burst of spikes, the current underlying an AHP in progress could be terminated or reduced by photolysis-induced production of calcium buffer from diazo-4 within the cell. This action was rapid (within the setting time of the flash artifact, i.e. < 10 ms) despite the fact that the manipulation occurred 400-500 ms following the end of Ca2+ entry. 5. Partial block of the slow AHP by buffer production was accompanied by an increase in the time to peak of the event. 6. The time to peak of the slow AHP could also be manipulated by experiments which altered the spatial distribution of Ca2+ entry, such as production of calcium spikes or dendritic depolarization by glutamate in the presence of tetrodotoxin. 7. The Ca2+-dependent K+ current responsible for the slow AHP responds immediately to increase or decreases in cytoplasmic Ca2+. It seems likely, therefore, that the slow AHP is controlled solely by changes in free Ca2+ and that the time course is governed by the redistribution of cytoplasmic Ca2+ following activity-induced entry through voltage- or receptor-operated channels.

•Li, H. & Henry, J.L. (2000). Adenosine action on interneurons and synaptic transmission onto interneurons in rat hippocampus in vitro. European Journal of Pharmacology 407(3): 237-244. To investigate the action of adenosine on interneurons as well as on excitatory synaptic transmission onto interneurons in the hippocampus, intracellular recordings were made from electrophysiologically identified interneurons in the CA1

Page 38 AHP, BK- and SK-channel references region of the hippocampal slice in vitro. The effects of adenosine and the preferential adenosine A1 receptor agonist, chloroadenosine, were examined. Application of 50 µM adenosine and 20 µM chloroadenosine to the bath produced a hyperpolarization of 5.6±1.6 (n=5) and 6.1±1.4 mV (n=6), respectively, as well as a decrease in membrane input resistance of 18.1±3.5% (n=5) and 18.5±1.4% (n=6), respectively. Adenosine depressed the postsynaptic potentials (PSPs) elicited in the interneurons by stimulation of fibers by 73±6.8% (n=5). The amplitude and the duration of the afterhyperpolarization which followed the spike of the action potential were attenuated by 48±6.9% and 31±8.6%, respectively (n=5). Chloroadenosine depressed the evoked PSPs in these interneurons by 61.2±2.7% (n=6) and depressed the duration and the amplitude of the afterhyperpolarization by 85.2±4.5% and by 72.6±4.8%, respectively (n=6). The data show that adenosine and chloroadenosine directly inhibit hippocampal CA1 interneurons by blocking the synaptic input, by hyperpolarizing the membrane potential and by depressing the afterhyperpolarization following individual action potential spikes. It is proposed that adenosine A1 receptors are present at pre- and/or postsynaptic sites of interneuron synapses in the hippocampal CA1 region. The present findings demonstrate that adenosine A1 receptor activation in CA1 interneurons is able to modulate the excitatory synaptic input to, and excitability of, these neurons. Thus, as adenosine is released during ischemia and epilepsy, adenosine may protect both interneurons and pyramidal cells from glutamate excitotoxicity through activation of adenosine A1 receptors on these neurons in the hippocampus.

•Magee, J.C. (1998). Dendritic hyperpolarization-activated currents modify the integrative properties of hippocampal CA1 pyramidal neurons. Journal of Neuroscience 18(19): 7613-7624. Step hyperpolarizations evoked slowly activating, noninactivating, and slowly deactivating inward currents from membrane patches recorded in the cell-attached patch configuration from the soma and apical dendrites of hippocampal CA1 pyramidal neurons. The density of these hyperpolarization-activated currents (Ih) increased over sixfold from soma to distal dendrites. Activation curves demonstrate that a significant fraction of Ih channels is active near rest and that the range is hyperpolarized relatively more in the distal dendrites. Ih activation and deactivation kinetics are voltage-and temperature-dependent, with time constants of activation and deactivation decreasing with hyperpolarization and depolarization, respectively. Ih demonstrated a mixed Na+-K+ conductance and was sensitive to low

Page 39 AHP, BK- and SK-channel references concentrations of external CsCl. Dual whole-cell recordings revealed regional differences in input resistance (Rin) and membrane polarization rates (taumem) across the somatodendritic axis that are attributable to the spatial gradient of Ih channels. As a result of these membrane effects the propagation of subthreshold voltage transients is directionally specific. The elevated dendritic Ih density decreases EPSP amplitude and duration and reduces the time window over which temporal summation takes place. The backpropagation of action potentials into the dendritic arborization was impacted only slightly by dendritic Ih, with the most consistent effect being a decrease in dendritic action potential duration and an increase in afterhyperpolarization. Overall, Ih acts to dampen dendritic excitability, but its largest impact is on the subthreshold range of membrane potentials where the integration of inhibitory and excitatory synaptic inputs takes place.

•Magee, J.C. & Carruth, M. (1999). Dendritic voltage-gated ion channels regulate the action potential firing mode of hippocampal CA1 pyramidal neurons. J Neurophysiol 82(4): 1895-1901. The role of dendritic voltage-gated ion channels in the generation of action potential bursting was investigated using whole cell patch-clamp recordings from the soma and dendrites of CA1 pyramidal neurons located in hippocampal slices of adult rats. Under control conditions somatic current injections evoked single action potentials that were associated with an afterhyperpolarization (AHP). After localized application of 4-aminopyridine (4-AP) to the distal apical dendritic arborization, the same current injections resulted in the generation of an afterdepolarization (ADP) and multiple action potentials. This burst firing was not observed after localized application of 4-AP to the soma/proximal dendrites. The dendritic 4-AP application allowed large-amplitude Na(+)-dependent action potentials, which were prolonged in duration, to backpropagate into the distal apical dendrites. No change in action potential backpropagation was seen with proximal 4- AP application. Both the ADP and action potential bursting could be inhibited by the bath application of nonspecific concentrations of divalent Ca(2+) channel blockers (NiCl and CdCl). Ca(2+) channel blockade also reduced the dendritic action potential duration without significantly affecting spike amplitude. Low concentrations of TTX (10-50 nM) also reduced the ability of the CA1 neurons to fire in the busting mode. This effect was found to be the result of an inhibition of backpropagating dendritic action potentials and could be overcome through the coordinated injection

Page 40 AHP, BK- and SK-channel references of transient, large-amplitude depolarizing current into the dendrite. Dendritic current injections were able to restore the burst firing mode (represented as a large ADP) even in the presence of high concentrations of TTX (300-500 µM). These data suggest the role of dendritic Na(+) channels in bursting is to allow somatic/axonal action potentials to backpropagate into the dendrites where they then activate dendritic Ca(2+) channels. Although it appears that most Ca(2+) channel subtypes are important in burst generation, blockade of T- and R-type Ca(2+) channels by NiCl (75 µM) inhibited action potential bursting to a greater extent than L-channel (10 µM ) or N-, P/Q-type (1 µM omega- MVIIC) Ca(2+) channel blockade. This suggest that the Ni-sensitive voltage-gated Ca(2+) channels have the most important role in action potential burst generation. In summary, these data suggest that the activation of dendritic voltage-gated Ca(2+) channels, by large-amplitude backpropagating spikes, provides a prolonged inward current that is capable of generating an ADP and burst of multiple action potentials in the soma of CA1 pyramidal neurons. Dendritic voltage-gated ion channels profoundly regulate the processing and storage of incoming information in CA1 pyramidal neurons by modulating the action potential firing mode from single spiking to burst firing.

•Mannaioni, G., Attucci, S., Missanelli, A., Pellicciari, R.C., orradetti, R. & Moroni, F. (1999). Biochemical and electrophysiological studies on (S)-(+)-2-(3'- carboxybicyclo(1.1.1)pentyl)- (CBPG), a novel mGlu5 receptor agonist endowed with mGlu1 receptor antagonist activity. Neuropharmacology 38(7): 917-926. The pharmacological profile of (S)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)- glycine (CBPG) and of other group 1 metabotropic glutamate (mGlu) receptor agents were studied in BHK cells transfected with mGlu receptor subtypes or in native receptors in brain slices by measuring second messenger responses. The mGlu receptor-mediated changes in the electrophysiological properties of CA1 pyramidal cells of the hippocampus were also evaluated. In mGlu5a receptor transfected cells, CBPG behaved as a partial agonist, while in mGlu1alpha receptor transfected cells, it behaved as a glutamate antagonist. No effect was found on cAMP formation in cells transfected with mGlu2 receptors or mGlu4 receptors. In brain slices, CBPG neither affected phospholipase D-coupled glutamate receptors nor did it modify the responses to ionotropic receptor stimulation (at concentrations up to 1 mM). When tested in CA1 pyramidal cells of the hippocampus, CBPG (50-100 µM) caused depolarization, increased cell input resistance, and decreased action potential

Page 41 AHP, BK- and SK-channel references frequency adaptation and afterhyperpolarization. DHPG (3-100 µM), an agonist of both mGlu1 and mGlu5 receptors, and CHPG (1000 µM), a low affinity mGlu5 agonist, produced qualitatively similar effects. The actions of CBPG or CHPG were not modified by AIDA (300 microM), a selective mGlu1 receptor antagonist. Our results suggest that CBPG could be a useful tool for discriminating between mGlu1 receptor and mGlu5 receptor effects and that mGlu5 receptors are the receptors which are mainly responsible for the direct excitatory effects of mGlu receptor agonists on CA1 pyramidal cells.

•Mannaioni, G., Marino, M.J., Valenti, O., Traynelis, S.F. & Conn, P.J. (2001). Metabotropic glutamate receptors 1 and 5 differentially regulate CA1 pyramidal cell function. Journal of Neuroscience 21(16): 5925-5934. The activation of group I metabotropic glutamate receptors (mGluRs) produces a variety of actions that lead to alterations in excitability and synaptic transmission in the CA1 region of the hippocampus. The group I mGluRs, mGluR1 and mGluR5, are activated selectively by (S)-3,5-dihydroxyphenylglycine (DHPG). To identify which of these mGluR subtypes are responsible for the various actions of DHPG in area CA1, we took advantage of two novel subtype-selective antagonists. (S)-(+)-alpha-amino-a-methylbenzeneacetic acid (LY367385) is a potent competitive antagonist that is selective for mGluR1, whereas 2-methyl-6-(phenylethynyl)- pyridine (MPEP) is a potent noncompetitive antagonist that is selective for mGluR5. The use of these compounds in experiments with whole-cell patch-clamp recording and Ca(2+)-imaging techniques revealed that each group I mGluR subtype plays distinct roles in regulating the function of CA1 pyramidal neurons. The block of mGluR1 by LY367385 suppressed the DHPG-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the direct depolarization of CA1 hippocampal neurons. In addition, the increase in the frequency of spontaneous IPSCs (sIPSCs) caused by the DHPG-induced depolarization of inhibitory interneurons also was blocked by LY367385, as was the DHPG-induced inhibition of transmission at the Schaffer collateral-->CA1 synapse. On the other hand, the block of mGluR5 by MPEP antagonized the DHPG-induced suppression of the Ca(2+)-activated potassium current (I(AHP)) and potentiation of the NMDA receptor. Finally, antagonism of the DHPG-induced suppression of evoked IPSCs required the blockade of both mGluR1 and mGluR5. These data suggest that mGluR1 and mGluR5 play distinct roles in the regulation of the excitability of hippocampal CA1 pyramidal neurons.

Page 42 AHP, BK- and SK-channel references

•Marrion, N.V. & Tavalin, S.J. (1998). Selective activation of Ca2+-activated K+ channels by co-localised Ca2+ channels in hippocampal neurons. Nature 395: 900- 905. Calcium entry through voltage-gated calcium channels can activate either large- (BK) or small- (SK) conductance calcium-activated potassium channels. In hippocampal neurons, activation of BK channels underlies the falling phase of an action potential and generation of the fast afterhyperpolarization (AHP). In contrast, SK channel activation underlies generation of the slow AHP after a burst of action potentials. The source of calcium for BK channel activation is unknown, but the slow AHP is blocked by dihydropyridine antagonists, indicating that L-type calcium channels provide the calcium for activation of SK channels. It is not understood how this specialized coupling between calcium and potassium channels is achieved. Here we study channel activity in cell-attached patches from hippocampal neurons and report a unique specificity of coupling. L-type channels activate SK channels only, without activating BK channels present in the same patch. The delay between the opening of L-type channels and SK channels indicates that these channels are 50-150 nm apart. In contrast, N-type calcium channels activate BK channels only, with opening of the two channel types being nearly coincident. This temporal association indicates that N and BK channels are very close. Finally, P/Q- type calcium channels do not couple to either SK or BK channels. These data indicate an absolute segregation of coupling between channels, and illustrate the functional importance of submembrane calcium microdomains.

•Martin, E.D., Arague, A. & Buno, W. (2001). Synaptic regulation of the slow Ca2+-activated K+ current in hippocampal CA1 pyramidal neurons: implication in epileptogenesis. Journal of Neurophysiology 86(6): 2878-2886. The slow Ca2+-activated K+ current (sI(AHP)) plays a critical role in regulating neuronal excitability, but its modulation during abnormal bursting activity, as in epilepsy, is unknown. Because synaptic transmission is enhanced during epilepsy, we investigated the synaptically mediated regulation of the sI(AHP) and its control of neuronal excitability during epileptiform activity induced by 4- aminopyridine (4AP) or 4AP+Mg2+-free treatment in rat hippocampal slices. We used electrophysiological and photometric Ca2+ techniques to analyze the sI(AHP) modifications that parallel epileptiform activity. Epileptiform activity was characterized by slow, repetitive, spontaneous depolarizations and action potential bursts and was associated with increased frequency and amplitude of spontaneous

Page 43 AHP, BK- and SK-channel references excitatory postsynaptic currents and a reduced sI(AHP.) The metabotropic glutamate receptor (mGluR) antagonist (S)-alpha-methyl-4-carboxyphenylglycine did not modify synaptic activity enhancement but did prevent sI(AHP) inhibition and epileptiform discharges. The mGluR-dependent regulation of the sI(AHP) was not caused by modulated intracellular Ca2+ signaling. Histamine, isoproterenol, and (±)-1- aminocyclopentane-trans-1,3-dicarboxylic acid reduced the sI(AHP) but did not increase synaptic activity and failed to evoke epileptiform activity. We conclude that 4AP or 4AP+Mg-free-induced enhancement of synaptic activity reduced the sI(AHP) via activation of postsynaptic group I/II mGluRs. The increased excitability caused by the lack of negative feedback provided by the sI(AHP) contributes to epileptiform activity, which requires the cooperative action of increased synaptic activity.

•Melyan, Z., Wheal, H.V. & Lancaster, B. (2002). Metabotropic-mediated kainate receptor regulation of IsAHP and excitability in pyramidal cells. Neuron 34(1): 107-114. Kainate receptors (KARs) on CA1 pyramidal cells make no detectable contribution to EPSCs. We report that these receptors have a metabotropic function, as shown previously for CA1 interneurons. Brief kainate exposure caused long-lasting inhibition of a postspike potassium current (I(sAHP)) in CA1 pyramidal cells. The pharmacological profile was independent of AMPA receptors or the GluR5 subunit, indicating a possible role for the GluR6 subunit. KAR inhibition of I(sAHP) did not require ionotropic action or network activity, but was blocked by the inhibitor of pertussis toxin-sensitive G proteins, N-ethylmaleimide (NEM), or the PKC inhibitor calphostin C. These data suggest how KARs, putatively containing GluR6, directly increase excitability of CA1 pyramidal cells and help explain the propensity for seizure activity following KAR activation.

•Moyer, J.R., Jr., Power, J.M., Thompson, L.T. & Disterhoft, J.F. (2000). Increased excitability of aged rabbit CA1 neurons after trace eyeblink conditioning. Journal of Neuroscience 20(14): 5476-5482. Cellular properties of CA1 neurons were studied in hippocampal slices 24 hr after acquisition of trace eyeblink conditioning in young adult and aging rabbits. Aging rabbits required significantly more trials than young rabbits to reach a behavioral criterion of 60% conditioned responses in an 80 trial session. Intracellular recordings revealed that CA1 neurons from aging control rabbits had

Page 44 AHP, BK- and SK-channel references significantly larger, longer lasting postburst afterhyperpolarizations (AHPs) and greater spike frequency adaptation (accommodation) relative to those from young adult control rabbits. After learning, both young and aging CA1 neurons exhibited increased postsynaptic excitability compared with their respective age-matched control rabbits (naive and rabbits that failed to learn). Thus, after learning, CA1 neurons from both age groups had reduced postburst AHPs and reduced accommodation. No learning-related differences were seen in resting membrane potential, membrane time constant, neuron input resistance, or action potential characteristics. Furthermore, comparisons between CA1 neurons from trace- conditioned aging and trace-conditioned young adult rabbits revealed no statistically significant differences in postburst AHPs or accommodation, indicating that similar levels of postsynaptic excitability were attained during successful acquisition of trace eyeblink conditioning, regardless of rabbit age. These data represent the first in vitro demonstration of learning-related excitability changes in aging rabbit CA1 neurons and provide additional evidence for involvement of changes in postsynaptic excitability of CA1 neurons in both aging and learning.

•Moyer, J.R., Jr., Thompson, L.T., Black, J.P. & Disterhoft, J.F. (1992). Nimodipine increases excitability of rabbit CA1 pyramidal in an age- and concentration-dependent manner. Journal of Neurophysiology 68: 2100-2109. 1. Cellular properties were studied before and after bath application of the dihydropyridine L-type calcium channel antagonist nimodipine in aging and young rabbit hippocampal CA1 pyramidal cells in vitro. Various concentrations of nimodipine, ranging from 10 nM to 10 µM, were tested to investigate age- and concentration-dependent effects on cellular excitability. Drug studies were performed on a population of neurons at similar holding potentials to equate voltage-dependent effects. The properties studied under current-clamp conditions included steady-state current-voltage relations (I-V), the amplitude and integrated area of the postburst afterhyperpolarization (AHP), accommodation to a prolonged depolarizing current pulse (spike frequency adaptation), and single action-potential waveform characteristics following synaptic activation. 2. Numerous aging-related differences in cellular properties were noted. Aging hippocampal CA1 neurons exhibited significantly larger postburst AHPs (both the amplitude and the integrated area were enhanced). Aging CA1 neurons also exhibited more hyperpolarized resting membrane potentials with a concomitant decrease in input resistance. When cells were grouped to equate resting potentials, no differences in

Page 45 AHP, BK- and SK-channel references input resistance were noted, but the AHPs were still significantly larger in aging neurons. Aging CA1 neurons also fired fewer action potentials during a prolonged depolarizing current injection than young CA1 neurons. 3. Nimodipine decreased both the peak amplitude and the integrated area of the AHP in an age- and concentration- dependent manner. At concentrations as low as 100 nM, nimodipine significantly reduced the AHP in aging CA1 neurons. In young CA1 neurons, nimodipine decreased the AHP only at 10 µM. No effects on input resistance or action-potential characteristics were seen. 4. Nimodipine increased excitability in an age- and concentration-dependent manner by decreasing spike frequency accommodation (increasing the number of action potentials during prolonged depolarizing current injection). In aging CA1 neurons, this effect was significant at concentrations as low as 10 nM. In young CA1 neurons, nimodipine decreased accommodation only at higher concentrations (> or = 1.0 µM). 5. We conclude that aging CA1 neurons were less excitable than young neurons. In aging hippocampus, nimodipine restores excitability, as measured by size of the AHP and degree of accommodation, to levels closely resembling those of young adult CA1 neurons. These actions of nimodipine on aging CA1 hippocampal neurons may partly underlie the drug's notable ability to improve associative learning in aging rabbits and other mammals.

•Moyer, J.R., Jr., Thompson, L.T. & Disterhoft, J.F. (1996). Trace eyeblink conditioning increases CA1 excitability in a transient and learning-specific manner. Journal of Neuroscience 16: 5536-5546. Time-dependent, learning-related changes in hippocampal excitability were evaluated by recording from rabbit CA1 pyramidal neurons in slices prepared at various times after acquisition of trace eyeblink conditioning. Increased excitability (reduced postburst afterhyperpolarizations and reduced spike-frequency adaptation) was seen as early as 1 hr after acquisition to behavioral criterion, was maximal in neurons studied 24 hr later, and returned to baseline within 7 d, whereas behavioral performance remained asymptotic for months. Neurons were held at -67 mV to equate voltage-dependent effects. No learning-related effects were observed on input resistance, action-potential amplitude or duration, or resting membrane potential. The excitability changes were learning-specific, because they were not seen in neurons from very slow learning (exhibited < 30% conditioned responses after 15 training sessions) or from pseudoconditioned control rabbits. Neurons from rabbits that displayed asymptotic behavioral performance after long-term retention testing (an additional training session 14 d after learning) were also

Page 46 AHP, BK- and SK-channel references indistinguishable from control neurons. Thus, the increased excitability of CA1 neurons was not performance- or memory-dependent. Rather, the time course of increased excitability may represent a critical window during which learning- specific alterations in postsynaptic excitability of hippocampal neurons are important for consolidation of the learned association elsewhere in the brain.

•Mynlieff, M. (1999). Identification of different putative neuronal subtypes in cultures of the superior region of the hippocampus using electrophysiological parameters. Neuroscience 93(2): 479-486. Cultured neurons offer many advantages over a slice preparation for whole- cell patch-clamp studies, such as better control over the environment and space clamp control. However, heterogeneous cultures of neurons present problems in distinguishing the cell type from which recordings are made. The present study uses correlations with data obtained in the hippocampal slice preparation to determine the feasibility of "identifying" different neuronal subtypes in cultures obtained from the superior region of postnatal two- to 13-day-old rat hippocampus. Whole- cell patch-clamp recording in the current-clamp mode after 24-96 h in culture was used to determine if the action potential duration would be a useful criterion in distinguishing cell types. Single action potentials were elicited by a 0.1-0.2 ms, 2-4 nA depolarizing pulse. The average membrane potential and input resistance were - 46.8±1.2 mV (n = 58) and 576±56 MΩ (n = 57), respectively. A frequency distribution of the action potential duration measured at half-maximal amplitude showed four distinct groups of neurons (group 1, 1.36±0.03 ms, n = 17; group 2, 2.19±0.05 ms, n = 20; group 3, 3.17±0.10 ms, n = 16; group 4, 4.36±0.13, n = 5). Based on correlations with previous studies using intracellular recording in identified cells in slices, the data suggest that group 1 represents basket cells, group 2 represents vertical cells, group 3 represents a combination of stellate cells and pyramidal cells, and group 4 represents another unidentified class of cells. Further analysis of the fast afterhyperpolarization allows distinction between pyramidal cells and stellate cells in group 3. In contrast to the interneurons in a slice preparation, these cells offer good voltage control and environmental control. Future studies will record from these cells in current-clamp mode to quickly characterize the action potential before switching to voltage-clamp recording to characterize the currents present in the different types of interneurons.

•Nicoll, R.A. (1988). The coupling of neurotransmitter receptors to ion channels in

Page 47 AHP, BK- and SK-channel references the brain. Science 241: 545-551. Recent studies on the action of neurotransmitters on hippocampal pyramidal cells indicate that different neurotransmitter receptors that use either the same or different coupling mechanisms converge onto the same ion channel. Conversely, virtually all of the neurotransmitters act on at least two distinct receptor subtypes coupled to different ion channels on the same cell. The existence of both convergence and divergence in the action of neurotransmitters results in a remarkable diversity in neuronal signaling.

•Norris, C.M., Halpain, S. & Foster, T.C. (1998). Reversal of age-related alterations in synaptic plasticity by blockade of L-type Ca2+ channels. Journal of Neuroscience 18(9): 3171-3179. The role of L-type Ca2+ channels in the induction of synaptic plasticity in hippocampal slices of aged (22-24 months) and young adult (4-6 months) male Fischer 344 rats was investigated. Prolonged 1 Hz stimulation (900 pulses) of Schaffer collaterals, which normally depresses CA3/CA1 synaptic strength in aged rat slices, failed to induce long-term depression (LTD) during bath application of the L-channel antagonist nifedipine (10 microM). When 5 Hz stimulation (900 pulses) was used to modify synaptic strength, nifedipine facilitated synaptic enhancement in slices from aged, but not young, adult rats. This enhancement was pathway-specific, reversible, and impaired by the NMDA receptor (NMDAR) antagonist DL-2-amino-5- phosphonopentanoic acid (AP5). Induction of long-term potentiation (LTP) in aged rats, using 100 Hz stimulation, occluded subsequent synaptic enhancement by 5 Hz stimulation, suggesting that nifedipine-facilitated enhancement shares mechanisms in common with conventional LTP. Facilitation of synaptic enhancement by nifedipine likely was attributable to a reduction ( approximately 30%) in the Ca2+-dependent K+-mediated afterhyperpolarization (AHP), because the K+ channel blocker apamin (1 µM) similarly reduced the AHP and promoted synaptic enhancement by 5 Hz stimulation. In contrast, apamin did not block LTD induction using 1 Hz stimulation, suggesting that, in aged rats, the AHP does not influence LTD and LTP induction in a similar way. The results indicate that, during aging, L-channels can (1) facilitate LTD induction during low rates of synaptic activity and (2) impair LTP induction during higher levels of synaptic activation via an increase in the Ca2+-dependent AHP.

•Nouranifar, R., Blitzer, R.D., Wong, T. & Landau, E. (1998). Metabotropic

Page 48 AHP, BK- and SK-channel references glutamate receptors limit adenylyl cyclase-mediated effects in rat hippocampus via protein kinase C. Neuroscience Letters 244(2): 101-105. Glutamate receptors of the metabotropic type (mGluRs) activate protein kinase C in hippocampus, but few physiological functions of this pathway are known. The present data show that mGluRs utilize protein kinase C to inhibit another second messenger system, the adenylyl cyclase pathway, in neurons of the CA1 area of hippocampus. Activation of mGluRs prevented beta-adrenergic receptors, which couple to adenylyl cyclase, from blocking the slow Ca2+-dependent afterhyperpolarization (AHP). Since the afterhyperpolarization modulates neuronal responsiveness, crosstalk between protein kinase C and the adenylyl cyclase pathway is likely to have physiological consequences. Moreover, mGluRs themselves block the afterhyperpolarization, so the observed interference with the beta-adrenergic response constitutes a hierarchical relationship in which mGluRs are dominant over beta-adrenergic receptors.

•Oh, M.M., Gamelli, A.E., Wu, W.W., Sametsky, E.A. & Disterhoft, J.F. (2001). Morris watermaze learning enhances neuronal excitability of CA1 hippocampal pyramidal neurons in rats. Society for Neuroscience Abstracts 31(921.1): 475. Previous studies from our laboratory have demonstrated enhanced neuronal excitability (i.e. reduced postburst AHP) of hippocampal pyramidal neurons from both rabbits and rats that have acquired a temporal, hippocampus-dependent task (trace eyeblink conditioning) compared to those from controls. This reduction was learning- dependent, as AHP was not reduced in neurons from rabbits that were trained but failed to acquire the task. Thus, we examined the CA1 pyramidal neurons from 3-6 mo F344 X BN rats after Morris watermaze training to determine if neuronal excitability is increased after learning a hippocampus-dependent, spatial task. The rats were randomly assigned to one of 3 groups: trained (WM), swim control (SC), and naive. WM consisted of 2 sessions/day for 2 days; 4 trials in a session. Every 8th trial was a probe trial. SC consisted of equal number of sessions and trials; however, each SC was time-yoked to a WM rat. Naive rats were handled for equal number of days. Hippocampal slices were prepared from each rat 1 d after the last trial. Biophysical properties of CA1 neurons were measured using current-clamp recording technique at a holding potential of -68±3 mV. The AHP amplitude and area of CA1 neurons from ventral hippocampus of WM rats were significantly reduced as compared to those from SC and naive rats: AHP amplitude (~25%); AHP area (~35%).

Page 49 AHP, BK- and SK-channel references

No differences were observed for dorsal CA1 neurons. Thus, hippocampal pyramidal neurons exhibit enhanced neuronal excitability as a cellular consequence of hippocampally-dependent learning across species and tasks.

•Oh, M.M., Power, J.M., Thompson, L.T., Moriearty, P.L. & Disterhoft, J.F. (1999). Metrifonate increases neuronal excitability in CA1 pyramidal neurons from both young and aging rabbit hippocampus. Journal of Neuroscience 19(5): 1814-1823. The effects of metrifonate, a second generation inhibitor, were examined on CA1 pyramidal neurons from hippocampal slices of young and aging rabbits using current-clamp, intracellular recording techniques. Bath perfusion of metrifonate (10-200 µM) dose-dependently decreased both postburst afterhyperpolarization (AHP) and spike frequency adaptation (accommodation) in neurons from young and aging rabbits (AHP: p < 0.002, young; p < 0.050, aging; accommodation: p < 0.024, young; p < 0.001, aging). These reductions were mediated by muscarinic cholinergic transmission, because they were blocked by addition of atropine (1 µM) to the perfusate. The effects of chronic metrifonate treatment (12 mg/kg for 3 weeks) on CA1 neurons of aging rabbits were also examined ex vivo. Neurons from aging rabbits chronically treated with metrifonate had significantly reduced spike frequency accommodation, compared with vehicle-treated rabbits. Chronic metrifonate treatment did not result in a desensitization to metrifonate ex vivo, because bath perfusion of metrifonate (50 µM) significantly decreased the AHP and accommodation in neurons from both chronically metrifonate- and vehicle- treated aging rabbits. We propose that the facilitating effect of chronic metrifonate treatment on acquisition of hippocampus-dependent tasks such as trace eyeblink conditioning by aging subjects may be caused by this increased excitability of CA1 pyramidal neurons.

•Pallotta, B.S., Blatz, A.L. & Magleby, K.L. (1992). Recording from calcium- activated potassium channels. Methods in Enzymology (Ion Channels). Rudy, B. & Iverson, L.E. San Diego, CA, Academic Press Inc. 207: 194-207.

•Pedarzani, P., Mosbacher, J., Rivard, A., Cingolani, L.A., Alberi, S., Oliver, D., Stocker, M., Adelman, J.P. & Fakler, B. (2000). Control of AHP and electrical activity in central neurons by tuning interaction between calmodulin and Ca2+-activated SK channels. Society for Neuroscience Abstracts 30. In central neurons action potentials are followed by afterhyperpolarizations

Page 50 AHP, BK- and SK-channel references

(AHPs) generated by the activation of small-conductance Ca2+-activated K+ channels (SK channels). By shaping the neuronal firing pattern, these AHPs contribute to the regulation of excitability and to the encoding function of neurons. SK channels are constitutive complexes between SK-a subunits and calmodulin. They display a unique mechanism of channel gating in which calcium binding to calmodulin triggers dynamic interactions between subunits that subsequently result in channel opening, while the unbinding of calcium from calmodulin results in channel closure. To study the impact of SK channel activity on neuronal function and its molecular mechanism, we have combined measurements on heterologously expressed wild-type and mutated SK channels and fluorescence emission measurements, with recordings of native SK- mediated currents and of firing patterns of hippocampal and neocortical neurons. Here we show that by stabilizing the Ca2+-dependent interaction between a subunits of various SK channels and calmodulin, 1-ethyl-2-benzimidazolinone (EBIO) slows down channel closure observed upon Ca2+ decrease and reduces the apparent sensitivity for Ca2+ into the lower nanomolar range. Recordings from CA1 pyramidal neurons in hippocampal slices show that EBIO increases both medium and slow AHP currents, resulting in a strong and dose-dependent reduction in excitability and electrical activity, effects also observed in networks derived from dissociated cortical neurons. These results underscore the importance of SK channels for shaping the electrical response patterns of central neurons, and suggest that modulating SK channel gating may be a potent mechanism for controlling excitability in the CNS.

•Pedarzani, P., Mosbacher, J., Rivard, A., Cingolani, L.A., Oliver, D., Stocker, M., Adelman, J.P. & Fakler, B. (2001). Control of electrical activity in central neurons by modulating the gating of small conductance Ca2+-activated K+ channels. Journal of Biological Chemistry 276: 9762-9769. In most central neurons, action potentials are followed by an afterhyperpolarization (AHP) that controls firing pattern and excitability. The medium and slow components of the AHP have been ascribed to the activation of small conductance Ca2+-activated potassium (SK) channels. Cloned SK channels are heteromeric complexes of SK alpha-subunits and calmodulin. The channels are activated by Ca2+ binding to calmodulin that induces conformational changes resulting in channel opening, and channel deactivation is the reverse process brought about by dissociation of Ca2+ from calmodulin. Here we show that SK channel gating is effectively modulated by 1-ethyl-2-benzimidazolinone (EBIO). Application of

Page 51 AHP, BK- and SK-channel references

EBIO to cloned SK channels shifts the Ca2+ concentration-response relation into the lower nanomolar range and slows channel deactivation by almost 10-fold. In hippocampal CA1 neurons, EBIO increased both the medium and slow AHP, strongly reducing electrical activity. Moreover, EBIO suppressed the hyperexcitability induced by low Mg2+ in cultured cortical neurons. These results underscore the importance of SK channels for shaping the electrical response patterns of central neurons and suggest that modulating SK channel gating is a potent mechanism for controlling excitability in the central nervous system.

•Pedarzani, P. & Storm, J.F. (1993). PKA mediates the effects of monoamine transmitters on the K+ current underlying the slow spike frequency adaptation in hippocampal neurons. Neuron 11(6): 1023-1035. The Ca2+-activated K+ current IAHP, which underlies spike frequency adaptation in cortical pyramidal cells, can be modulated by multiple transmitters and probably contributes to state control of the forebrain by ascending monoaminergic fibers. Here, we show that the modulation of this current by norepinephrine, serotonin, and histamine is mediated by protein kinase A in hippocampal CA1 neurons. Two specific protein kinase A inhibitors, Rp-cAMPS and Walsh peptide, suppressed the effects of these transmitters on IAHP and spike frequency adaptation. The effects of the cyclic AMP analog 8CPT-cAMP were also inhibited, whereas muscarinic and metabotropic glutamate receptor agonists had full effect. Intracellular application of protein kinase A catalytic subunit or a phosphatase inhibitor mimicked the effects of monoamines or 8CPT-cAMP. These results demonstrate that monoaminergic modulation of neuronal excitability in the mammalian CNS is mediated by protein phosphorylation.

•Pedarzani, P. & Storm, J.F. (1995). Dopamine modulates the slow Ca2+-activated K+ current IAHP via cyclic AMP-dependent protein kinase in hippocampal neurons. Journal of Neurophysiology 74(6): 2749-2753. 1. The effects of dopamine on the slow Ca2+-dependent K+ current (IAHP; AHP, afterhyperpolarization) and spike frequency adaptation were studied by whole cell voltage-clamp and sharp microelectrode current-clamp recordings in rat CA1 pyramidal neurons in rat hippocampal slices. 2. Dopamine suppressed IAHP in a dose- dependent manner, under whole cell voltage-clamp conditions. Similarly, under current-clamp conditions, dopamine inhibited spike frequency adaptation and suppressed the slow afterhyperpolarization. 3. The effect of dopamine on IAHP was

Page 52 AHP, BK- and SK-channel references mimicked by a D1 receptor agonist and blocked by dopamine receptor antagonists only in a minority of the cells. 4. Dopamine suppressed IAHP after blocking or desensitizing the beta-adrenergic receptors and, hence, did not act by cross- reacting with this receptor type. 5. The effects of dopamine on IAHP and spike frequency adaptation were suppressed by blocking the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent kinase (PKA) with Rp-cAMPS and, hence, are probably mediated by the activation of this kinase. 6. We conclude that dopamine increases hippocampal neuron excitability, like other monoamine neurotransmitters, by suppressing IAHP and spike frequency adaptation, via cAMP and protein kinase A. The receptor type mediating this effect of dopamine remains to be defined.

•Pedarzani, P. & Storm, J.F. (1996). Interaction between alpha- and beta- adrenergic receptor agonists modulating the slow Ca2+-activated K+ current IAHP in hippocampal neurons. European Journal of Neuroscience 8(10): 2098-2110. Noradrenaline inhibits the Ca2+-activated K+ current IAHP, which underlies the slow afterhyperpolarization and spike frequency adaptation in hippocampal and neocortical neurons. The resulting increase in excitability probably contributes to the state control of the forebrain during arousal and attention. The modulation of IAHP by noradrenaline has previously been shown to be mediated by beta 1 receptors, cyclic AMP and protein kinase A, but not by alpha receptors. We have now tested the possibility that alpha receptors also contribute to IAHP modulation through interaction with beta receptors, by the use of whole-cell recordings in CA1 pyramidal cells of rat hippocampal slices. The alpha-receptor agonist 6-fluoro- noradrenaline strongly potentiated the effect of isoproterenol on IAHP. The synergistic effect of 6-fluoro-noradrenaline and isoproterenol was blocked by the beta-receptor antagonist timolol, but the receptor type mediating the effect of 6- fluoro-noradrenaline could not be unequivocally identified by using alpha-receptor antagonists. The effect of high concentrations of noradrenaline on IAHP was only partly blocked by the beta-receptor antagonist timolol, and was further reduced by blocking alpha receptors, again suggesting a contribution from alpha receptors. In contrast, the effect of low concentrations of noradrenaline seemed to be potentiated by the alpha-receptor antagonist phentolamine in 57% of the cells, suggesting concentration-dependent antagonistic interaction between alpha and beta receptors. Further tests indicated that the cross-talk between 6-fluoro- noradrenaline and isoproterenol occurs upstream from cyclic AMP production, and that protein kinase A serves as a final common path for the modulation of IAHP by

Page 53 AHP, BK- and SK-channel references noradrenaline, and by the combination of 6-fluoro-noradrenaline and isoproterenol.

•Pedarzani, P. & Storm, J.F. (1996). Evidence that Ca/calmodulin-dependent protein kinase mediates the modulation of the Ca2+-dependent K+ current, IAHP, by acetylcholine, but not by glutamate, in hippocampal neurons. Pflugers Arch 431(5): 723-728. Muscarinic and metabotropic glutamate receptor agonists increase the excitability of hippocampal and other cortical neurons by suppressing the Ca2+- activated K+current, IAHP, which underlies the slow afterhyperpolarization (AHP) and spike frequency adaptation. We have examined the mechanism of action of a (carbachol) and a metabotropic glutamate receptor agonist (1- Aminocyclopentane-trans-1,3-dicarboxylic acid; t-ACPD) on IAHP in hippocampal CA1 neurons in slices, by using highly specific protein kinase inhibitors. We found that inhibition of protein kinase A (PKA) with the adenosine 3',5'-cyclic monophosphate (cAMP) analogue Rp-adenosine-3',5'-cyclic phosphorothioate Rp-cAMPS, did not prevent the muscarinic and glutamatergic suppression of IAHP. In contrast, two specific peptide inhibitors of Ca2+/calmodulin-dependent protein kinase II (CaM-K II), each partially blocked the effect of carbachol, but not the effect of t-ACPD on IAHP. We conclude that CaM-K II, but not PKA, is involved in mediating the muscarinic suppression of IAHP, although other pathways may also contribute. In contrast, neither CaM-K II nor PKA seems to mediate the metabotropic glutamate receptor action on IAHP.

•Pedarzani, P., Krause, M., Haug, T., Storm, J.F. & Stuhmer, W. (1998). Modulation of the Ca2+-activated K+ current sIAHP by a phosphatase-kinase balance under basal conditions in rat CA1 pyramidal neurons. J Neurophysiol 79: 3252-3256. The slow Ca2+-activated K+ current, sIAHP, underlying spike frequency adaptation, was recorded with the whole cell patch-clamp technique in CA1 pyramidal neurons in rat hippocampal slices. Inhibitors of serine/threonine protein phosphatases (microcystin, calyculin A, cantharidic acid) caused a gradual decrease of sIAHP amplitude, suggesting the presence of a basal phosphorylation- dephosphorylation turnover regulating sIAHP. Because selective calcineurin (PP-2B) inhibitors did not affect the amplitude of sIAHP, protein phosphatase 1 (PP-1) or 2A (PP-2A) are most likely involved in the basal regulation of this current. The ATP analogue, ATP-gamma-S, caused a gradual decrease in the sIAHP amplitude,

Page 54 AHP, BK- and SK-channel references supporting a role of protein phosphorylation in the basal modulation of sIAHP. When the protein kinase A (PKA) inhibitor adenosine-3', 5'-monophosphorothioate, Rp- isomer (Rp-cAMPS) was coapplied with the phosphatase inhibitor microcystin, it prevented the decrease in the sIAHP amplitude that was observed when microcystin alone was applied. Furthermore, inhibition of PKA by Rp-cAMPS led to an increase in the sIAHP amplitude. Finally, an adenylyl cyclase inhibitor (SQ22, 536) and adenosine 3',5'-cyclic monophosphate-specific type IV phosphodiesterase inhibitors (Ro 20-1724 and rolipram) led to an increase or a decrease in the sIAHP amplitude, respectively. These findings suggest that a balance between basally active PKA and a phosphatase (PP-1 or PP-2A) is responsible for the tonic modulation of sIAHP, resulting in a continuous modulation of excitability and firing properties of hippocampal pyramidal neurons.

•Poolos, N.P. & Johnston, D. (1999). Calcium-activated potassium conductances contribute to action potential repolarization at the soma but not the dendrites of hippocampal CA1 pyramidal neurons. Journal of Neuroscience 19(13): 5205-5212. Evidence is accumulating that voltage-gated channels are distributed nonuniformly throughout neurons and that this nonuniformity underlies regional differences in excitability within the single neuron. Previous reports have shown that Ca2+, Na+, A-type K+, and hyperpolarization-activated, mixed cation conductances have varying distributions in hippocampal CA1 pyramidal neurons, with significantly different densities in the apical dendrites compared with the soma. Another important channel mediates the large-conductance Ca2+-activated K+ current (IC), which is responsible in part for repolarization of the action potential (AP) and generation of the afterhyperpolarization that follows the AP recorded at the soma. We have investigated whether this current is activated by APs retrogradely propagating in the dendrites of hippocampal pyramidal neurons using whole-cell dendritic patch-clamp recording techniques. We found no IC activation by back-propagating APs in distal dendritic recordings. Dendritic APs activated IC only in the proximal dendrites, and this activation decayed within the first 100-150 µm of distance from the soma. The decay of IC in the proximal dendrites occurred despite AP amplitude, plus presumably AP-induced Ca2+ influx, that was comparable with that at the soma. Thus we conclude that IC activation by action potentials is nonuniform in the hippocampal pyramidal neuron, which may represent a further example of regional differences in neuronal excitability that are determined by the nonuniform distribution of voltage-gated channels in dendrites.

Page 55 AHP, BK- and SK-channel references

•Power, J.M., Oh, M.M. & Disterhoft, J.F. (2001). Metrifonate decreases sI(AHP) in CA1 pyramidal neurons in vitro. Journal of Neurophysiology 85(1): 319- 322. Metrifonate, a cholinesterase inhibitor, has been shown to enhance learning in aging rabbits and rats, and to alleviate the cognitive deficits observed in Alzheimer's disease patients. We have previously determined that bath application of metrifonate reduces the spike frequency adaptation and postburst afterhyperpolarization (AHP) in rabbit CA1 pyramidal neurons in vitro using sharp electrode current-clamp recording. The postburst AHP and accommodation observed in current clamp are the result of four slow outward potassium currents (sI(AHP), I(AHP), I(M), and I(C)) and the hyperpolarization activated mixed cation current, I(h). We recorded from visually identified CA1 hippocampal pyramidal neurons in vitro using whole cell voltage-clamp technique to better isolate and characterize which component currents of the AHP are affected by metrifonate. We observed an age-related enhancement of the slow component of the AHP tail current (sI(AHP)), but not of the fast decaying component of the AHP tail current (I(AHP), I(M), and I(C)). Bath perfusion of metrifonate reduced sI(AHP) at concentrations that cause a reduction of the AHP and accommodation in current-clamp recordings, with no apparent reduction of I(AHP), I(M), and I(C). The functional consequences of metrifonate administration are apparently mediated solely through modulation of the sI(AHP).

•Rimini, R., Rimland, J.M. & Terstappen, G.C. (2000). Quantitative expression analysis of the small conductance calcium-activated potassium channels, SK1, SK2 and SK3, in human brain. Molecular Brain Research 85(1-2): 218-220. Small conductance calcium-activated potassium (SK) channels are important in controlling neuronal excitability and three SK channels have been identified to date. In the present study, we report the first quantitative analysis of SK1, SK2 and SK3 expression in human brain using TaqMan RT-PCR on a range of human brain and peripheral tissue samples. SK1 expression is restricted to the brain whereas SK2 and SK3 are more widely expressed.

•Sah, P. (1996). Ca2+-activated K+ currents in neurones: Types, physiological roles and modulation. Trends in Neuroscience 19: 150-154. Action potentials in neurones are followed by a hyperpolarization, which can

Page 56 AHP, BK- and SK-channel references last up to several seconds. This hyperpolarization has several phases that are mediated by the activation of different types of Ca2+-activated K+ currents. Patch- clamp studies have revealed two families of Ca2+-activated K+ channels of small (SKCa) and high (BKCa) conductance. Activation of BKCa channels contributes to action-potential repolarization, while SKCa channels are thought to underlie the afterhyperpolarization (AHP). In addition, AHPs in neurones can be divided into two distinct types that are easily separated by kinetic and pharmacological criteria. It is now clear that only one type of AHP can be explained by activation of SKCa channels while a new type of Ca2+-activated K+ channel underlies the other. Modulation of this channel by a range of transmitters is a key determinant of the excitability of many neurones.

•Sah, P. & Bekkers, J.M. (1996). Apical dendritic location of slow afterhyperpolarization current in hippocampal pyramidal neurons: Implications for the integration of long-term potentiation. Journal of Neuroscience 16(15): 4537- 4542. Trains of action potentials in hippocampal pyramidal neurons are followed by a prolonged afterhyperpolarization (AHP) lasting several seconds, which is attributable to the activation of a slow calcium-activated potassium current ((sI)AHP). Here we examine the location of (sI)AHP on CA1 pyramidal neurons by comparing it with two GABAergic inhibitory postsynaptic currents (IPSCs) with known somatic and dendritic locations. Whole-cell patch-clamp recordings were made for CA1 pyramidal neurons in acute hippocampal slices. Stepping the membrane potential at the peak of (sI)AHP produced a relaxation ("switchoff") of the AHP current with a time constant of 7.4 ± 0.4 ms (mean ± SEM). The switchoff time constants for somatic and dendritic GABAA IPSCs were 3.5 ± 0.5 ms and 8.8 ± 0.3 ms, respectively. This data, together with cable modeling, indicates that active (sI)AHP channels are distributed over the proximal dendrites within approximately 200 micrometers of the soma. Excitatory postsynaptic potentials (EPSPs) evoked in stratum (s.) radiatum had their amplitudes shunted more by the AHP than did EPSPs evoked in s. oriens, suggesting that active AHP channels are restricted to the apical dendritic tree. Blockade of the AHP during a tetanus, which in control conditions elicited a decremental short-term potentiation (STP), converted STP to long-term potentiation (LTP). Thus, activation of the AHP increases the threshold for induction of LTP. These results suggest that in addition to its established role in spike frequency adaptation, the AHP works as an adjustable gain control, variably

Page 57 AHP, BK- and SK-channel references hyperpolarizing and shunting synaptic potentials arising in the apical dendrites.

•Sah, P. & Clements, J.D. (1999). Photolytic manipulation of [Ca2+]i reveals slow kinetics of potassium channels underlying the afterhyperpolarisation in hippocampal pyramidal neurons. Journal of Neuroscience 19: 3657-3664. The identity of the potassium channel underlying the slow, apamin-insensitive component of the afterhyperpolarization current (sIAHP) remains unknown. We studied sIAHP in CA1 pyramidal neurons using simultaneous whole-cell recording, calcium fluorescence imaging, and flash photolysis of caged compounds. Intracellular calcium concentration ([Ca2+]i) peaked earlier and decayed more rapidly than sIAHP. Loading cells with low concentrations of the calcium chelator EGTA slowed the activation and decay of sIAHP. In the presence of EGTA, intracellular calcium decayed with two time constants. When [Ca2+]i was increased rapidly after photolysis of DM-Nitrophen, both apamin-sensitive and apamin-insensitive outward currents were activated. The apamin-sensitive current activated rapidly (<20 ms), whereas the apamin-insensitive current activated more slowly (180 ms). The apamin- insensitive current was reduced by application of serotonin and carbachol, confirming that it was caused by sIAHP channels. When [Ca2+]i was decreased rapidly via photolysis of diazo-2, the decay of sIAHP was similar to control (1. 7 s). All results could be reproduced by a model potassium channel gated by calcium, suggesting that the channels underlying sIAHP have intrinsically slow kinetics because of their high affinity for calcium.

•Sah, P. & Faber, L.E.S. (2002). Channels underlying neuronal calcium-activated potassium currents. Progress in Neurobiology 66(5): 345-353. In many cell types rises in cytosolic calcium, either due to influx from the extracellular space, or by release from an intracellular store activates calcium dependent potassium currents on the plasmalemma. In neurons, these currents are largely activated following calcium influx via voltage gated calcium channels active during the action potentials. Three types of these currents are known: I(c), I(AHP) and I(sAHP). These currents can be distinguished by clear differences in their pharmacology and kinetics. Activation of these potassium currents modulates action potential time course and the repetitive firing properties of neurons. Single channel studies have identified two types of calcium-activated potassium channel which can also be separated on biophysical and pharmacological grounds and have been named BK and SK channels. It is now clear that BK channels underlie I(c) whereas SK channels

Page 58 AHP, BK- and SK-channel references underlie I(AHP). The identity of the channels underlying I(sAHP) are not known. In this review, we discuss the properties of the different types of calcium-activated potassium channels and the relationship between these channels and the macroscopic currents present in neurons.

•Sah, P., French, C.R. & Gage, P.W. (1985). Effects of noradrenaline on some potassium currents in CA1 neurones in rat hippocampal slices. Neuroscience Letters 60(3): 295-300. Pyramidal (CA1) cells in rat hippocampal slices were voltage clamped using a single electrode voltage clamp. In the presence of tetrodotoxin (TTX), depolarizing pulses from holding potentials of -60 to -70 mV elicited a slow inward calcium (Ca2+) current and two outward potassium (K+) currents: an A current and a slower, Ca2+-dependent K+ current. Noradrenaline (NA) (20 µM) depressed the amplitude of the K+ currents without affecting the Ca2+ current. The effect of NA could be blocked with and was mimicked by isoprenaline, suggesting that NA depresses the K+ currents by binding to beta-receptors.

•Sah, P. & Isaacson, J.S. (1995). Channels underlying the slow afterhyperpolarization in hippocampal pyramidal neurons: Neurotransmitters modulate the open probability. Neuron 15: 435-441. The slow afterhyperpolarization in hippocampal pyramidal neurons is mediated by a calcium-activated potassium current (IAHP) and is a target for variety of different neurotransmitters. The characteristics of the channels underlying IAHP and how they are modulated by neurotransmitters are, however, unknown. In this study, we have examined the properties of the channels underlying IAHP using fluctuation analysis of the macroscopic current. Our results indicate that this channel has a unitary conductance of 2-5 pS and a mean open time of about 2 ms. When the peak amplitude of IAHP was maximal, these channels have an open probability of 0.4. Noradrenaline and carbachol reduced IAHP amplitude by lowering open channel probability. These result indicate that a novel calcium-activated potassium channel underlies IAHP. This channel is modulated in a similar fashion by two different transmitter systems that utilize distinct protein kinases.

•Sailer, C.A., Kaufmann, W.A., Trieb, M. & Knaus, H.G. (2001). SK2 channels in the rodent brain: Regional distribution and co-localization with marker antibodies. Society for Neuroscience Abstracts 31(382.1): 196.

Page 59 AHP, BK- and SK-channel references

Small-conductance Ca2+-activated K+ (SK) channels are important for excitability control, afterhyperpolarization, and have been implicated in regulation of the functional state in vertebrate neurons. To characterize the cellular and subcellular distribution of SK2 channels in more detail, we raised sequence-specific antibodies against unique recognition domains. The most promising antibodies were then subjected to immunohistochemistry and used to perform a detailed distribution study of SK2 channels in rat and mouse brain sections. SK2 immunoreactivity, as detected by all antibodies, was associated with subregions of the hippocampal formation, layer V neurons of neocortex, habenula and reticular thalamic nucleus. Moderate staining was observed in the amygdala, fornix, mammilary bodies as well as in various nuclei of the thalamus and the brainstem. To investigate the subcellular distribution pattern and cell-type specific expression of SK2 channels, double-labelling immunofluorescence experiments with established glial and neuronal marker antibodies were performed in brain slices. Evidence for somato-dendritic localization of SK2 protein is going to be presented for a number of defined neurons.

•Sametsky, E.A., Wu, W.W., Oh, M.M., Moskal, J.R. & Disterhoft, J.F. (2001). Dorso-ventral gradient of excitability in hippocampal pyramidal neurons. Society for Neuroscience Abstracts 31(382.7): 196. The hippocampal formation possesses heterogeneity and functional differentiation along its septo-temporal axis despite its uniform lamellar structure. To determine whether CA1 pyramidal neurons differ in their biophysical properties, whole-cell voltage-clamp and intracellular current-clamp recordings were obtained from dorsal (DH), middle (MH) and ventral (VH) hippocampi of young rabbits and rats. In voltage-clamp, AHP currents were evoked by a voltage step to -5mV/100 ms from a holding potential of -55 mV and were isolated pharmacologically (TTX, TEA, CsCl, 4-AP, CNQX, D-AP5 and PTX). CA1 neurons from rabbits did not differ in resting membrane potential (~ -75 mV) and membrane resistance (60-100 M). The peak amplitude of AHP current (IAHP and IM) tended to increase from the DH to the VH [mean 209 pA (DH), 258 pA (MH), and 388 pA (VH)]. There was a similar, significant gradient (p<0.05) in the slow AHP current (sIAHP) measured 1 sec after voltage pulse offset [127 pA (DH), 178 pA (MH), and 300 pA (VH)]. In current-clamp recording from rats, the AHP was evoked by a 100ms depolarizing current step to elicit 4 action potentials from a holding potential of -673mV. There were significant increases in peak amplitude (p<0.001) and area (p<0.05) of the AHP in the

Page 60 AHP, BK- and SK-channel references

VH (n=12) compared to DH (n=12). There were no differences observed for duration or latency of the AHP, resting membrane potential, input resistance, nor for the spike-frequency adaptation. These data may help to explain lesion data indicating that, e.g., the dorsal hippocampus is preferentially involved in spatial learning. Supported by: BY NIH AG08796, MH11737, MH12858

•Saviane, C. & Cherubini, E. (2001). Changes of intracellular calcium concentration down-regulate a voltage-dependent potassium current in CA3 pyramidal neurons of organotypic hippocampal cultures. Society for Neuroscience Abstracts 31(382.16): 196. K+ channels play a crucial role in regulating excitability and temporal coding of neurons. An increase in [Ca2+]i activates a class of K+ channels (BK and SK), responsible for action potential repolarization and spike frequency adaptation. Here we show that [Ca2+]i down-regulates a voltage-dependent potassium current responsible for the timing of action potential initiation. Whole-cell recordings in current clamp mode were performed from CA3 pyramidal neurons in organotypic hippocampal cultures. Depolarizing current steps (800 ms duration) from the resting membrane potential (-572 mV, n=5) activated action potential firing with a delay of 251106 ms (n=5) from the beginning of the pulse. A 10 mV membrane hyperpolarization produced an increase in the delay of the first spike of 37761 ms (n=5) for the same current steps. Bath application of Cd2+ (200 M) induced a block of the slow afterhyperpolarization (AHP) following the train of action potentials without changes in spike duration. In Cd2+, a further delay of 37746 ms (n=3) in the appearance of the first spike was observed. In calcium-free solution (with 1 mM EGTA) a similar block of AHP was observed, but in contrast with Cd2+ this phenomenon was associated with a broadening of the action potential. The same current steps produced cell firing that in 3/4 cells was delayed of 177104 ms. Application of 4-aminopyridine (50 M), in the presence of Cd2+ or in calcium-free solution, strongly reduced the delay of the first spike and increased its duration, suggesting the involvement of a voltage-dependent potassium current similar to ID.

•Savic, N., Pedarzani, P. & Sciancalepore, M. (2001). Medium afterhyperpolarization and firing pattern modulation in interneurons of stratum radiatum in the CA3 hippocampal region. Journal of Neurophysiology 85(5): 1986- 1997. Stratum (st.) radiatum interneurons represent a heterogeneous class of

Page 61 AHP, BK- and SK-channel references hippocampal cells with as yet poorly characterized physiological properties. Intracellular staining with biocytin, in situ hybridization, and patch-clamp recording have been combined to investigate the morphological and electrophysiological properties of these cells in the CA3 hippocampal region in young rats [postnatal days 10 to 21 (P10-21)]. Labeled cells presented a heterogeneous morphology with various soma shapes, often found multipolar, and dendritic arborizations confined to st. radiatum. The passive membrane properties of these st. radiatum interneurons showed instead no significant differences between P10 and P21. Low resting potential, high-input resistance, and short time constants characterized CA3 st. radiatum interneurons, which were silent at rest. Action potentials, elicited by brief current pulses, were lower and shorter than in pyramidal cells and followed by a Ca2+-dependent medium-duration afterhyperpolarizing potential (mAHP). Prolonged depolarizing current injection generated trains of action potentials that fired at constant frequency after a slight accommodation. The maximum steady-state firing rate was 31 ± 4 (SD) Hz. Hyperpolarizing current pulses revealed a prominent inward rectification characterized by a "sag," followed by a depolarizing rebound that triggered action potentials. Sag and anodal brake excitation were blocked by Cs(+), suggesting that they were mediated by a hyperpolarization-activated cation conductance (I(h)). In the presence of tetrodotoxin and tetraethylammonium, biphasic tail currents were elicited in voltage clamp after a depolarizing step inducing Ca2+ influx. Tail currents presented a fast Ca2+-activated and apamin- sensitive component (I(AHP)) and were further reduced by carbachol. The presence of I(AHP) was consistent with the high expression level of the apamin-sensitive SK2 subunit transcript in CA3 st. radiatum interneurons as detected by in situ hybridization. Different pharmacological agents were shown to affect the afterhyperpolarizing potential as well as the firing properties of st. radiatum interneurons. Exposure to Ca2+-free solutions mainly affected the late phase of repolarization and strongly reduced the mAHP. The mAHP was also attenuated by carbachol and by apamin, suggesting it to be partly mediated by I(AHP). Reduction of the mAHP increased the interneuron firing frequency. In conclusion, st. radiatum interneurons of CA3 hippocampal region represent a class of nonpyramidal cells with action potentials followed by an AHP of relatively short duration, partially generated by apamin and carbachol-sensitive conductances involved in the regulation of the cell firing rate.

•Schnee, M.E. & Brown, B.S. (1998). Selectivity of (DuP 996), a

Page 62 AHP, BK- and SK-channel references neurotransmitter release enhancer, in blocking voltage-dependent and calcium- activated potassium currents in hippocampal neurons. Journal of Pharmacology & Experimental Therapeutics 286(2): 709-717. Linopirdine [DuP 996, 3, 3-bis(4-pyridinylmethyl)-1-phenylindolin-2-one], a putative cognition enhancing drug, increases acetylcholine release in rat brain tissue and improves performance in animal models of learning and memory. The mechanism whereby linopirdine enhances acetylcholine release has been proposed to involve inhibition of the M-type K+ current (IM). Our study examines the selectivity of linopirdine for IM by determining its effects on other ionic currents present in rat hippocampal CA1 neurons using patch clamp techniques. Linopirdine was found to block voltage-gated, calcium-activated and leak K+ currents in a dose- dependent manner. Of the seven currents measured, linopirdine was most selective for IM with an IC50 of 2.4 ± 0.4 µM, followed by IC (measured as a medium afterhyperpolarization tail current, ImAHP) with an IC50 of 16.3 ± 2.4 µM. Both IM and IC were completely suppressed by linopirdine. At a concentration of 100 µM, linopirdine weakly inhibited the K+ leak current, IL, the transient outward current, IA, the delayed rectifier, IK, and the slow component of IAHP, by 28 ± 8, 37 ± 10, 36 ± 9 and 52 ± 10 percent, respectively. The mixed Na+/K+ inward rectifying current, IQ, was essentially unaffected by linopirdine (IC50 >300 µM). These results indicate that linopirdine selectively blocks IM at concentrations ≤ 3 µM, the approximate EC50 for acetylcholine release enhancement. Inhibition of other voltage-gated and calcium-activated K+ currents could also contribute to enhanced neurotransmitter release by linopirdine at intermediate (IC) and high (IL, IA, IK, IsAHP) concentrations.

•Schweitzer, P., Madamba, S. & Siggins, G.R. (1998). Somatostatin increases a voltage-insensitive K+ conductance in rat CA1 hippocampal neurons. Journal of Neurophysiology 79(3): 1230-1238. Somatostatin (SST) is a neuropeptide involved in several central processes. In hippocampus, SST hyperpolarizes CA1 pyramidal neurons and augments the K+ M current (IM). However, the limited involvement of IM at resting potential in these cells suggests that the peptide also may modulate another channel to hyperpolarize hippocampal pyramidal neurons (HPNs). We studied the effect of SST on noninactivating conductances of rat CA1 HPNs in a slice preparation. Using MK886, a specific inhibitor of the enzymatic pathway that leads to the augmentation of IM by SST, we have uncovered and characterized a second conductance activated by the

Page 63 AHP, BK- and SK-channel references peptide. SST did not affect IM when applied with MK886 or the amplitudes of the slow Ca2+-dependent K+ afterhyperpolarization-current and the cationic Q current but still caused an outward current, indicating that SST acts upon another conductance. In the presence of MK886, SST elicited an outward current that reversed around -100 mV and that displayed a linear current-voltage relationship. Reversal potentials obtained in different external K+ concentrations are consistent with a conductance carried solely by K+ ions. The slope of the current-voltage relationship increased proportionately with the extracellular K+ concentration and remained linear. This suggests that SST opens a voltage-insensitive leak current (IK(L)) in HPNs not an inwardly rectifying K+ current as reported in other neuron types. A low concentration of extracellular Ba2+ (150 µM) only slightly decreased the SST-induced effect in a voltage-independent manner, whereas a high concentration of Ba2+ (2 mM) completely blocked it. Extracellular Cs+ (2 mM) did not affect the outward SST current but inhibited the inward component. We conclude that SST inhibits HPNs by activating two different K+ conductances: the voltage-insensitive IK(L) and the voltage-dependent IM. The hyperpolarizing effect of SST at resting membrane potential appears to be mainly carried by IK(L), whereas IM dominates at slightly depolarized potentials.

•Shah, M.M. & Haylett, D.G. (2000). Ca2+ channels involved in the generation of the slow afterhyperpolarization in cultured rat hippocampal pyramidal neurons. Journal of Neurophysiology 83(5): 2554-2561. The advantages of using isolated cells have led us to develop short-term cultures of hippocampal pyramidal cells, which retain many of the properties of cells in acute preparations and in particular the ability to generate afterhyperpolarizations after a train of action potentials. Using perforated-patch recordings, both medium and slow afterhyperpolarization currents (mI(AHP) and sI(AHP), respectively) could be obtained from pyramidal cells that were cultured for 8-15 days. The sI(AHP) demonstrated the kinetics and pharmacologic characteristics reported for pyramidal cells in slices. In addition to confirming the insensitivity to 100 nM apamin and 1 mM TEA, we have shown that the sI(AHP) is also insensitive to 100 nM charybdotoxin but is inhibited by 100 µM D-tubocurarine. Concentrations of nifedipine (10 µM) and nimodipine (3 µM) that maximally inhibit L-type calcium channels reduced the sI(AHP) by 30 and 50%, respectively. However, higher concentrations of nimodipine (10 µM) abolished the sI(AHP), which can be partially explained by an effect on action potentials. Both nifedipine and nimodipine at

Page 64 AHP, BK- and SK-channel references maximal concentrations were found to reduce the HVA calcium current in freshly dissociated neurons to the same extent. The N-type calcium channel inhibitor, omega-conotoxin GVIA (100 nM), irreversibly inhibited the sI(AHP) by 37%. Together, omega-conotoxin (100 nM) and nifedipine (10 µM) inhibited the sI(AHP) by 70%. 10 µM ryanodine also reduced the sI(AHP) by 30%, suggesting a role for calcium-induced calcium release. It is concluded that activation of the sI(AHP) in cultured hippocampal pyramidal cells is mediated by a rise in intracellular calcium involving multiple pathways and not just entry via L-type calcium channels. Axoclamp

•Shah, M.M. & Haylett, D.G. (2002). K+ currents generated by NMDA receptor activation in rat hippocampal pyramidal neurons. Journal of Neurophysiology 87(6): 2983-2989. Long lasting outward currents mediated by Ca2+-activated K(+) channels can be induced by Ca2+ influx through N-methyl-D-aspartate (NMDA)-receptor channels in voltage-clamped hippocampal pyramidal neurons. Using specific inhibitors, we have attempted to identify the channels that underlie these outward currents. At a holding potential of -50 mV, applications of 1 mM NMDA to the soma of cultured hippocampal pyramidal neurons induced the expected inward currents. In 44% of cells tested, these were followed by outward currents (average amplitude 60 ± 7 pA) that peaked 2.5 s after the initiation of the inward NMDA currents and decayed with a time constant of 1.4 s. In 43% of those cells exhibiting an outward current, SK channel inhibitors, UCL 1848 (100 nM) and apamin (100 nM) abolished the outward current. In the remainder of the cells, the outward currents were either insensitive or only partly inhibited (44 ± 4%) by 100 nM UCL 1848. In these cells, the outward currents were reduced by the slow afterhyperpolarization (sAHP) inhibitors, muscarine (3 µM; 43 ± 9%), UCL 1880 (3 µM; 34 ± 10%), and UCL 2027 (3 µM; 57 ± 6%). Neither the BK channel inhibitor, charybdotoxin (100 nM), nor the Na(+)/K(+) ATPase inhibitor, ouabain (100 µM), reduced these outward currents. Irrespective of the pharmacology, the time course of the outward current did not differ. Interestingly, no correlation was observed between the presence of a slow apamin- insensitive afterhyperpolarization and an outward current insensitive to SK channel blockers following NMDA-receptor activation. It is concluded that an NMDA- mediated rise in [Ca2+](i) can result in the activation of apamin-sensitive SK channels and of the channels that underlie the sAHP. The activation of these channels may, however, depend on their location relative to NMDA receptors as well as on the spatial Ca2+ buffering within individual neurons.

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•Shah, M.M., Miscony, Z., Javadzadeh-Tabatabaie, M., Ganellin, C.R. & Haylett, D.G. (2001). Clotrimazole analogues: Effective blockers of the slow afterhyperpolarization in cultured rat hippocampal pyramidal neurones. British Journal of Pharmacology 132: 889-898. 1. The pharmacology of the slow afterhyperpolarization (sAHP) was studied in cultured rat hippocampal pyramidal neurones. 2. Clotrimazole, its in vivo metabolite, 2-chlorophenyl-bisphenyl-methanol (CBM) and the novel analogues, UCL 1880 and UCL 2027, inhibited the sI(AHP) with similar IC50s (1-2 µM). 3. Clotrimazole and CBM also inhibited the high voltage-activated (HVA) Ca2+ current in pyramidal neurones with IC50s of 4.7 µM and 2.2 µM respectively. UCL 1880 was a less effective Ca2+ channel blocker, reducing the HVA Ca2+ current by 50% at 10 µM. At concentrations up to 10 µM, UCL 2027 had no effect on the Ca2+ current, indicating that its effects on the sI(AHP) were independent of Ca2+ channel block. 4. Clotrimazole also inhibited both the outward holding current (IC50=2.8 µM) present at a potential of - 50 mV and the apamin-sensitive medium AHP (mAHP; IC50 approximately amp;10 µM). The other clotrimazole analogues tested had smaller effects on these two currents. The present work also shows that 100 nM UCL 1848, an inhibitor of apamin-sensitive conductances, abolishes the mAHP. 5. Currents were recorded from HEK293 cells transfected with hSK1 and rSK2. The SK currents were very sensitive to inhibition by UCL 1848 but were not significantly reduced by the sI(AHP) inhibitor, UCL 2027 (10 µM). 10 µM UCL 1880 reduced the hSK1 current by 40%. 6. UCL 2027 appears to be the first relatively selective blocker of the sAHP to be described. Furthermore, the ability of UCL 2027 to block the sAHP with minimal effect on SK1 channel activity questions the role of this channel in the sAHP.

•Shao, L.R., Halvorsrud, R., Borg-Graham, L.F. & Tavalin, S.J. (1999). The role of BK-type Ca2+-dependent K+ channels in spike broadening during repetitive firing in rat hippocampal pyramidal cells. Journal of Physiology (London) 521: 135- 146. 1. The role of large-conductance Ca2+-dependent K+ channels (BK-channels; also known as maxi-K- or slo-channels) in spike broadening during repetitive firing was studied in CA1 pyramidal cells, using sharp electrode intracellular recordings in rat hippocampal slices, and computer modelling. 2. Trains of action potentials elicited by depolarizing current pulses showed a progressive, frequency-dependent spike broadening, reflecting a reduced rate of repolarization. During a 50 ms long 5

Page 66 AHP, BK- and SK-channel references spike train, the spike duration increased by 63.6 ± 3.4 % from the 1st to the 3rd spike. The amplitude of the fast after-hyperpolarization (fAHP) also rapidly declined during each train. 3. Suppression of BK-channel activity with (a) the selective BK-channel blocker iberiotoxin (IbTX, 60 nM), (b) the non-peptidergic BK- channel blocker (2-10 µM), or (c) calcium-free medium, broadened the 1st spike to a similar degree (approximately 60 %). BK-channel suppression also caused a similar change in spike waveform as observed during repetitive firing, and eliminated (occluded) most of the spike broadening during repetitive firing. 4. Computer simulations using a reduced compartmental model with transient BK-channel current and 10 other active ionic currents, produced an activity-dependent spike broadening that was strongly reduced when the BK-channel inactivation mechanism was removed. 5. These results, which are supported by recent voltage-clamp data, strongly suggest that in CA1 pyramidal cells, fast inactivation of a transient BK-channel current (ICT), substantially contributes to frequency-dependent spike broadening during repetitive firing.

•Shinohara, S. & Kawasaki, K. (1997). Electrophysiological changes in rat hippocampal pyramidal neurons produced by cholecystokinin octapeptide. Neuroscience 78(4): 1005-1016. Effects of cholecystokinin octapeptide (CCK-8) were investigated in CA1 pyramidal neurons of rat hippocampal slice cultures using the whole-cell patch-clamp technique. In the current-clamp mode, CCK-8 (100 nM) produced slight depolarizaton (2.1 ± 0.3 mV) and reduced the amplitude of afterhyperpolarization following a train of spikes. CCK-8 (10 nM-1 µM) concentration-dependently reduced the amplitude of afterhyperpolarization. CCK-4, a selective agonist for CCK(B) receptors, also attenuated the amplitude of afterhyperpolarization. CCK-8-induced suppression was completely abolished by (+)L-365,260, a selective CCK(B) receptor antagonist, but not by (-)L-364,718, a selective CCK(A) receptor antagonist. Similarly, CCK-8 reduced the tail currents following a depolarizing pulse. The tail currents were characterized as Ca2+-activated K+ currents. When neurons were held at a holding potential of -40 mV, CCK-8 elicited inward currents with a reduction of membrane conductance. This current had a relatively linear current voltage relationship and was reversed in polarity at membrane potentials close to the K+ equilibrium potential, suggesting that CCK-8 decreases leak K+ currents. Moreover, voltage- activated Ca2+ currents were partially blocked by CCK-8, and this effect was enhanced by intracellular application of GTPgammaS (300 µM) or a protein

Page 67 AHP, BK- and SK-channel references phosphatase inhibitor, okadaic acid (100 nM), and attenuated by GDPbetaS (300 µM) or a protein kinase inhibitor, staurosporin (400 nM). In acutely-prepared hippocampal slices from neonatal rats, CCK-8 also depolarized CA1 pyramidal neurons and suppressed afterhyperpolarization following a train of action potentials. These results indicate that CCK-8 increases neuronal excitability by suppressing leak K+ currents and Ca2+-activated K+ currents in CA1 pyramidal neurons of the hippocampus through activation of CCK(B) receptors.

•Soh, H., Jung, W., Uhm, D.Y. & Chung, S. (2001). Modulation of large conductance calcium-activated potassium channels from rat hippocampal neurons by glutathione. Neuroscience Letters 298(2): 115-118. We have investigated the modulation of neuronal large conductance Ca2+- activated K+ channels by glutathione. Single channel recordings were made from cultured neonatal rat hippocampal neurons by using excised inside-out patch clamp method. Glutathione, a physiological sulfhydryl specific reducing reagent, increased channel activities in concentration dependent manner with half activation concentration of 710 µM. Conversely, oxidized form of glutathione inhibited channel activities with half inhibition concentration of 520 µM. Our results provide direct evidence that when neuronal large conductance Ca2+-activated K+ channels are exposed to reducing or oxidizing environments, channel activities are increased or decreased in opposite directions due to the redox modification. This may constitute an important regulatory mechanism of neuronal Ca2+-activated K+ channel activities.

•Stocker, M., Krause, M. & Pedarzani, P. (1999). An apamin-sensitive Ca2+- activated K+ current in hippocampal pyramidal neurons. Proceedings of the National Academy of Sciences USA 96(8): 4662-7. In hippocampal and other cortical neurons, action potentials are followed by afterhyperpolarizations (AHPs) generated by the activation of small-conductance Ca2+-activated K+ channels (SK channels). By shaping the neuronal firing pattern, these AHPs contribute to the regulation of excitability and to the encoding function of neurons. Here we report that CA1 pyramidal neurons express an AHP current that is suppressed by apamin and is involved in the control of repetitive firing. This current presents distinct kinetic and pharmacological features, and it is modulated differently than the apamin-insensitive slow AHP current. Furthermore, our in situ hybridizations show that the apamin-sensitive SK subunits are expressed in CA1 pyramidal neurons, providing a potential molecular correlate to the apamin-sensitive

Page 68 AHP, BK- and SK-channel references

AHP current. Altogether, these results clarify the discrepancy between the reported high density of apamin-binding sites in the CA1 region and the apparent lack of an apamin-sensitive current in CA1 pyramidal neurons, and they may explain the effects of this toxin on hippocampal synaptic plasticity and learning.

•Stocker, M. & Pedarzani, P. (2000). Differential distributions of three Ca2+- activated K+ channel subunits, SK1, SK2, and SK3 in the adult rat central nervous system. Molecular Cellular Neuroscience 15: 476-493. Ca2+-activated, voltage-independent K(+) channels are present in most neurons and mediate the afterhyperpolarizations (AHPs) following action potentials. They present distinct physiological and pharmacological properties and play an important role in controlling neuronal firing frequency and spike frequency adaptation. We used in situ hybridization to characterize the distribution patterns of the three cloned SK channel subunits (SK1-3), the prime candidates likely to underlie Ca2+- dependent AHPs in the central nervous system. We found high levels of expression in regions presenting prominent AHP currents, such as, for example, neocortex and CA1-3 layers of the hippocampus (SK1 and SK2), reticularis thalami (SK1 and SK2), supraoptic nucleus (SK3), and inferior olivary nucleus (SK2 and SK3). Our results reveal the functional role of SK channels with defined subunit compositions in some neurons and open the way to the identification of the molecular determinants of AHP currents in many brain regions.Ca2+-activated, voltage-independent K(+) channels are present in most neurons and mediate the afterhyperpolarizations (AHPs) following action potentials. They present distinct physiological and pharmacological properties and play an important role in controlling neuronal firing frequency and spike frequency adaptation. We used in situ hybridization to characterize the distribution patterns of the three cloned SK channel subunits (SK1-3), the prime candidates likely to underlie Ca2+-dependent AHPs in the central nervous system. We found high levels of expression in regions presenting prominent AHP currents, such as, for example, neocortex and CA1-3 layers of the hippocampus (SK1 and SK2), reticularis thalami (SK1 and SK2), supraoptic nucleus (SK3), and inferior olivary nucleus (SK2 and SK3). Our results reveal the functional role of SK channels with defined subunit compositions in some neurons and open the way to the identification of the molecular determinants of AHP currents in many brain regions.

•Storm, J.F. (1987). Action potential repolarization and a fast after- hyperpolarization in rat hippocampal pyramidal cells. Journal of Physiology 385:

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733-759. 1. The repolarization of the action potential, and a fast after- hyperpolarization (AHP) were studied in CA1 pyramidal cells (n = 76) in rat hippocampal slices (28-37 °C). Single spikes were elicited by brief (1-3 ms) current pulses, at membrane potentials close to rest (-60 to -70 mV). 2. Each action potential was followed by four after potentials: (a) the fast AHP, lasting 2-5 ms; (b) an after depolarization; (c) a medium AHP (50-100 ms); and (d) a slow AHP (1-2 s). Both the fast AHP and the slow AHP (but not the medium AHP were inhibited by Ca2+-free medium or Ca2+-channel blockers (Co2+, Mn2+ or Cd2+); but TEA (0.5-2 nM) blocked only the fast AHP and noradrenaline (2-5 µM) only the slow AHP This suggests that two Ca2+-activated K+ currents were involved: a fast, TEA-sensitive one (IC) underlying the fast AHP, and a slow noradrenaline-sensitive one (IAHP) underlying the slow a.h.p. 3. Like the fast a.h.p., spike repolarization seems to depend on a Ca2+-dependent K+ current of the fast, TEA-sensitive kind (IC). The repolarization was slowed by Ca2+-free medium, Co2+, Mn2+, Cd2+, or TEA, but not by noradrenaline. Charybdotoxin (CTX; 30 nM), a scorpion toxin which blocks the large-conductance Ca2+ activated K+ channel in muscle, had a similar effect to TEA. The effects of TEA and Cd2+ (or Mn2+) showed mutual occlusion. Raising the external K+ concentration reduced the fast AHP and slowed the spike repolarization, whereas Cl- loading of the cell was ineffective. 4. The transient K+ current, IA, seems also to contribute to spike repolarization, because: (a) 4-aminopyridine (4-AP; 0.1 mM), which blocks IA, slowed the spike repolarization; (b) depolarizing pre- pulses, which inactivate IA, had a similar effect; (c) hyperpolarizing pre-pulses speeded up the spike repolarization; (d) the effects of 4-AP and pre-pulses persisted during Ca2+ blockade (like IA); and (e) depolarizing pre-pulses reduced the effect of 4-AP. 5. Pre-pulses or 4-AP broadened the spike less, and in a different manner, than Ca2+-free medium, Cd2+, Co2+, Mn2+, TEA or CTX. The former broadening was uniform, with little effect on the fast a.h.p., whereas the latter affected mostly the last two-thirds of the spike repolarization and abolished the fast AHP. This suggests different roles for Ic and Ia during the action potential. 6. IN the presence of Mn2+, 4-AP and carbachol (to block Ic, IAHP, Ia, and the M- current) high concentrations of TEA (4-30 mM) slowed the spike repolarization further, suggesting that another current, perhaps a delayed rectifier (Ik), plays a role when the spike is broadened. 7. In addition to outward currents, an inward Ca2+ current seemed to be active during the falling phase of the spike. Thus, in 2-5 mM TEA, the spike developed a shoulder which was blocked by Cd2+ or Mn2+. 8. In

Page 70 AHP, BK- and SK-channel references conclusion, both a Ca2+-dependent (Ic) and a transient (Ia) K+ current seem to repolarize the action potential in hippocampal neurons, as previously reported in autonomic ganglia. Thus, the mechanism of spike repolarization in vertebrate nerve cells may differ from that in squid axon.

•Storm, J.F. (1987). Intracellular injection of a Ca2+ chelator inhibits spike repolarization in hippocampal neurons. Brain Research 435: 387-392. The Ca-dependence of spike repolarization and afterhyperpolarizations (AHPs) in CA1 pyramidal cells, was tested with intracellular electrodes containing the Ca2+ buffers EGTA or 1,2-bis(o-aminophenoxy)ethane- N,N,N',N' tetraacetic acid (BAPTA). EGTA blocked only the slow AHP; but the fast acting Ca chelator BAPTA also inhibited spike repolarization and the fast AHP. This supports the hypothesis that a fast Ca-activated K+-current contributes to spike repolarization.

•Storm, J.F. (1987). Phorbol esters broaden the action potential in CA1 hippocampal pyramidal cells. Neuroscience Letters 75: 71-74. Intracellular recordings were made from CA1 pyramidal cells in rat hippocampal slices. Single action potentials were elicited by injection of brief current pulses. Bath application of phorbol esters (4 beta-phorbol 12,13-diacetate, 0.3-5 µM; or 4 beta-phorbol-12,13-dibutyrate, 5-10 µM) broadened the action potential in each of the cells tested (n = 9). The broadening reflected slowing of the repolarization, whereas the upstroke of the spike was unchanged. This effect may enhance transmitter release from synaptic terminals, and contribute to enhancement of synaptic transmission through activation of protein kinase C, a mechanism which has been associated with long term potentiation.

•Storm, J.F. (1988). Temporal integration by a slowly inactivating K+ current in hippocampal neurons. Nature 336: 379-381. A central aspect of neuronal function is how each nerve cell translated synaptic input into a sequence of action potentials that carry information along the axon, coded as spike frequency. When transduction from a graded depolarizing input to spikes is studied by injecting a depolarizing current, there is often a remarkably long delay to the first action potential, both in mammalian and molluscan neurons. Here, I report that the delayed excitation in rat hippocampal neurons is due to a slowly inactivating potassium current, ID. ID co-exists with other voltage-gated K+ currents, including a fast A current and a slow delayed rectifier current. As ID

Page 71 AHP, BK- and SK-channel references activates in the subthreshold range, and takes tens of seconds to recover from inactivation, it enables the cell to integrate separate depolarizing inputs over long times. ID also makes the encoding properties of the cell exceedingly sensitive to the prevailing membrane potential.

•Storm, J.F. (1989). An after-hyperpolarization of medium duration in rat hippocampal pyramidal cells. Journal of Physiology 409: 171-190. 1. In hippocampal pyramidal cells, action potentials are followed by three after-hyperpolarizations (AHPs): a fast AHP (fAHP) lasting 2-5 ms, a medium AHP (mAHP) lasting 50-100 ms, and a slow AHP (sAHP) lasting more than 1 s. The mechanism underlying the mAHP was studied in CA1 cells (n = 46) in rat hippocampal slices, using injection of depolarizing current to elicit discharge. 2. The current underlying the mAHP was studied by single-electrode voltage clamp in two ways. Either the voltage clamp was activated following a burst of spikes, thus recording the early tail current underlying the mAHP (hybrid clamp), or, after blocking the spikes with tetrodotoxin, the early tail current following a depolarizing voltage clamp command (to -20 to -45 mV for 100-400 ms) was measured. In both cases, the early tail current (measured at -60 mV) showed the following characteristics: (a) it decayed exponentially with a time constant of about 50 ms; (b) it was substantially reduced by the muscarinic agonist carbachol (40-50 microM); (c) it was moderately reduced (by 20% or less) by Ca2+-free medium and Ca2+ channel blockers (Cd2+, Mn2+), which abolished the fAHP and the sAHP; (d) it was partly blocked by tetraethylammonium (TEA, 1-10 mM) both before and during Ca2+ channel blockade; (e) it was resistant to noradrenaline (5-10 microM), which blocked the sAHP, and to apamin (100 nM). 3. The mAHP itself, recorded under current clamp, showed properties corresponding to those of the early tail current. 4. Unlike the current underlying the sAHP, which was reduced and reversed by hyperpolarization, the early tail current appeared to be reduced only at potentials down to -80 mV, and to increase at more negative potentials. The early tail current and mAHP-like undershoot at hyperpolarized potentials was blocked by external Cs+, but not by carbachol, in contrast to the early tail current and mAHP at -60 mV. 5. It was concluded that two currents contribute to the mAHP: IM (a voltage-gated muscarine-sensitive K+ current) and IC (a Ca2+-dependent TEA-sensitive K+ current). TEA reduced both the IM (5 mM) and the IC (1 mM) component of the mAHP. When the cell is hyperpolarized, a third current, IQ (a Ca+-sensitive mixed Na+-K+ inward current activated by hyperpolarization), masks the reversal of the

Page 72 AHP, BK- and SK-channel references mAHP by causing a depolarizing sag which resembles the decay of the mAHP.

•Storm, J.F. (1990). Potassium currents in hippocampal pyramidal cells. Advances in Brain Research (Understanding the Brain Through the Hippocampus) 83: 161-187. The hippocampal pyramidal cells provide an example of how multiple potassium (K) currents co-exist and function in central mammalian neurones. The data come from CA1 and CA3 neurones in hippocampal slices, cell cultures and acutely dissociated cells from rats and guinea-pigs. Six voltage- or calcium(Ca)-dependent K currents have so far been described in CA1 pyramidal cells in slices. Four of them (IA, ID, IK, IM) are activated by depolarization alone; the two others (IC, IAHP) are activated by voltage-dependent influx of Ca ions (IC may be both Ca- and voltage-gated). In addition, a transient Ca-dependent K current (ICT) has been described in certain preparations, but it is not yet clear whether it is distinct from IC and IA. (1) IA activates fast (within 10 ms) and inactivates rapidly (time constant typically 15-50 ms) at potentials positive to -60 mV; it probably contributes to early spike-repolarization, it can delay the first spike for about 0.1 s, and may regulate repetitive firing. (2) ID activates within about 20 ms but inactivates slowly (seconds) below the spike threshold (-90 to -60 mV), causing a long delay (0.5-5 s) in the onset of firing. Due to its slow recovery from inactivation (seconds), separate depolarizing inputs can be "integrated". ID probably also participates in spike repolarization. (3) IK activates slowly (time constant, tau, 20-60 ms) in response to depolarizations positive to -40 mV and inactivates (tau about 5s) at -80 to -40 mV; it probably participates in spike repolarization. (4) IM activates slowly (tau about 50 ms) positive to -60 mV and does not inactivate; it tends to attenuate excitatory inputs, it reduces the firing rate during maintained depolarization (adaptation) and contributes to the medium after-hyperpolarization (mAHP); IM is suppressed by acetylcholine (via muscarinic receptors), but may be enhanced by somatostatin. (5) IC is activated by influx of Ca ions during the action potential and is thought to cause the final spike repolarization and the fast AHP (although ICT may be involved). Like IM, it also contributes to the medium AHP and early adaptation. It differs from IAHP by being sensitive to tetraethylammonium (TEA, 1 mM), but insensitive to noradrenaline and muscarine. Large-conductance (BK; about 200 pS) Ca-activated K channels, which may mediate IC, have been recorded. (6) IAHP is slowly activated by Ca-influx during action potentials, causing spike- frequency adaptation and the slow AHP. Thus, IAHP exerts a strong negative feedback control of discharge activity.

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•Tanabe, M., Gähwiler, B.H. & Gerber, U. (1998). L-type Ca2+ channels mediate the slow Ca2+-dependent afterhyperpolarization current in rat CA3 pyramidal cells in vitro. Journal of Neurophysiology 80(5): 2268-2273. Single-electrode voltage-clamp recordings were obtained from CA3 pyramidal cells in rat hippocampal organotypic slice cultures, and the slow Ca2+-dependent K+ current or afterhyperpolarization current (IAHP) was elicited with brief depolarizing voltage jumps. The slow IAHP was suppressed by the selective L-type Ca2+ channel antagonists isradipine (2 microM) or nifedipine (10 µM). In contrast, neither omega-conotoxin MVIIA (1 µM) nor omega-agatoxin IVA (200 nM), N-type and P/Q-type Ca2+ channel antagonists, respectively, attenuated this slow outward current. The slow IAHP was significantly reduced by thapsigargin (10 µM), a Ca2+ ATPase inhibitor that depletes intracellular Ca2+ stores, and by ryanodine (10-100 µM), which blocks Ca2+-induced Ca2+ release from intracellular compartments. At this concentration thapsigargin did not modify high-threshold Ca2+ current, which was, however, blocked by isradipine. Thus, in hippocampal CA3 pyramidal cells, Ca2+ influx through L-type Ca2+ channels is necessary to trigger the slow IAHP. Furthermore, intracellular Ca2+-activated Ca2+ stores represent a critical component in the transduction pathway leading to the generation of the slow IAHP.

•Thompson, L.T., Gant, J.C. & Lea, P. (2000). Postsynaptic plasticity in young and aging rat CA1 neurons and spatial learning: Further emphasis on accommodation. Society for Neuroscience Abstracts 30(73.12). Although Hebbian synaptic (i.e. release or receptor mediated) mechanisms have been keenly studied for their roles in learning and memory, considerable evidence demonstrates strongly conserved postsynaptic K+ channel-mediated plasticity in multiple forms of learning and memory consolidation. Following new learning, long after this transient plasticity has decayed to a basal state and when no evidence of learning-dependent synaptic plasticity can be found, a strong relationship persists between excitability and learning rate. The aging process also alters neuronal excitability. Past work has emphasized the role of the post-burst afterhyperpolarization (AHP); the important role of spike frequency accommodation in determining burst firing activity and reciprocal transhippocampal signalling to cortical and noncortical areas also deserves additional attention. Intracellular and whole-cell methods were utilized in a submerged ventral brain slice preparation to examine synaptic and nonsynaptic excitability measures at intervals after learning a

Page 74 AHP, BK- and SK-channel references complex radial-arm maze spatial task. No learning-related plasticity in synaptic or passive membrane properties were observed in CA1 neurons. Plasticity in the post- burst AHP was nonlinearly related to that of spike frequency adaptation or accommodation. Decreased accommodation, which transiently accompanies learning, was also most pronounced in rats that demonstrated the most rapid learning. Conversely, the strongest accommodation was observed in aging neurons from learning-impaired rats. The implications of altered accommodation in modulating hippocampal output are discussed.

•Thompson, L.T., Moyer, J.R., Jr. & Disterhoft, J.F. (1996). Transient changes in excitability of rabbit CA3 neurons with a time-course appropriate to support memory consolidation. Journal of Neurophysiology 76: 1836-1849. 1. The excitability of CA3 pyramidal neurons was assessed with intracellular recordings in hippocampal slices from behaviorally naive rabbits. CA3 pyramidal neurons had large (-13.1 ± 0.3 mV; mean ± SE) postburst afterhyperpolarizations (AHPs) and exhibited robust spike-frequency adaptation (accommodation) to prolonged (800-ms) depolarizing current injection at resting potentials of -68 mV. AHP and accommodation measures differed in scale but not in kind from those obtained in stable recordings from CA1 pyramidal neurons in the same slices or from the same rabbits, with CA3 neurons having larger longer AHPs but fewer spikes during accommodation. 2. Groups of rabbits were trained in a simple, associative- learning task, trace eye-blink conditioning, which required an intact hippocampus for successful acquisition. Memory consolidation in this task also involves the hippocampus, whereas long-term retention of the learned response does not. The time course and magnitude of learning-specific changes in excitability were assessed in 201 CA3 pyramidal neurons. 3. Learning increased the excitability of CA3 pyramidal neurons soon after acquisition (within 1-24 h). The mean postburst AHP was reduced to approximately half (-6.4 ± 0.3 mV) the basal amplitude of the AHP observed in naive controls. The area and duration of the postburst AHP similarly were reduced. Approximately half of all pyramidal neurons tested soon after learning exhibited significantly reduced AHPs, whereas none exhibited enhanced AHPs. 4. Trace conditioning also reduced accommodation of CA3 pyramidal neurons 1- 24 h after learning. Neurons from successfully trained rabbits fired significantly more action potentials (5.6 ± 1.5) in response to prolonged depolarization than did neurons from naive controls (4.1 ± 0.2). The magnitude of the learning-specific change in accommodation was less than that for the AHP. Approximately 45% of

Page 75 AHP, BK- and SK-channel references neurons tested exhibited significantly reduced accommodation soon after learning. 5. Both learning-specific changes in CA3 increased neuronal excitability. Both changes were highly time dependent. AHPs were reduced maximally 1-24 h after learning, then increased, returning to basal (naive) levels within 7 days and remaining basal thereafter. The decay rate of accommodation to basal levels preceded that of the AHP by several days. 6. Other membrane properties, including action potential characteristics, resting potential, and input resistance, were unchanged by learning. The restriction of the observed changes to two interrelated measures of excitability concurs with earlier reports that learning-specific changes in the mammalian hippocampus are linked to changes in a limited number of membrane conductances. 7. Learning, not long-term memory or performance of the learned behavior, was linked to the excitability changes. Neurons from rabbits that failed to acquire the task after considerable training exhibited no excitability changes. Neurons from pseudoconditioned rabbits were indistinguishable from neurons of behaviorally naive controls. Finally, neurons from rabbits that explicitly demonstrated long-term retention of the conditioned response were indistinguishable from those of naive controls. 8. Behavioral changes persisted for extremely long periods, but the observed changes in hippocampal excitability were transient and greatest soon after learning. Excitability was enhanced for a period of a few days, a period demonstrated in other eyeblink studies to be required for memory consolidation. Because hippocampal excitability then returned to basal levels but memory of the learned task persisted, postconsolidation memory traces (the "engram") must be extrahippocampal.

•Tombaugh, G.C. (1998). Intracellular pH buffering shapes activity-dependent Ca2+ dynamics in dendrites of CA1 interneurons. J Neurophysiol 80(4): 1702-1712. Voltage-gated calcium (Ca) channels are highly sensitive to cytosolic H+, and Ca2+ influx through these channels triggers an activity-dependent fall in intracellular pH (pHi). In principle, this acidosis could act as a negative feedback signal that restricts excessive Ca2+ influx. To examine this possibility, whole cell current-clamp recordings were taken from rat hippocampal interneurons, and dendritic Ca2+ transients were monitored fluorometrically during spike trains evoked by brief depolarizing pulses. In cells dialyzed with elevated internal pH buffering (high beta), trains of >15 action potentials (Aps) provoked a significantly larger Ca2+ transient. Voltage-clamp analysis of whole cell Ca currents revealed that differences in cytosolic pH buffering per se did not alter baseline Ca channel

Page 76 AHP, BK- and SK-channel references function, although deliberate internal acidification by 0.3 pH units blunted Ca currents by approximately 20%. APs always broadened during a spike train, yet this broadening was significantly greater in high beta cells during rapid but not slow firing rates. This effect of internal beta on spike repolarization could be blocked by cadmium. High beta also 1) enhanced the slow afterhyperpolarization (sAHP) seen after a spike train and 2) accelerated the decay of an early component of the sAHP that closely matched a sAHP conductance that could be blocked by apamin. Both of these effects on the sAHP could be detected at high but not low firing rates. These data suggest that activity-dependent pHi shifts can blunt voltage-gated Ca2+ influx and retard submembrane Ca2+ clearance, suggesting a novel feedback mechanism by which Ca2+ signals are shaped and coupled to the level of cell activity.

•Toro, L. & Stefani, E. (1991). Ca2+-activated K+ channels: Metabolic regulation. Journal of Bioenergetics & Biomembranes 23: 561-576. Calcium-activated potassium (KCa) channels are highly modulated by a large spectrum of metabolites. Neurotransmitters, hormones, lipids, and nucleotides are capable of activating and/or inhibiting KCa channels. Studies from the last few years have shown that metabolites modulate the activity of KCa channels via: (1) a change in the affinity of the channel for Ca2+ (K 1/2 is modified), (2) a parallel shift in the voltage axis of the activation curves, or (3) a change in the slope (effective valence) of the voltage dependence curve. The shift of the voltage dependence curve can be a direct consequence of the change in the affinity for Ca2+. Recently, the mechanistic steps involved in the modulation of KCa channels are being uncovered. Some interactions may be direct on KCa channels and others may be mediated via G-proteins, second messengers, or phosphorylation. The information given in this review highlights the possibility that KCa channels can be activated or inhibited by metabolites without a change in the intracellular Ca2+ concentration.

•Tzounopoulos, T. & Bissonnette, J.M. (2000). A role for the AHP in metaplasticity. Society for Neuroscience Abstracts 30. Long-term potentiation of synaptic transmission in the hippocampus is the leading experimental model for the synaptic changes that may underlie learning and memory. Interestingly, the ability of a synapse for plastic changes itself displays marked variation and plasticity. This higher form of plasticity, called "metaplasticity" can occur concurrently with synaptic plasticity via identical induction mechanisms. Here, we report that the afterhyperpolarization (AHP, more

Page 77 AHP, BK- and SK-channel references specifically, its apamin sensitive component) by affecting the degree of activation of NMDA receptors affects the degree and direction of synaptic plasticity in the CA1 area in the hippocampus. The intermediate frequency where no lasting change in transmission occurs, the modification threshold, is shifted to the left in the presence of apamin. This effect appears to be postsynaptic since apamin does not affect basal synaptic transmission, paired-pulse facilitation, and post-tetanic potentiation. Additionally, blockade of the AHP does not affect metaplasticity in mossy fibers where plasticity is independent of NMDA receptor activation. These findings suggest a new and important synaptic role for potassium channels that underlie the AHP. The AHP is a very popular target of modulatory pathways, including neurotransmitters. Recognition of the presence of metaplasticity and its mechanisms will provide not only new light on how the brain stores information but also new interpretation on old data, such as the effect of certain neurotransmitters in neuronal excitability.

•Tzounopoulos, T., Linardatos, E. & Stackman, R.W. (2001). Enhanced synaptic plasticity and learning in mice lacking the afterhyperpolarization. Society for Neuroscience Abstracts 31(537.26). Blockade of the medium afterhyperpolarization (mAHP) by apamin facilitates the induction of synaptic plasticity in CA1 hippocampal neurons. We tested whether this facilitation enhances hippocampal-dependent spatial learning in the water maze. To do this, the mAHP was blocked either pharmacologically (apamin) or genetically (SK3 transgenic mice). C57BL6/J mice received apamin (0.4 mg/kg, IP; n=11) or 0.9% saline (n=12) 30 min prior to daily training to learn the spatial location of a submerged escape platform. Immediately after the 4th, 12th and 20th trial, each mouse received a 30-sec probe test of spatial memory retention. Apamin-treated mice exhibited a significant preference for the training quadrant during the probe trial interpolated after the 4th trial. Saline-treated mice required 12 training trials to develop this degree of preference. These results suggest that apamin- induced blockade of SK channels facilitated hippocampal-dependent spatial learning. To test whether SK3 channels contribute to hippocampal-dependent spatial learning and the enhancing effects of apamin, wild-type (WT) mice and conditional SK3 knockout mice were tested using the same water maze paradigm. No group differences were found in escape latency, or in performance during the interpolated probe trials. These results suggest that apamin-induced enhancement of spatial learning may be a consequence of facilitating hippocampal synaptic plasticity and

Page 78 AHP, BK- and SK-channel references may not be due to SK3 channels.

•Velumian, A.A. & Carlen, P.L. (1999). Differential control of three after- hyperpolarizations in rat hippocampal neurones by intracellular calcium buffering. Journal of Physiology (London) 517(Pt. 1): 201-216. 1. The whole-cell recording technique, combined with internal perfusion, was used to study the effects of intracellular Ca2+ buffering on fast, medium and slow after-hyperpolarizations (fAHP, mAHP and sAHP) in hippocampal CA1 pyramidal neurones in rat brain slices at room temperature. 2. The action potentials and the fAHP were unaffected by 100 mµM to 3 mM concentrations of the internally applied fast Ca2+ chelator BAPTA. At higher (10-15 mM) concentrations, BAPTA inhibited the fAHP and prolonged the decay of the action potential, suggesting that the corresponding large-conductance Ca2+-activated K+ channels are located close to the sites of Ca2+ entry during an action potential. Addition of Ca2+ to the BAPTA- containing solution (at a ratio of 4.5 [Ca2+] : 10 [BAPTA]) to maintain the control level of [Ca2+]i did not prevent the effects of high concentrations of BAPTA. 3. The mAHP, activated by a train of action potentials, was inhibited by internally applied BAPTA within the range of concentrations used (100 µM to 15 mM), and this effect could not be reversed or prevented by addition of Ca2+ to the BAPTA- containing solution. The inhibition of the mAHP by BAPTA could also be observed after blockade of the hyperpolarization-activated IQ type mixed Na+-K+ current (also known as Ih) component of the mAHP by bath-applied 3-5 mM Cs+, suggesting that the inhibition of the mAHP by BAPTA is due to inhibition of the depolarization- activated IM (muscarinic) type K+ current. 4. The sAHP, activated by a train of action potentials, was potentiated by 100-300 microM internally applied BAPTA, both with and without added Ca2+. At 1-2 mM or higher concentrations, the potentiation of the sAHP by BAPTA without added Ca2+ was transient and was followed by a fast decrease. With added Ca2+, however, BAPTA caused a persistent potentiation of the sAHP with more than a 10-fold increase in duration for periods exceeding 1 h even at concentrations of the buffer as high as 10-15 mM. Earlier reports showing a blockade of the sAHP by BAPTA, based on experiments without added Ca2+, were apparently due to a sharp reduction in intracellular free [Ca2+] and to a high intracellular concentration of the free buffer. 5. Internally applied BAPTA caused a prolongation of the spike discharge during an 800 ms-long depolarizing current step. At 100-300 µM BAPTA, but not at 1-2 mM or higher concentrations, this effect could be reversed by addition of Ca2+. The effects of

Page 79 AHP, BK- and SK-channel references

BAPTA on the spike discharge occurred in parallel with the changes in the sAHP time course, which was more prolonged at higher concentrations of the buffer. 6. The concentration-dependent differential control of the three types of AHP in hippocampal neurones by BAPTA is related to modulation of intracellular Ca2+ diffusion by a fast acting mobile Ca2+ buffer.

•Vergara, C., Latorre, R., Marrion, N.V. & Adelman, J.P. (1998). Calcium- activated potassium channels. Current Opinions in Neurobiology 8(3): 321-9. Calcium-activated potassium channels are fundamental regulators of neuronal excitability, participating in interspike interval and spike- frequency adaptation. For large-conductance calcium-activated potassium (BK) channels, recent experiments have illuminated the fundamental biophysical mechanisms of gating, demonstrating that BK channels are voltage gated and calcium modulated. Structurally, BK channels have been shown to possess an extracellular amino- terminal domain, different from other potassium channels. Domains and residues involved in calcium- gating, and perhaps calcium binding itself, have been identified. For small- and intermediate-conductance calcium-activated potassium channels, SK and IK channels, clones have only recently become available, and they show that SK channels are a distinct subfamily of potassium channels. The biophysical properties of SK channels demonstrate that kinetic differences between apamin-sensitive and apamin-insensitive slow afterhyperpolarizations are not attributable to intrinsic gating differences between the two subtypes. Interestingly, SK and IK channels may prove effective drug targets for diseases such as myotonic muscular dystrophy and sickle cell anemia.

•Wadsworth, J.D., Doorty, K.B., Ganellin, C.R. & Strong, P.N. (1996). Photolabile derivatives of 125I-apamin: Defining the structural criteria required for labeling high and low molecular mass polypeptides associated with small conductance Ca2+-activated K+ channels. Biochemistry 35(24): 7917-27. The structure of apamin-sensitive Ca2+-activated K+ channels has been investigated using high-affinity, photolabile azidoaryl derivatives of 125I-[alpha- formyl-Cys1]apamin and 125I-[epsilon-formyl-Lys4]-apamin. Labeling patterns suggest that similar structural constraints are required for labeling analogous polypeptides associated with distinct channel subtypes. When photoprobes are coupled at the epsilon-amino- Lys4 position of apamin, comparable low molecular mass (approximately 30 kDa) polypeptides are efficiently labeled on either brain or

Page 80 AHP, BK- and SK-channel references liver plasma membranes, irrespective of the structure of the photoprobe. However, when photoprobes are coupled at the alpha-amino-Cys1 position of apamin, the pattern of labeling on both brain and liver plasma membranes varies, depending upon the length of the spacer arm incorporated into the photoprobe. Spacer arms of approximately 8-9 A efficiently label only high molecular mass polypeptides (86, 59 kDa), accompanied by weak, variable labeling of a 44-kDa component. A shorter spacer arm (5.7 A) results in feeble labeling of 86- and 59-kDa polypeptides and barely detectable labeling of 44- and approximately 30- kDa polypeptides. In contrast, a long spacer arm (12.8 A) efficiently labels only approximately 30-kDa polypeptides. These findings point to close similarities in the topography of the 125I-apamin binding site present on pharmacologically distinct subtypes of apamin- sensitive Ca2+- activated K+ channels and indicates that heterooligomeric association of high and low molecular mass polypeptide subunits may be a general structural feature of members belonging to this family of K+ channels.

•Wang, Z.F. & Shi, Y.L. (2001). Toosendanin-induced inhibition of small- conductance calcium-activated potassium channels in CA1 pyramidal neurons of rat hippocampus. Neuroscience Letters 303(1): 13-16. The effect of toosendanin (TSN) on small-conductance calcium-activated potassium channels (SK(Ca)) in pyramidal neurons of rat hippocampal CA1 region was observed using the inside-out configuration of patch-clamp technique. The results showed that TSN (1.7–170 µM) inhibited the SK(Ca) activity by reducing the open probability and open frequency significantly in a concentration-dependent manner, and the effects were partially reversible. Elevating Ca2+ concentration at the intracellular side of the patch ([Ca2+](i)) from 1 to 10 µM decreased the inhibitory efficacy. Analysis of the channel kinetics indicated that TSN increased the slow closed time constant significantly, while open time and unitary conductance of channel did not change. These data provide a further explanation for TSN-induced facilitation of neurotransmitter release and antibotulismic effects of the drug.

•Weiss, C., Preston, A.R., Oh, M.M., Schwarz, R.D., Welty, D. & Disterhoft, J.F. (2000). The M1 muscarinic agonist CI-1017 facilitates trace eyeblink conditioning in aging rabbits and increases the excitability of CA1 pyramidal neurons. Journal of Neuroscience 20(2): 783-790. The M1 muscarinic agonist CI-1017 was administered intravenously to aging rabbits on a daily basis before and during hippocampally dependent trace eyeblink

Page 81 AHP, BK- and SK-channel references conditioning sessions. Circulating levels of CI-1017 were significantly related to the drug dose. The drug was found to significantly increase the rate and amount of learning in a dose-dependent manner with no significant effects on the amplitude, area, or latency of conditioned responses. There was no evidence of pseudoconditioning at the highest drug concentration, and the minimally effective dose produced only mild and temporary hypersalivation as a side effect. CI-1017 (10 µM) was also found to increase the excitability of CA1 pyramidal neurons recorded from hippocampal slices from young and aging naive rabbits as measured by changes in spike-frequency adaptation and the postburst afterhyperpolarization. These biophysical changes were reversed with either atropine (1 µM) or (1 µM). These results suggest that M1 agonists ameliorate age-related learning and memory impairments at least in part by reducing the afterhyperpolarization and spike- frequency adaptation of hippocampal pyramidal neurons and that M1 agonists may be an effective therapy for reducing the cognitive deficits that accompany normal aging and/or Alzheimer's disease.

•Wolfart, J., Franz, O., Neuhoff, H. & Roeper, J. (2000). Functional role and molecular identity of SK channels in dopaminergic midbrain neurons. Society for Neuroscience Abstracts 30. Small conductance calcium-activated potassium (SK) channels contribute to an important part of neuronal signalling: the spike afterhyperpolarization (AHP). Therefore, SK-channels have been implicated in regulation of the pacemaker frequency in dopaminergic (DA) midbrain neurons. Our aim is to identify the molecular composition of SK channels and their functional contribution to the pacemaker frequency in DA neurons. AHP currents (I-AHPs) of DA neurons were studied using standard whole-cell and gramicidin-perforated patch- clamp recordings in brain slices from 12-14 day old C57Bl/6 mice. I-AHP amplitudes recorded at –80 mV were in the range of 20-300 pA (mean=97±8 pA, n=68). In most cells, I-AHPs decayed bi-exponentially with time constants of 138±9 ms and 1103±76 ms, respectively (n=38). The faster I-AHPs were sensitive to d-tubocurarine (IC50 =0.077 mM, Hill coef.=0.97, n=44) and low nanomolar concentrations of apamin (n=20), while the slower I-AHPs were unaffected by these toxins. Under conditions of intact calcium handling in gramicidin-perforated whole-cell recordings, selective inhibition of the fast I-AHPs accelerated the spiking frequency of DA neurons in a frequency-dependent manner. This indicates that apamin/d-tubocurarine-sensitive SK channels are directly involved in controlling the pacemaker of DA neurons. We

Page 82 AHP, BK- and SK-channel references currently investigate the mRNA and protein expression profiles of SK channel isoforms in single DA neurons using single-cell RT-mPCR and immunocytochemistry.

•Wu, J. & Okada, Y.C. (1999). Roles of a potassium afterhyperpolarization current in generating neuromagnetic fields and field potentials in longitudinal CA3 slices of the guinea-pig. Clinical Neurophysiology 110(11): 1858-1867. OBJECTIVES: Roles of a calcium-dependent potassium conductance of slow afterhyperpolarization (AHP) type (gK(AHP)) in generating magnetoencephalographic (MEG) signals were studied in hippocampal longitudinal CA3 slices of the guinea pig. METHODS: The roles of gK(AHP) were experimentally inferred from effects of its blocker, carbamylcholine-chloride (carbachol, CCh), on MEG signals. The MEG signals were compared with extracellular field potentials and intracellular potentials of the pyramidal cells in the slice. RESULTS: CCh profoundly affected MEG waveforms. CCh reduced the initial spike of the evoked MEG signals independently of stimulation of the cell layer and apical dendrites. The slow wave of the evoked MEG signals was reduced by the somatic stimulation, but was enhanced by the apical stimulation. Elevated extracellular calcium and bath-applied tetraethylammonium (TEA) enhanced the CCh effects. CCh also increased spontaneous MEG signals. These effects on MEG and field potentials could be interpreted on the basis of synaptic and intracellular effects of CCh. CONCLUSIONS: Our results indicate that abnormality in this subtle calcium-dependent potassium channel may profoundly influence MEG and EEG signals.

•Wu, W.W., Oh, M.M. & Disterhoft, J.F. (2002). Age-related biophysical alterations of hippocampal pyramidal neurons: implications for learning and memory. Ageing Research Reviews 1(2): 181-207. Normal brain aging is associated with deficits in learning and memory. The hippocampus, a structure critical for proper learning and memory functions, is frequently implicated in aging-related learning deficits. There are many reports of learning-related changes in hippocampal pyramidal neurons from animals that were trained in hippocampus-dependent learning paradigms. One consistent finding in hippocampal pyramidal neurons is a learning-related increase in postsynaptic neuronal excitability, resulting from a reduction in the postburst afterhyperpolarization (AHP). The hippocampus, as well as the ability to acquire hippocampus-dependent tasks, is particularly affected by aging. Correspondingly, hippocampal neurons also display an age-related decrease in excitability, resulting

Page 83 AHP, BK- and SK-channel references from an enhanced AHP. The correlation between neuronal excitability and learning ability strongly suggests that changes in the AHP are critically involved in learning and age-related learning deficits. Additional support for this argument comes from in vitro studies that examined the effect of compounds that facilitated learning in aging animals on the properties of CA1 pyramidal neurons. Many of these compounds increased the excitability of CA1 pyramidal neurons by reducing the AHP. Subsequent voltage-clamp recordings showed that AHP reduction by these compounds mainly reflects the reduction of two of its currents, the I(AHP) and the sI(AHP). Conversely, age-related AHP enhancements primarily impact the I(AHP) and the sI(AHP). Given that the I(AHP) accounts for a small portion of the total AHP, and that the sI(AHP) is the AHP current that most critically modulates neuronal excitability, changes in neuronal excitability seen in learning and in aging are predominantly caused by changes in the sI(AHP). The fact that the sI(AHP) receives neuromodulation from many transmitter systems important for learning and sensitive to aging lends further support for its role in age-related learning deficits. In this article, we review: (1) two hippocampus-dependent learning tasks, trace eyeblink conditioning and Morris water maze training, that are used extensively in our laboratory to examine learning and aging-related learning deficits; (2) aging-related changes in several important neurotransmitter systems, and how the these changes impact learning and memory functions during aging; and (3) changes in the AHP and the sI(AHP) in hippocampal pyramidal neurons in relation to compromised neurotransmission, as well as to learning, in aging animals. The correlations between a reduction in the sI(AHP) in learning, and an enhancement in the sI(AHP) in aging provide compelling evidence that this current plays a critical role in cognitive functions, and further suggest that the key modulators of the AHP are good candidates for future therapeutic interventions in age-related neurodegenerative diseases.

•Wu, W.W., Sametsky, E.A., Oh, M.M., Power, J.M. & Disterhoft, J.F. (2001). Nimodipine causes similar reductions in the sIAHP of CA1 hippocampal pyramidal neurons in young and aging rabbits. Society for Neuroscience Abstracts 31(382.9): 196. Previous behavioral and electrophysiological studies from our laboratory have implicated changes in neuronal excitability, reflected by changes in the AHP, in learning and in age-related learning deficits. We have further shown a correlation between the AHP and the sIAHP, a component of the AHP, suggesting a role for the

Page 84 AHP, BK- and SK-channel references sIAHP in determining neuronal excitability. Because the sIAHP is a Ca2+-dependent K+ current, and previous studies have shown an enhanced L-type Ca2+ influx with age, we performed voltage-clamp experiments to examine if the increase in the sIAHP with age were due to an enhanced L-type Ca2+ influx. Hippocampal slices were prepared from <3 mo and >36 mo old rabbits. After achieving whole-cell configuration, TTX, TEA, CsCl, 4-AP, CNQX, D-AP5, and PTX were used to isolate the AHP. A 100 ms/50 mV voltage step from a holding potential of 55 mV was used to evoke the AHP, and the sIAHP was measured 1s after pulse offset. After acquiring baseline measurements, 10 M nimodipine was bath applied to block the L-type Ca2+ influx. Our data show enhanced area and amplitude of the sIAHP, and an enhanced amplitude of the IAHP, in CA1 neurons of aging rabbits (ps < 0.02). The average sIAHP amplitude of neurons from aging rabbits was larger than that of the young by ~57%. Nimodipine significantly reduced the sIAHP in neurons of both groups (ps < 0.0001) by ~25%; the remaining sIAHP of neurons from aging animals was larger than that of the young by ~35%. Our results suggest a partial contribution of L-type Ca2+ influx to the enhancement of the sIAHP with age. Supported by: NIH AG08796, MH11737, MH12858

•Young, S.R., Bianchi, R., Kim, D., Shin, H.S. & Wong, R.K.S. (2000). Transduction mechanisms underlying Group I mGluR-mediated block of afterhyperpolarization in CA3 pyramidal cells. Society for Neuroscience Abstracts 30. Simultaneous current clamp and optical recordings of [Ca2+]i transients were made from CA3 pyramidal cells in mouse and guinea pig hippocampal slices. After- hyperpolarizations (AHPs) observed after single action potentials consisted of fast and slow phases with half-duration times of about 1 sec and 3-4 sec respectively. Stimulation of group I mGluR with DHPG (10 mM) produced a reduction of the fast phase AHP with no noticeable effects on the slow phase. In addition, the agonist significantly broadened the foot of the action potential. Monitoring of [Ca2+]i revealed that the changes in AHP amplitude and action potential duration were not accompanied by alterations in the [Ca2+]i response. Recordings from CA3 neurons in slices prepared from phospholipase C b1 knockout (PLCb1 -/-) mice showed that the time courses of the AHP and action potentials were comparable to those of wild- type mice. Addition of DHPG suppressed the fast phase of the AHP in the PLCb1 -/- neurons but no longer affected the duration of the action potential. Action potential-dependent [Ca2+]i signals were not altered in the mutant mice. The results

Page 85 AHP, BK- and SK-channel references suggest that the suppression of the fast phase AHP in response to DHPG is directly mediated by G-protein action while the effect on action potential duration requires the involvement of PLCb1. Supported by: NIH Grant DHHS NS35481

•Yoshida, A., Oda, M. & Ikemoto, Y. (1991). Kinetics of the Ca2+-activated K+ channel in rat hippocampal neurons. Japanese Journal of Physiology 41(2): 297-315. The kinetics of the large-conductance Ca2+-activated K+ channel (235 pS in symmetrical 150 mM K+) were examined in the inside-out mode of the patch clamp technique. The open probability of the channel increased when [Ca2+]i, [Sr2+]i, or [Ba2+]i was increased. The [Ca2+]i-response relation was fitted with a Hill coefficient of 2 and half-maximum concentrations of 185, 80, 14.5, and 5.5 microM at -40, -20, +20, and +40 mV, respectively. The channel was blocked by TEA or Ba2+. The open-time histogram showed a single exponential component and the closed-time histogram showed at least two exponential components at various [Ca2+]i. Increasing [Ca2+]i decreased the time constant of the slow component of the closed-time histogram. Cell-attached patch recording revealed activation of the large-conductance Ca2+-activated K+ channel (BK channel) during the action potential. The deactivation time course was consistent with the fast after- hyperpolarization. A minimum model of the channel, close(2)-close(1)-open, where the transition from close(2) to close(1) requires the binding of 2 Ca2+, reconstructed quick activation of the channel if [Ca2+]i of 40 µM was assumed.

•Zahorodna, A. & Bijak, M. (1999). An antidepressant-induced decrease in the responsiveness of hippocampal neurons to group I metabotropic glutamate receptor activation. European Journal of Pharmacology 386(2-3): 173-179. , a serotonin and noradrenaline uptake inhibitor, is the prototypical . The effects of imipramine on neuronal responsiveness to the group I glutamate metabotropic (mGlu) receptor agonist (RS)- 3,5-dihydroxyphenylglycine (DHPG) were studied ex vivo, in the CA1 area of rat hippocampus, using extracellular and intracellular recording. DHPG increased the population spike amplitude, depolarized CA1 cells and decreased the slow afterhyperpolarization. Imipramine (20 µM) administered acutely in vitro did not change the effect of DHPG on population spikes. Repeated treatment with imipramine (10 mg/kg, twice daily, for 14 days) significantly attenuated the enhancing effect of DHPG (2.5 and 5 µM) on population spikes, as well as the DHPG-

Page 86 AHP, BK- and SK-channel references induced depolarization and the decrease in the slow afterhyperpolarization. Repeated treatment with imipramine had no effect on passive or active membrane properties of CA1 pyramidal cells. The results of the time-course experiment demonstrated that the imipramine-induced decrease in the responsiveness of CA1 cells to DHPG was apparent after a 7-day treatment; there was a further decrease after 14 days of treatment to a level which was not changed by longer (21-day) administration of imipramine. The attenuation of neuronal responsiveness to DHPG induced by a 14-day treatment was still detectable 7 days after imipramine withdrawal. It is concluded that repeated treatment with imipramine induces a decrease in the responsiveness of rat CA1 hippocampal neurons to group I mGlu receptor activation with a time course which correlates with the delayed onset of the therapeutic effect of antidepressants in humans. This suggests that alterations in mGlu receptors may contribute to antidepressant efficacy.

•Zahorodna, A., Palucha, A. & Bijak, M. (1999). Comparison of the effects of low and high concentrations of group I metabotropic receptor agonists on field potentials in the hippocampal CA1 region in vitro. Polish Journal of Pharmacology 50(4-5): 291-298. We compared the effects of low and high concentrations of the selective group I metabotropic glutamate receptor (mGluR) agonist (R,S)-3,5- dihydroxyphenylglycine (DHPG) and the nonselective mGluR agonist (1S,3R)-1- aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD) on extracellularly recorded potentials which were evoked in the rat hippocampal CA1 region by stimulation of the Schaffer collateral/commissural pathway and on intracellularly recorded electrophysiological properties of CA1 neurons, in vitro. At low concentrations (2.5 and 5 µM) DHPG and (1S,3R)-ACPD increased while at high concentrations (20 and 50 µM) they decreased population spike amplitudes. Simultaneous recordings of population spikes in the CA1 cell layer and field excitatory postsynaptic potentials (fEPSPs) in stratum radiatum of the CA1 area revealed that the enhancement of the population spike amplitude is not associated with any change in the fEPSP slope, but the decrease in population spikes is accompanied with a decrease in the fEPSP slope, suggesting that at high concentrations both agents may attenuate excitatory synaptic transmission in CA1 cells. DHPG and (1S,3R)-ACPD had a number of direct excitatory effects on CA1 pyramidal cells like a concentration-dependent depolarization and an inhibition of the slow afterhyperpolarization, which in all probability underlay the increase in the amplitude of population spikes. At high

Page 87 AHP, BK- and SK-channel references concentrations, both mGluR agonists strongly depolarized CA1 cells indicating that depolarization block of cell discharges may underlay the reduction in the population spike amplitude. Furthermore, robust cell discharges induced by the strong depolarizations, activate several secondary processes which may significantly contribute to the action of high concentrations of DHPG and (1S,3R)-ACPD. Therefore, the effects of low and high concentrations of the studied mGluR agonists may involve different mechanisms, at low concentrations the effects can be directly related to the activation of postsynaptically localized group I mGluRs while at higher concentrations the contribution of indirect effects may predominate.

DENTATE GRANULE NEURONS

•Aradi, I. & Holmes, W.R. (1999). Role of multiple calcium and calcium-dependent conductances in regulation of hippocampal dentate granule cell excitability. Journal of Computational Neuroscience 6(3): 215-235. We have constructed a detailed model of a hippocampal dentate granule (DG) cell that includes nine different channel types. Channel densities and distributions were chosen to reproduce reported physiological responses observed in normal solution and when blockers were applied. The model was used to explore the contribution of each channel type to spiking behavior with particular emphasis on the mechanisms underlying postspike events. T-type calcium current in more distal dendrites contributed prominently to the appearance of the depolarizing after- potential, and its effect was controlled by activation of BK-type calcium-dependent potassium channels. Coactivation and interaction of N-, and/or L-type calcium and AHP currents present in somatic and proximal dendritic regions contributed to the adaptive properties of the model DG cell in response to long-lasting current injection. The model was used to predict changes in channel densities that could lead to epileptogenic burst discharges and to predict the effect of altered buffering capacity on firing behavior. We conclude that the clustered spatial distributions of calcium related channels, the presence of slow delayed rectifier potassium currents in dendrites, and calcium buffering properties, together, might explain the resistance of DG cells to the development of epileptogenic burst discharges.

•Dietrich, D., Selke, K., Clusmann, H., Kral, T. & Schramm, J. (2001). Epileptic granule cells devoid of calbindin display increased fast AHP and no medium AHP. Society for Neuroscience Abstracts 31(558.12).

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It is well known that during the course of mesial temporal lobe epilepsy the majority of granule cells (GCs) loose their intracellular Ca2+-binding protein calbindin. However, the consequences of the reduced content of calbindin for excitability and integrative properties of GCs in situ are not known. Using immunohistochemistry, patch-clamp recordings and intracellular staining in hippocampal slices from epileptic patients we have analyzed the firing pattern of GCs with and without calbindin. GCs that contain normal levels of calbindin display a firing pattern indistinguishable from that of rodent GCs: action potentials are followed by a fast and a medium AHP and during the first 5-8 action potentials in a train, a prominent spike frequency adaptation develops. The amplitude of the Ca2+ dependent fast AHP remains constant during tonic firing at 20 Hz. In contrast, GCs which do not contain detectable amounts of calbindin could be clearly identified as such solely based on their distinct firing pattern: they have a significantly larger fast AHP amplitude and the medium AHP component is not detectable. Early spike frequency adaptation is absent in such cells devoid of calbindin. During tonic firing at 20 Hz the fast AHP amplitude gradually reduces by 30%. Decreasing Ca2+-entry by lowering extracellular Ca2+ restores the medium AHP and depresses the fast AHP amplitude back to normal levels. The results show that epileptic GCs without calbindin generate high frequency discharges which possibly result from decreased buffering of Ca2+-entry during action potentials.

•Hu, H. & Storm, J.F. (2001). BK channels contribute to action potential repolarization in granule cells of the rat dentate gyrus. Society for Neuroscience Abstracts 31(382.10): 196. Large-conductance Ca2+-activated K+ channels have been implicated in action potential (AP) repolarization and generation of the fast after-hyperpolarization (fAHP) in hippocampal pyramidal cells. We have now tested their role in another class of principal neurons of the hippocampal formation: the dentate gyrus(DG) granule cells. Using whole-cell somatic recordings from DG granule cells in hippocampal slices (34 -37°C), spike trains were evoked by injecting 50 ms depolarizing current pulses. The 1st spike in the train was followed by a pronounced fAHP, lasting 3-10 ms. A low dose of tetraethylammonium (TEA 0.5 mM, n=5) reversibly reduced the fAHP and slowed the AP repolarization. The selective BK channel blocker iberiotoxin (IbTX 100nM, n=5) had a similar, but irreversible, effect (30.34.7% increase in spike 90- 10% repolarization time; 39.916.0% reduction of the fAHP). The BK channel blocker

Page 89 AHP, BK- and SK-channel references paxilline (2 M, n=3) had similar effects. Calcium-free medium with 1-2 mM Mn2+ (n=5) also reversibly reduced the fAHP, although it had little effect on the overall AP duration, consistent with a dual effect on inward Ca2+ and outward BK currents. In cells dialysed with 10 mM EGTA (n=5), the fAHP and spike decay time were unaffected, although the Ca2+-activated slow AHP (sAHP) was abolished. Subsequent application of 100 nM IbTX to the EGTA-loaded granule cells also reduced fAHP and broadened the APs, consistent with rapid activation of BK channels, colocalized with Ca2+ channels, during the spike. In conclusion, our results indicate that a BK channel current contributes to the spike repolarization and the fAHP in DG granule cells.

•Podlogar, M., Dietrich, D., Selke, K., Kral, T., Clusmann, H. & Schramm, J. (2000). Perforated patch-analysis of firing properties in hippocampal granule cells: Minimal sAHP and tonic firing. Society for Neuroscience Abstracts 30. Afterhyperpolarizations (AHPs) represent major intrinsic inhibitory mechanisms in the regulation of neuronal excitability. The slow AHP (sAHP) is known as the most prominent AHP lasting for several seconds. Our study was designed to investigate the importance of the sAHP for the resistance of hippocampal granule cells (GCs) to discharge in a bursting pattern. In order to preserve the physiological cytoplasmic constitution we used the gramicidin perforated patch technique in rat hippocampal slices to characterize the sAHP in GCs in compare to the sAHP in pyramidal cells. In pyramidal neurons, an sAHP could be evoked by suprathreshold current injection as short as 50 ms (min. 10 APs). In 50% of these cells a significant sAHP occured during the current injection and lead to a total suppression of firing. A current injection of 500 ms (10 APs) was followed by an sAHP of 9.1 ± 1.3 mV with a half amplitude width of 2.7 ± 0.4 s (Rin 267 ± 55 MOhm). In contrast, we could detect only a minimal sAHP in GCs. This minimal sAHP only emerged after prolonged current injections (> 500 ms, min. 20 APs). During a current injection we never observed an sAHP and the cells also never stopped firing. The sAHP had a typical kinetic (half amplitude width 2.20 ± 0.62 s) but a very small amplitude of 1.8 ± 0.6 mV (500 ms, 20 - 30 APs, Rin 225 ± 81 MΩ). Accordingly, after an early component of spike frequency adaptation within the first 300 ms, the cells continued firing tonically with a constant frequency (maximal 50 - 100 Hz). We conclude that in GCs there is only a very small conductance change during the sAHP, which cannot account for their low excitability.

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•Valiante, T.A., Abdul-Ghani, M.A., Carlen, P.L. & Pennefather, P. (1997). Analysis of current fluctuations during after-hyperpolarization current in dentate granule neurones of the rat hippocampus. Journal of Physiology (London) 499(Pt. 1): 121-134. 1. We have studied macroscopic current fluctuations associated with the after-hyperpolarization current (IAHP) that follows a 200 ms voltage-clamp step to 0 mV in dentate granule (DG) neurones of the rat hippocampus. This maximally effective stimulus produced a peak IAHP of 205 ± 20 pA. Background noise was minimized by using the whole-cell single-electrode voltage-clamp configuration. 2. Conventional current-variance analysis was performed on IAHP to obtain estimates of the unitary AHP channel current (i) and the maximal attainable AHP current (Imax). A second approach, utilizing changes in the power spectrum of IAHP 'noise' during the decay of IAHP, was employed to yield an independent estimate of Imax as well as an estimate of the mean open-state duration of AHP channels. 3. Changes in the power spectrum during IAHP decay revealed that the mean channel open time is fixed at 6.9 ± 0.5 ms and that the decay is due to changes in channel closed-state duration. The same analysis gave a value for Imax of 320 ± 20 pA (n = 7). 4. Current-variance analysis suggests that channels responsible for generation of IAHP have a unitary current of 0.29± 0.08 pA at -45 mV in 5 mM extracellular potassium and an Imax of 400 ± 180 (n = 7). Thus, both methods indicate that about 1200 channels are available to generate IAHP in DG neurones and that about 60% are open at the peak of a maximal IAHP. 5. Computer simulations of IAHP currents in a model neurone show that dendritic current sources will result in an underestimation of i while Imax is underestimated to a lesser extent. Estimates of Imax obtained from power-spectrum analysis are more accurate and less affected by neuronal electrotonic structure than estimates of Imax based on current-variance analysis.

CORTICAL PYRAMIDAL NEURONS

•Abel, H.J., Callaway, J.C. & Foehring, R.C. (2001). Spike-induced calcium signals and afterhyperpolarizations in neocortical pyramidal neurons. Society for Neuroscience Abstracts 31(382.18): 196. In neocortical pyramidal cells, Ca2+ entry through voltage-gated channels during repetitive firing activates Ca2+-dependent K+ channels, resulting in an afterhyperpolarization (AHP) which is an important negative regulator of the cells

Page 91 AHP, BK- and SK-channel references firing behavior (e.g., spike frequency adaptation). This AHP consists of an apamin- sensitive medium component (mAHP) and an apamin-insensitive slow component (sAHP). These two AHP components differ in decay kinetics. The spike-induced build- up of [Ca2+]i may also serve to monitor the cells activity and control cellular processes such as gene transcription. Using whole-cell patch recording from slices of rat somatosensory neocortex with simultaneous fura-2 Ca2+ imaging, we determined the relationships between (1) the frequency and number of spikes and the amplitudes of the mAHP and sAHP and (2) between the frequency and number of spikes and [Ca2+]i in soma and dendrites. The mAHP was present after one spike but the sAHP required several spikes to be measurable. Both AHPs increased in size with additional spikes until a plateau level was reached. We found that [Ca2+]i in the proximal dendrites reaches a steady-state plateau whose amplitude is dependent on spike frequency (cf., Helmchen et al. (1996) Biophys. J. 70: 1069-1081). However, our data suggest that this relationship is exponential rather than linear. Finally, we compared the time course of [Ca2+]i decay with the time courses of the sAHP and mAHP to examine compartmentalization as a possible explanation for differences in kinetics between the two AHPs. Supported by: NS 33579 (to R.C.F.) and NS36843 (to J.C.C).

•Barkai, E. & Saar, D. (2001). Cellular correlates of olfactory learning in the rat piriform cortex. Review. Reviews in Neuroscience 12(2): 111-120. This review describes research that combines cellular physiology with behavioral neuroscience, to study the cellular mechanisms underlying learning and memory in the mammalian brain. Rats were trained with an olfactory conditioning paradigm, in which they had to memorize odors in order to be rewarded with drinking water. Such training results in rule learning, which enables enhanced acquisition of odor memory. Training results in the following learning-related physiological modifications in intrinsic and synaptic properties in olfactory (piriform) cortex pyramidal neurons: 1. increased neuronal excitability, indicated by reduced afterhyperpolarization, and 2. increased synaptic transmission, indicated by reduced paired-pulse facilitation. These modifications are correlated to enhanced learning capability rather than to storage of memory for specific odors. In addition, using a different paradigm of odor-training, it is shown that NMDA and beta-adrenergic receptors are involved at different stages of long-term memory consolidation.

•Bowen, T., Guy, C.A., Craddock, N., Cardno, A.G., Williams, N.M., Spurlock,

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G., Murphy, K.C., Jones, L.A., Gray, M., Sanders, R.D., McCarthy, G., Chandy, K.G., Fantino, E., Kalman, K., Gutman, G.A., Gargus, J.J., Williams, J., McGuffin, P., Owen, M.J. & O'Donovan, M.C. (1998). Further support for an association between a polymorphic CAG repeat in the hKCa3 gene and schizophrenia. Molecular Psychiatry 3(3): 266-269. A recent study has suggested that a polymorphism in the hKCa3 potassium channel may be associated with raised susceptibility to schizophrenia. Despite its modest statistical significance, the study is intriguing for two reasons. First, hKCa3 contains a polymorphic CAG repeat in its coding sequence, with large repeats more common in schizophrenics compared with controls. This is interesting in view of several repeat expansion detection (RED) studies that have reported an excess of large CAG repeats in psychotic probands. Second, the hKCa3 gene is a functional candidate gene because studies of antipsychotic and psychotogenic compounds suggest that glutamatergic systems modulated by SKCa channels may be important in schizophrenia pathogenesis. In the light of the above, we have tested the hypothesis of an association between schizophrenia and the hKCa3 CAG repeat polymorphism using a case control study design. Under the same model of analysis as the earlier study, schizophrenic probands had a higher frequency of alleles with greater than 19 repeats than controls (chi 2 = 2.820, P = 0.047, 1-tail). Our data therefore provide modest support for the hypothesis that polymorphism in the hKCa3 gene may contribute to susceptibility to schizophrenia.

•Brenner, R., Jegla, T.J., Wickenden, A., Liu, Y. & Aldrich, R.W. (2000). Cloning and functional characterisation of novel large conductance calcium-activated potassium channel beta-subunits, hKCNMB3 and hKCNMB4. Journal of Biological Chemistry 275(9): 6453-6461. We present the cloning and characterization of two novel calcium-activated potassium channel beta subunits, hKCNMB3 and hKCNMB4, that are enriched in the testis and brain, respectively. We compare and contrast the steady state and kinetic properties of these beta subunits with the previously cloned mouse beta1 (mKCNMB1) and the human beta2 subunit (hKCNMB2). Once inactivation is removed, we find that hKCNMB2 has properties similar to mKCNMB1. hKCNMB2 slows Hslo1 channel gating and shifts the current-voltage relationship to more negative potentials. hKCNMB3 and hKCNMB4 have distinct effects on slo currents not observed with mKCNMB1 and hKCNMB2. Although we found that hKCNMB3 does interact with Hslo channels, its effects on Hslo1 channel properties were slight,

Page 93 AHP, BK- and SK-channel references increasing Hslo1 activation rates. In contrast, hKCNMB4 slows Hslo1 gating kinetics, and modulates the apparent calcium sensitivity of Hslo1. We found that the different effects of the beta subunits on some Hslo1 channel properties are calcium-dependent. mKCNMB1 and hKCNMB2 slow activation at 1 µM but not at 10 µM free calcium concentrations. hKCNMB4 decreases Hslo1 channel openings at low calcium concentrations but increases channel openings at high calcium concentrations. These results suggest that beta subunits in diverse tissue types fine-tune slo channel properties to the needs of a particular cell.

•Chandy, K.G., Fantino, E., Wittekindt, O., Kalman, K., Tong, L.L., Ho, T.H., Gutman, G.A., Crocq, M.A., Ganguli, R., Nimgaonkar, V., Morris-Rosendahl, D.J. & Gargus, J.J. (1998). Isolation of a novel potassium channel gene hSKCa3 containing a polymorphic CAG repeat: A candidate for schizophrenia and bipolar disorder? Molecular Psychiatry 3(1): 32-7. Many human hereditary neurodegenerative diseases are caused by expanded CAG repeats, and anonymous CAG expansions have also been described in schizophrenia and bipolar disorder. We have isolated and sequenced a novel human cDNA encoding a neuronal, small conductance calcium- activated potassium channel (hSKCa3) that contains two arrays of CAG trinucleotide repeats. The second CAG repeat in hSKCa3 is highly polymorphic in control individuals, with alleles ranging in size from 12 to 28 repeats. The overall allele frequency distribution is significantly different in patients with schizophrenia compared to ethnically matched controls (Wilcoxon Rank Sum test, P=0.024), with CAG repeats longer than the modal value being over-represented in patients (Fisher Exact test, P=0.0035). A similar, non- significant, trend is seen for patients with bipolar disorder. These results provide evidence for a possible association between longer alleles in the hSKCa3 gene and both of these neuropsychiatric diseases, and emphasize the need for more extensive studies of this new gene. Small conductance calcium-activated K+ channels play a critical role in determining the firing pattern of neurons. These polyglutamine repeats may modulate hSKCa3 channel function and neuronal excitability, and thereby increase disease risk when combined with other genetic and environmental effects.

•Dickson, C.T., Mena, A.R. & Alonso, A. (1997). Electroresponsiveness of medial entorhinal cortex layer III neurons in vitro. Neuroscience 81(4): 937-950. The entorhinal cortex funnels sensory information from the entire cortical

Page 94 AHP, BK- and SK-channel references mantle into the hippocampal formation via the perforant path. A major component of this pathway originates from the stellate cells in layer II and terminates on the dentate granule cells to activate the hippocampal trisynaptic circuit. In addition, there is also a significant, albeit less characterized, component of the perforant path that originates in entorhinal layer III pyramidal cells and terminates directly in area CA1. As a step in understanding the functional role of this monosynaptic component of the perforant path, we undertook the electrophysiological characterization of entorhinal layer III neurons in an in vitro rat brain slice preparation using intracellular recording techniques with sharp micropipettes and under current-clamp conditions. Cells were also intracellularly injected with biocytin to assess their pyramidal cell morphology. Layer III pyramidal cells did not display either the rhythmic subthreshold membrane potential oscillations nor spike- cluster discharge that characterizes the spiny stellate cells from layer II. In contrast, layer III pyramidal cells displayed a robust tendency towards spontaneous activity in the form of regular tonic discharge. Analysis of the voltage-current relations also demonstrated, in these neurons, a rather linear membrane voltage behaviour in the subthreshold range with the exception of pronounced inward rectification in the depolarizing direction. Depolarizing inward rectification was unaffected by Ca(2+)-conductance block with but was abolished by voltage-gated Na(+)-conductance block with tetrodotoxin, suggesting that a persistent Na(+)- conductance provides much of the inward current sustaining tonic discharge. In addition, in the presence of tetrodotoxin, an intermediate threshold (approximately -50 mV) Ca(2+)-dependent rebound potential was also observed which could constitute another pacemaker mechanism. A high-threshold Ca(2+)-conductance was also found to contribute to the action potential as judged by the decrease in spike duration towards the peak observed during Ca(2+)-conductance block. On the other hand, Ca(2+)-conductance block increase spike duration at the base and abolished the monophasic spike afterhyperpolarization. Analysis of the input-output relations revealed firing properties similar to those of regularly spiking neocortical cells. Current-pulse driven spike trains displayed moderate adaptation and were followed by a Ca(2+)-dependent slow afterhyperpolarization. In summary, the intrinsic electroresponsiveness of entorhinal layer III pyramidal cells suggest that these neurons may perform a rather high-fidelity transfer function of incoming neocortical sensory information directly to the CA1 hippocampal subfield. The pronounced excitability of layer III cells, due to both Na+ and Ca2+ conductances, may also be related to their tendency towards degeneration in epilepsy.

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•Foehring, R.C., Schwindt, P.C. & Crill, W.E. (1989). Norepinephrine selectively reduces slow Ca2+- and Na+-mediated K+ currents in cat neocortical neurons. Journal of Neurophysiology 61: 245-256. 1. The effects of norepinephrine (NE) and related agonists and antagonists were examined on large neurons from layer V of cat sensorimotor cortex ("Betz cells") were examined in a brain slice preparation using intracellular recording, constant current stimulation and single microelectrode voltage clamp. 2. Application of NE (0.1-100 µM) usually caused a small depolarization from resting potential; hyperpolarizations were rare. Application of NE reversibly reduced rheobase and both the Ca2+- and Na+-dependent portions of the slow afterhyperpolarization (sAHP) that followed sustained firing evoked by constant current injection. The faster Ca2+-dependent medium afterhyperpolarization (mAHP), the fast afterhyperpolarization (fAHP), the action potential, and input resistance were unaffected. 3. The changes in excitability produced by NE application were most apparent during prolonged stimulation. The cells exhibited steady repetitive firing to currents that were formerly ineffective. The slow phase of spike frequency adaptation was reduced selectively and less habituation occurred during repeated long-lasting stimuli. The relation between firing rate and injected current became steeper if firing rate was averaged over several hundred milliseconds. 4. During voltage clamp in TTX, NE application selectively reduced the slow component of Ca2+-mediated K+ current. The faster Ca2+-mediated K+ current was unaffected, as were two voltage-dependent, transient K+ currents, the anomalous rectifier and leakage conductance measured at resting potential. Depolarizing voltage steps in the presence of Cd2+ revealed an apparent time- and voltage-dependent increase of the persistent Na+ current after NE application. The voltage-clamp results suggested ionic mechanisms for all effects seen during constant current stimulation except the depolarization from resting potential. The latter was insensitive to Cd2+ and TTX and occurred without a detectable change in membrane conductance. 5. NE application did not alter Ca2+ spikes evoked in the presence of TTX and 10 mM TEA. Inward Ca2+ currents examined during voltage clamp in TTX (with K+ currents reduced) became slightly larger after NE application. We conclude that NEs reduction of the slow Ca2+-mediated K+ current is not caused by reduction of Ca2+ influx. 6. Effects on membrane potential, rheobase, and the sAHP were mimicked by the beta-adrenergic agonist isoproterenol, but not by the alpha-adrenergic agonists clonidine or phenylephrine at higher concentrations.(ABSTRACT TRUNCATED AT

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400 WORDS)

•Jin, W., Sugaya, A., Tsuda, T., Ohguchi, H. & Sugaya, E. (2000). Relationship between large conductance calcium-activated potassium channel and bursting activity. Brain Research 860(1-2): 21-28. To elucidate the role of the large conductance calcium-activated potassium channel (BK(Ca) channel) in the production of bursting activity, which is characteristic of convulsions, effects of iberiotoxin (IbTX), a selective blocker of the BK(Ca) channel, on bursting activity, induced by various procedures were examined using primary cultured neurons from the cerebral cortex of mice. IbTX completely inhibited bursting activity induced by (PTZ), caffeine, 1,4,5-inositol triphosphate (IP3) and direct forced increase of intracellular calcium. Inherent spontaneous bursting activity in the cerebral cortical neurons of the El mouse, which shows a high susceptibility to convulsions was also completely inhibited by IbTX. Apamin, a specific blocker of the small conductance calcium-activated potassium channel (SK(Ca) channel) showed no inhibition of bursting activity. These findings suggest that the BK(Ca) channel is essential for the production of bursting activity, and also suggest the possibility of clinical use of blocking agents of the BK(Ca) channel against intractable epilepsy.

•Kitagawa, H., Nishimura, Y., Kumazawa, Y., Akamine, T. & Yamamoto, T. (2000). Activity-dependent slow hyperpolarization in cat sensorimotor cortex in vitro. Brain Research 869(1-2): 69-77. The synaptic regulatory mechanism of resting membrane potential of layer III and V pyramidal neurons was analyzed intracellularly in the slice preparation of cat sensorimotor cortex. During the tetanic stimulation of white matter, subthreshold membrane depolarization was induced, and after that, a slowly developing hyperpolarization was induced in the normal solution. When the membrane potential showed a slow change, spike duration and input resistance did not change and evoked single synaptic response did not reveal the enhancement of slow IPSPs. However, afterhyperpolarization following action potential was enhanced. The slow hyperpolarization and the enhancement of afterhyperpolarization were not observed in the cells treated with an NMDA receptor antagonist or a Ni(2+) (50-100 µM), or the cells hyperpolarized more than -80 mV before the tetanic stimulation.

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•Klink, R. & Alonso, A. (1993). Ionic mechanisms for the subthreshold oscillations and differential electroresponsiveness of medial entorhinal cortex layer II neurons. Journal of Neurophysiology 70(1): 144-157. 1. Layer II of the medial entorhinal cortex is composed of two electrophysiologically and morphologically distinct types of projection neurons: stellate cells (SCs), which are distinguished by rhythmic subthreshold oscillatory activity, and non-SCs. The ionic mechanisms underlying their differential electroresponsiveness, particularly in the subthreshold range of membrane potentials, were investigated in an "in vitro" slice preparation. 2. In both SCs and non-SCs, the apparent membrane input resistance was markedly voltage dependent, respectively decreasing or increasing at hyperpolarized or subthreshold depolarized potential levels. Thus the neurons displayed inward rectification in the hyperpolarizing and depolarizing range. 3. In the depolarizing range, inward rectification was blocked by tetrodotoxin (TTX, 1 µM) in both types of neurons and thus shown to depend on the presence of a persistent low-threshold Na+ conductance (gNap). However, in the presence of TTX, pronounced outward rectification became manifest in the subthreshold depolarizing range of membrane potentials (positive to -60 mV) in the SCs but not in the non-SCs. 4. The rhythmic subthreshold membrane potential oscillations that were present only in the SCs were abolished by TTX and not by Ca2+ conductance block with Cd2+ or Co2+. Subthreshold oscillations thus rely on the activation of voltage-gated Na+, and not Ca2+, conductances. The Ca2+ conductance block also had no effect on the subthreshold outward rectification. 5. Prominent time-dependent inward rectification in the hyperpolarizing range in the SCs persisted after Na(+)- and Ca2+ conductance block. This rectification was not affected by Ba2+ (1 mM), but was blocked by Cs+ (1-4 mM). Therefore, it is most probably generated by a hyperpolarization-activated cationic current (Q-like current). However, the Q-like current appears to play no major role in the generation of subthreshold rhythmic membrane potential oscillations, because these persisted in the presence of Cs+. 6. On the other hand, in the SCs, the fast, sustained, outward rectification that strongly developed (after Na+ conductance block) at the oscillatory voltage level was not affected by Cs+ but was blocked by Ba2+ (1 mM). Barium was also effective in blocking the subthreshold membrane potential oscillations. 7. In the non-SCs, which do not generate subthreshold rhythmic membrane potential oscillations or manifest subthreshold outward rectification in TTX, Ca2+ conductance block abolished spike repolarization and caused the development of long-lasting Na(+)-

Page 98 AHP, BK- and SK-channel references dependent plateau potentials at a high suprathreshold voltage level. At this level, where prominent delayed rectification is present, the Na+ plateaus sustained rhythmic membrane potential oscillations.(ABSTRACT TRUNCATED AT 400 WORDS)

•Klink, R. & Alonso, A. (1993). Differential electroresponsiveness of stellate and pyramidal-like cells of medial entorhinal cortex layer II. Journal of Neurophysiology 70(1): 128-143. 1. The electroresponsive properties of neurons from layer II of the rat medial entorhinal cortex (MEC) were studied by intracellular recording under current clamp in an in vitro brain slice preparation. From a total of 184 cells that fulfilled our criteria for recording stability, two groups of projection neurons were distinguished on the basis of their intrinsic biophysical properties and morphological characteristics (demonstrated by intracellular biocytin injection; n = 34). 2. Stellate cells (SCs) were the most abundant (69%). They were highly electroresponsive, and minimal changes (1-3 mV) of membrane potential generated an active response. Subthreshold depolarizing or hyperpolarizing current pulse injection always caused the membrane potential to attain an early peak and then sag to a lower level. Depolarization-induced "sags" were larger and determined early firing in all cells. The voltage-current relationship of SCs was markedly non-linear, demonstrating robust inward rectification in the hyperpolarizing and depolarizing range. 3. SCs generated persistent rhythmic subthreshold voltage oscillations on DC depolarization positive to -60 mV. The mean frequency of the oscillations was 8.6 Hz (theta range) at a membrane potential of approximately -55 mV, at which level occasional single spiking also occurred. At slightly more positive potentials, a striking 1- to 3-Hz repetitive bursting pattern emerged. This consisted of nonadapting trains of spikes ("clusters") interspersed with subthreshold oscillations that had a mean frequency of 21.7 Hz (beta range). 4. Nonstellate cells (39%; mostly pyramidal-like) displayed time-dependent inward rectification that was less pronounced than that of SCs, and minimal depolarization-induced sags. On threshold depolarization, firing was always preceded by a slowly rising ramp depolarization and thus occurred with a long delay. Inward rectification in the depolarizing range was very pronounced. However, non-SCs did not generate persistent rhythmic subthreshold oscillatory activity or spike clusters. 5. Of the electrophysiological parameters quantified, spike threshold, spike duration, depolarizing afterpotential amplitude and apparent membrane time constant demonstrated statistically

Page 99 AHP, BK- and SK-channel references significant differences between SCs and non-SCs. 6. The repetitive hiring properties in response to square current pulses of short duration (< 500 ms) were also different between SCs and non-SCs. First, most SCs displayed a bilinear frequency-current (f-I) relationship for only the first interspike interval, whereas most non-SCs displayed a bilinear relationship for all intervals. Second, SCs had a much steeper primary f-I slope for early intervals than non-SCs. Finally, SCs displayed more pronounced and faster spike frequency adaptation than non- SCs.(ABSTRACT TRUNCATED AT 400 WORDS)

•Klink, R. & Alonso, A. (1997). Muscarinic modulation of the oscillatory and repetitive firing properties of entorhinal cortex layer II neurons. Journal of Neurophysiology 77(4): 1813-1828. Neurons in layer II of the entorhinal cortex (EC) are key elements in the temporal lobe memory system because they integrate and transfer into the hippocampal formation convergent sensory input from the entire cortical mantle. EC layer II also receives a profuse cholinergic innervation from the basal forebrain that promotes oscillatory dynamics in the EC network and may also implement memory function. To understand the cellular basis of cholinergic actions in EC, we investigated by intracellular recording in an in vitro rat brain slice preparation the muscarinic modulation of the electroresponsive properties of the two distinct classes of medial EC layer II projection neurons, the stellate cells (SCs) and non- SCs. In both SCs and non-SCs, muscarinic receptor activation with carbachol (CCh, 10-50 µM) caused atropine-sensitive (300 nM) membrane depolarization. In SCs, the CCh-induced membrane depolarization was associated with subthreshold membrane potential oscillations and "spike cluster" discharge, which are typically expressed by these cells on depolarization. CCh, however, caused a decrease of the dominant frequency of the membrane potential oscillations from 9.2 ± 1.1 (SD) Hz to 6.3 ± 1.1 Hz, as well as a decrease of the intracluster firing frequency from 18.1 ± 1.7 Hz to 13.6 ± 1.3 Hz. In addition, spike cluster discharge was less robust, and the cells tended to shift into tonic firing during CCh. In contrast to SCs, in non-SCs, CCh drastically affected firing behavior by promoting the development of voltage- dependent, long-duration (1-5 s) slow bursts of action potentials that could repeat rhythmically at slow frequencies (0.2-0.5 Hz). Concomitantly, the slow afterhyperpolarization (sAHP) was replaced by long-lasting plateau postdepolarizations. In both SCs and non-SCs, CCh also produced conspicuous changes on the action potential waveform and its afterpotentials. Notably, CCh

Page 100 AHP, BK- and SK-channel references significantly decreased spike amplitude and rate of rise, which suggests muscarinic modulation of a voltage-dependent Na+ conductance. Finally, we also observed that whereas CCh abolished the sAHP in both SCs and non-SCs, the membrane-permeant analogues of adenosine 3',5'-cyclic monophosphate, 8-(4-chlorophenylthio)-adenosine- cyclic monophosphate and 8-bromo-adenosine-cyclic-monophosphate, abolished the sAHP in SCs but not in non-SCs. The data demonstrate that cholinergic modulation further differentiates the intrinsic electroresponsiveness of SCs and non-SCs, and add support to the presence of two parallel processing systems in medial EC layer II that could thereby differentially influence their hippocampal targets. The results also indicate an important role for the cholinergic system in tuning the oscillatory dynamics of entorhinal neurons.

•Klink, R. & Alonso, A. (1997). Ionic mechanisms of muscarinic depolarization in entorhinal cortex layer II neurons. Journal of Neurophysiology 77(4): 1829-1843. The mechanisms underlying direct muscarinic depolarizing responses in the stellate cells (SCs) and non-SCs of medial entorhinal cortex layer II were investigated in tissue slices by intracellular recording and pressure-pulse applications of carbachol (CCh). Subthreshold CCh depolarizations were largely potentiated in amplitude and duration when paired with a short DC depolarization that triggered cell firing. During Na+ conductance block, CCh depolarizations were also potentiated by a brief DC depolarization that allowed Ca2+ influx and the potentiation was more robust in non-SCs than in SCs. Also, in non-SCs, CCh depolarizations could be accompanied by spikelike voltage oscillations at a slow frequency. In both SCs and non-SCs, the voltage-current (V-I) relations were similarly affected by CCh, which caused a shift to the left of the steady-state V-I relations over the entire voltage range and an increase in apparent slope input resistance at potentials positive to about -70 mV. CCh responses potentiated by Ca2+ influx demonstrated a selective increase in slope input resistance at potentials positive to about -75 mV in relation to the nonpotentiated responses. K+ conductance block with intracellular injection of Cs+ (3 µM) and extracellular Ba2+ (1 mM) neither abolished CCh depolarizations nor resulted in any qualitatively distinct effect of CCh on the V-I relations. CCh depolarizations were also undiminished by block of the time-dependent inward rectifier Ih, with extracellular Cs . However, CCh depolarizations were abolished during Ca2+ conductance block with low-Ca2+ (0.5 mM) solutions containing Cd2+, Co2+, or Mn2+, as well as by intracellular Ca2+ chelation with bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid.

Page 101 AHP, BK- and SK-channel references

Inhibition of the Na+-K+ ATPase with strophanthidin resulted in larger CCh depolarizations. On the other hand, when NaCl was replaced by N-methyl-D- glucamine, CCh depolarizations were largely diminished. CCh responses were blocked by 0.8 microM pirenzepine, whereas hexahydro-sila-difenidolhydrochloride,p- fluoroanalog (p-F-HHSiD) and himbacine were only effective antagonists at 5- to 10-fold larger concentrations. Our data are consistent with CCh depolarizations being mediated in both SCs and non-SCs by m1 receptor activation of a Ca2+- dependent cationic conductance largely permeable to Na+. Activation of this conductance is potentiated in a voltage-dependent manner by activity triggering Ca2+ influx. This property implements a Hebbian-like mechanism whereby muscarinic receptor activation may only be translated into substantial membrane depolarization if coupled to postsynaptic cell activity. Such a mechanism could be highly significant in light of the role of the entorhinal cortex in learning and memory as well as in pathologies such as temporal lobe epilepsy.

•Pineda, J.C., Galarraga, E. & Foehring, R.C. (1999). Different Ca2+ source for slow AHP in completely adapting and repetitive firing pyramidal neurons. Neuroreport 10(9): 1951-1956. Intracellular recordings and organic and inorganic Ca2+ channel blockers were used in a neocortical brain slice preparation to test whether high-voltage-activated (HVA) Ca2+ channels are differentially coupled to Ca2+-dependent afterhyperpolarizations (AHPs) in sensorimotor neocortical pyramidal neurons. For the most part, spike repolarization was not Ca2+ dependent in these cells, although the final phase of repolarization (after the fast AHP) was sensitive to block of N- type current. Between 30 and 60% of the medium afterhyperpolarization (mAHP) and between approximately 80 and 90% of the slow AHP (sAHP) were Ca2+ dependent. Based on the effects of specific organic Ca2+ channel blockers (dihydropyridines, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC), the sAHP is coupled to N-, P-, and Q-type currents. P-type currents were coupled to the mAHP. L-type current was not involved in the generation of either AHP but (with other HVA currents) contributes to the inward currents that regulate interspike intervals during repetitive firing. These data suggest different functional consequences for modulation of Ca2+ current subtypes.

•Pineda, J.C., Waters, R.S. & Foehring, R.C. (1998). Specificity in the interaction of HVA Ca2+ channel types with Ca2+-dependent AHPs and firing

Page 102 AHP, BK- and SK-channel references behavior in neocortical pyramidal neurons. Journal of Neurophysiology 79(5): 2522- 2534. Intracellular recordings and organic and inorganic Ca2+ channel blockers were used in a neocortical brain slice preparation to test whether high-voltage-activated (HVA) Ca2+ channels are differentially coupled to Ca2+-dependent afterhyperpolarizations (AHPs) in sensorimotor neocortical pyramidal neurons. For the most part, spike repolarization was not Ca2+ dependent in these cells, although the final phase of repolarization (after the fast AHP) was sensitive to block of N- type current. Between 30 and 60% of the medium afterhyperpolarization (mAHP) and between approximately 80 and 90% of the slow AHP (sAHP) were Ca2+ dependent. Based on the effects of specific organic Ca2+ channel blockers (dihydropyridines, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC), the sAHP is coupled to N-, P-, and Q-type currents. P-type currents were coupled to the mAHP. L-type current was not involved in the generation of either AHP but (with other HVA currents) contributes to the inward currents that regulate interspike intervals during repetitive firing. These data suggest different functional consequences for modulation of Ca2+ current subtypes.

•Saar, D., Grossman, Y. & Barkai, E. (1998). Reduced after-hyperpolarization in rat piriform cortex pyramidal neurons is associated with increased learning capability during operant conditioning. European Journal of Neuroscience 10(4): 1518-1523. Learning-related cellular modifications were studied in the rat piriform cortex. Water-deprived rats were divided to three groups: 'trained' rats were trained in a four-arm maze to discriminate positive cues in pairs of odours, 'control' rats were 'pseudo-trained' by random water rewarding, and 'naive' rats were water-deprived only. In one experimental paradigm, the trained group was exposed to extensive training with rats learning to discriminate between 35 and 50 pairs of odours. Piriform cortex pyramidal neurons from 'trained', 'control' and 'naive' rats did not differ in their passive membrane properties and single spike characteristics. However, the after-hyperpolarizations (AHPs) that follow six-spike trains were reduced after 'extensive training' by 43% and 36% compared with 'control' and 'naive', respectively. This effect was not observed in the piriform cortex of another group of rats, in which hyperexcitability was induced by chemical kindling. In another experimental paradigm rats were trained only until they demonstrated 'rule learning', usually after discriminating between one and two pairs

Page 103 AHP, BK- and SK-channel references of odours ('mild training'). In this experiment, a smaller, yet significant, reduction (20%) in AHPs was observed. AHP reduction was apparent in most of the sampled neurons. AHP remained reduced up to 3 days after the last training session. 5 days or more after the last training session, AHP amplitude recovered to pre-training value and did not differ between 'trained' rats and the others. Accordingly, training suspension for 5 days or more resulted in slower learning of novel odours. We suggest that increased neuronal excitability, manifested as reduced AHP, is related to the ability of the cortical network to enter a 'learning mode' which creates favourable conditions for enhanced learning capability.

•Saar, D., Grossman, Y. & Barkai, E. (2001). Long-lasting cholinergic modulation underlies rule learning in rats. Journal of Neuroscience 21(4): 1385-1392. We studied the role of acetylcholine (ACh) in creating learning-related long- lasting modifications in the rat cortex. Rats were trained to discriminate positive and negative cues in pairs of odors, until they demonstrated rule learning and entered a mode of high capability for learning of additional odors. We have previously reported that pyramidal neurons in olfactory (piriform) cortex from trained rats had reduced spike afterhyperpolarization (AHP) for 3 d after rule learning. In the present study we examined the mechanism underlying this long- lasting modification. The cholinergic agonist carbachol reduced both slow AHP and firing adaptation in neurons from pseudotrained rats, but had no effect on neurons from trained rats, suggesting pre-existing cholinergic effect. Intracellular application of the calcium chelator BAPTA abolished the difference in slow AHP and in adaptation between groups, suggesting that the difference resulted from reduction in the ACh-sensitive, Ca2+-dependent potassium current, I(AHP). At the behavioral level, application of the muscarinic blocker before each training session delayed rule learning but had no effect on further acquisition of odor memory. We suggest that intense ACh activity during rule learning enhances neuronal excitability in the piriform cortex by reducing I(AHP) and that the effect outlasts the stage of rule learning, so that ACh activity is not crucial for further odor learning.

•Schwindt, P.C., Spain, W.J. & Crill, W.E. (1988). Influence of anomalous rectifier activation on afterhyperpolarizations of neurons from cat sensorimotor cortex in vitro. Journal of Neurophysiology 59: 468-481. 1. Large neurons from layer V of cat sensorimotor cortex (Betz cells) were

Page 104 AHP, BK- and SK-channel references studied to determine the influence of the anomalous rectifier current (IAR) on slow afterhyperpolarizations (AHPs). The neurons were examined using intracellular recording and single-microelectrode voltage clamp in an in vitro brain slice preparation. 2. A faster medium-duration AHP (mAHP) and slower AHP (sAHP) followed repetitive firing. The amplitude of the mAHP often increased or remained constant during membrane potential hyperpolarization. The membrane potential trajectory resulting solely from IAR activation was similar to the mAHP. 3. Postrepetitive firing voltage clamp was used to measure directly slowly decaying K+ currents (IK) and IAR at different membrane potentials. IK exhibited both a fast and slow decay. The time constants of the fast decay of IK and IAR activation were similar. IAR increased with hyperpolarization or raised extracellular K+ concentration [( K+]o), whereas both the fast and slow components of IK reversed or nulled near -100 mV and behaved as pure K+ currents in response to raised [K+]o. 4. To determine the precise contribution of IK and IAR to the AHP waveform, theoretical AHPs were computed using a quantitative model based on voltage-clamp measurements. The calculated AHPs were qualitatively similar to measured AHPs. The amplitude of the mAHP showed little change with hyperpolarization because of the increasing dominance of IAR at more negative membrane potentials. The sAHP was little affected by IAR activation. 5. Several model parameters subject to biological variation among Betz cells were varied in the calculations to determine their importance in the AHP waveform. With IK parameters held constant, the amplitude and time course of the mAHP depended on resting potential, membrane time constant, the kinetics of the anomalous rectifier conductance (GAR), and the maximum value of GAR. IAR activation could result in a biphasic AHP even when the fast decay of IK was omitted from the calculations. 6. A wider variation of model parameters revealed behavior that may be relevant to other neurons. Certain values of membrane or IAR activation time constants resulted in a monophasic AHP even when the fast decay of IK was present. The decay of a biphasic AHP could reflect either the onset of IAR or the fast decay of IK, depending on the relative value of their time constants. Procedures are outlined to discriminate between these possibilities using current clamp methods.(ABSTRACT TRUNCATED AT 400 WORDS)

•Schwindt, P.C., Spain, W.J., Foehring, R.C., Chubb, M.C. & Crill, W.E. (1988). Slow conductances in neurons from cat sensorimotor cortex in vitro and their role in slow excitability changes. Journal of Neurophysiology 59: 450-467.

Page 105 AHP, BK- and SK-channel references

1. The electrophysiological and pharmacological properties of slow afterpotentials in large layer V neurons from cat sensorimotor cortex were studied in an in vitro slice preparation using intracellular recording and single- microelectrode voltage clamp. These properties were used to assess the role of afterpotential mechanisms in prolonged excitability changes. 2. The mean duration of a slow afterhyperpolarization (sAHP) was 13.5 s following 100 spikes evoked at 100 Hz. Its time course was best described by two exponential components, which decayed with time constants of several hundred milliseconds (the early sAHP) and several seconds (the late sAHP). The amplitude of both the early and late components were sensitive to membrane potential and raised extracellular K+ concentration [( K+]o). 3. The early sAHP was reduced when divalent cations were substituted for Ca2+, whereas the late sAHP was unaffected. We conclude that a Ca2+-mediated K+ conductance is responsible for much of the early sAHP. In the presence of tetrodotoxin (TTX), 1-s voltage-clamp steps were used to evoke slow AHPs or outward ionic currents. These AHPs and currents were abolished in Ca2+- free perfusate, but they had a maximum duration of only a few seconds. Thus the slowest outward currents we could observe during voltage clamp in TTX were responsible only for the early sAHP. 4. The possible role of an electrogenic Na+-K+ pump in the late sAHP was examined by applying ouabain to the slice. Ouabain did not reduce selectively the late sAHP, and its effect was best explained by a decrease in intracellular K+ concentration and an increase in [K+]o. 5. Muscarinic and beta- adrenergic agonists reduced or abolished the entire (early and late) sAHP. Neither type of agonist affected the Ca2+-dependent, apamin-sensitive medium-duration afterhyperpolarization (35). We conclude that both the Ca2+-mediated K+ conductance underlying the early sAHP and the Ca2+-independent mechanisms underlying the late sAHP are sensitive to at least two classes of transmitter agonists. 6. We focused on the muscarinic effects. When concentrations greater than 5 microM were employed, the entire (early and late) sAHP was replaced by a slow afterdepolarization (sADP). Muscarine reduced the sAHP directly by reducing the underlying outward ionic currents and indirectly by causing the sADP. The sADP was Ca2+-mediated, since it was abolished by Ca2+-free perfusate but not by TTX. 7. The ionic currents underlying the sAHP and the sADP influenced excitability for seconds following evoked repetitive firing.(ABSTRACT TRUNCATED AT 400 WORDS)

•Schwindt, P.C., Spain, W.J., Foehring, R.C., Stafstrom, C.E., Chubb, M.C. &

Page 106 AHP, BK- and SK-channel references

Crill, W.E. (1988). Multiple potassium conductances and their functions in neurons from cat sensorimotor cortex in vitro. Journal of Neurophysiology 59: 424-449. 1. Potassium conductances were studied in large layer V neurons using an in vitro slice preparation of cat sensorimotor cortex. The kinetics and pharmacological sensitivity of K+ currents were studied directly using single microelectrode voltage clamp and indirectly by evoking single or multiple spikes and recording the spike repolarization and subsequent afterhyperpolarizations (AHPs). 2. A fast-decaying afterhyperpolarization (fAHP) and a subsequent medium-duration afterhyperpolarization (mAHP) followed a single spike. The amplitude and duration of the mAHP increased when multiple spikes were evoked at a fast rate (e.g., 100 Hz), and a slower afterhyperpolarization (sAHP) appeared only after sustained repetitive firing. 3. All AHPs were reduced by membrane potential hyperpolarization and raised extracellular K+ concentration, suggesting they were caused by an increased K+ conductance. Only the mAHP and sAHP reversed at the estimated value of potassium equilibrium potential (-100 mV), whereas the mean reversal potential of the fAHP was nearly identical to the mean value of resting potential (-71 mV). 4. Mechanisms underlying spike repolarization, the fAHP, and the mAHP were investigated. Two rapidly activating outward currents, a fast-inactivating current and a slowly inactivating delayed rectifier, were detected by voltage clamp. Both currents were reduced rapidly by tetraethylammonium (TEA). The fast transient current was reduced slowly after divalent cations were substituted for Ca2+ (through a mechanism unrelated to blockade of Ca2+ channels), whereas the delayed rectifier was unaffected. 5. Spike duration was increased and the fAHP was abolished only by blocking agents that reduced the fast outward currents. Effects of extracellular and intracellular TEA were similar. Effects of TEA and Ca2+-free perfusate were additive and resembled the effects of intracellular Cs+. The addition of apamin, d- tubocurare, or Cd2+ was ineffective. We conclude that the two fast outward currents reflect pharmacologically and kinetically separate K+ conductances that are primarily responsible for spike repolarization and the fAHP. 6. Voltage-clamp studies revealed two additional outward currents, which were persistent and Ca2+- mediated. Each current activated and deactivated slowly, but the kinetics of one component were approximately 10 times slower than the other. The decay of these currents gave rise to AHPs resembling the mAHP and the early sAHP. 7. Neither the mAHP nor the sAHP was reduced by TEA. The mAHP was reduced when divalent cations were substituted for Ca2+ or when Cd2+, apamin, or d-tubocurare were added.(ABSTRACT TRUNCATED AT 400 WORDS)

Page 107 AHP, BK- and SK-channel references

AMYGDALA

•Faber, E.S.L. & Sah, P. (2001). Role of the slow AHP during synaptic stimulation in lateral amygdala neurones. Society for Neuroscience Abstracts 31(713.3). The amygdala has been shown to be important in placing emotional significance to sensory input. We have previously shown in projection neurones in the lateral amygdala (LA) that single or trains of action potentials are followed by a slow afterhyperpolarisation (AHP). The slow AHP contributes to spike frequency adaptation and can be blocked by neurotransmitters such as noradrenaline. The current study aimed to investigate the role of the slow AHP in synaptic integration these cells. Visualised whole cell patch clamp recordings were made from 400 m coronal brain slices in vitro from Wistar rats (3-4 weeks old of either sex). The pipette solution contained (mM) KMeSO4 135, NaCl 8, HEPES 10, Mg2ATP 2 and Na3GTP 0.3, and the slices were perfused in oxygenated aCSF at 30-32°C. Synaptic responses were evoked by stimulation of the external capsule with a bipolar stimulating electrode. Somatic 100 ms 400 pA current injections from resting potential evoked between one and five action potentials followed by a slow AHP lasting up to 6 seconds (n=6). Similarly five brief (5 ms) current injections sufficient to evoke single spikes (1 nA) were followed by a slow AHP identical to that evoked by a single current injection (n=6). In contrast a train of 10 stimuli applied at 100 Hz, sufficient to evoke summating EPSPs with one to ten action potentials riding on top failed to evoke a slow AHP in 16 out of 18 cells. These findings show that synaptic activity that evokes action potential firing does not always initiate the slow AHP in LA neurones.

•Faber, E.S.L. & Sah, P. (2002). Physiological role of calcium-activated potassium currents in the rat lateral amygdala. Journal of Neuroscience 22(5): 1618-1628. Principal neurons in the lateral nucleus of the amygdala (LA) exhibit a continuum of firing properties in response to prolonged current injections ranging from those that accommodate fully to those that fire repetitively. In most cells, trains of action potentials are followed by a slow afterhyperpolarization (AHP) lasting several seconds. Reducing calcium influx either by lowering concentrations of extracellular calcium or by applying nickel abolished the AHP, confirming it is mediated by calcium influx. Blockade of large conductance calcium-activated

Page 108 AHP, BK- and SK-channel references potassium channel (BK) channels with paxilline, iberiotoxin, or TEA revealed that BK channels are involved in action potential repolarization but only make a small contribution to the fast AHP that follows action potentials. The fast AHP was, however, markedly reduced by low concentrations of 4-aminopyridine and alpha- dendrotoxin, indicating the involvement of voltage-gated potassium channels in the fast AHP. The medium AHP was blocked by apamin and UCL1848, indicating it was mediated by small conductance calcium-activated potassium channel (SK) channels. Blockade of these channels had no effect on instantaneous firing. However, enhancement of the SK-mediated current by 1-ethyl-2-benzimidazolinone or paxilline increased the early interspike interval, showing that under physiological conditions activation of SK channels is insufficient to control firing frequency. The slow AHP, mediated by non-SK BK channels, was apamin-insensitive but was modulated by carbachol and noradrenaline. Tetanic stimulation of cholinergic afferents to the LA depressed the slow AHP and led to an increase in firing. These results show that BK, SK, and non-BK SK-mediated calcium-activated potassium currents are present in principal LA neurons and play distinct physiological roles.

•Faber, E.S.L., Callister, R.J. & Sah, P. (2001). Morphological and electrophysiological properties of principal neurons in the rat lateral amygdala in vitro. Journal of Neurophysiology 85: 714-723. In this study, we characterize the electrophysiological and morphological properties of spiny principal neurons in the rat lateral amygdala using whole cell recordings in acute brain slices. These neurons exhibited a range of firing properties in response to prolonged current injection. Responses varied from cells that showed full spike frequency adaptation, spiking three to five times, to those that showed no adaptation. The differences in firing patterns were largely explained by the amplitude of the afterhyperpolarization (AHP) that followed spike trains. Cells that showed full spike frequency adaptation had large amplitude slow AHPs, whereas cells that discharged tonically had slow AHPs of much smaller amplitude. During spike trains, all cells showed a similar broadening of their action potentials. Biocytin-filled neurons showed a range of pyramidal-like morphologies, differed in dendritic complexity, had spiny dendrites, and differed in the degree to which they clearly exhibited apical versus basal dendrites. Quantitative analysis revealed no association between cell morphology and firing properties. We conclude that the discharge properties of neurons in the lateral nucleus, in response to somatic current injections, are determined by the differential distribution of ionic

Page 109 AHP, BK- and SK-channel references conductances rather than through mechanisms that rely on cell morphology.

RECOMBINANT AND RECONSTITUTED CHANNELS

•Campos Rosa, J., Beckwith-Hall, B.M., Galanakis, D., Ganellin, C.R., Dunn, P.M. & Jenkinson, D.H. (1997). Bis-quinolinium cyclophanes: A novel class of potent blockers of the apamin-sensitive Ca2+-activated K+ channel. Bioorg. Med. Chem. Letters 7: 7-10. Dequalinium [1,1'-(decane-1, 10-diyl)bis(2-methyl-4-aminoquinolinium)] is an effective blocker of the small conductance Ca2(+)-activated K+ channel. It has been shown that the number of methylene groups in the alkyl chain linking the two quinolinium rings of this type of molecule is not critical for activity. To further investigate the role of the linker, analogues of dequalinium have been synthesized, in which the alkyl chain has been replaced by CH2XCH2 where X is a rigid or semirigid group containing aromatic rings. The compounds have been tested for blockade of the slow after-hyperpolarization on rat sympathetic neurons. The most potent compounds have X = phenanthryl, fluorenyl, cis-stilbene, and C6H4(CH2)nC6H4, where n = 0-4. The conformational preferences of the compounds were investigated using the XED/COSMIC molecular modeling system. Although there is some dependence of the potency of the analogue on the conformational properties of the linker (X), overall, X groups having substantial structural differences are tolerated. It seems that X provides a support for the two quinolinium groups and does not interact with the channel directly. The intramolecular separation between the quinolinium rings, which is provided by rigid groups X, is not critical for activity; this may be attributed to the residual conformational mobility of the heterocycles and to the extensive delocalization of the positive charge. These two factors may permit favorable contacts between the quinolinium groups and the channel over a range of intramolecular separations.

•Campos Rosa, J., Galanakis, D., Ganellin, C.R. & Dunn, P.M. (1996). Synthesis, molecular modeling, and K+ channel-blocking activity of dequalinium analogues having semirigid linkers. Journal of Medicinal Chemistry 39(21): 4247-54. Dequalinium [1,1'-(decane-1, 10-diyl)bis(2-methyl-4-aminoquinolinium)] is an effective blocker of the small conductance Ca2(+)-activated K+ channel. It has been shown that the number of methylene groups in the alkyl chain linking the two quinolinium rings of this type of molecule is not critical for activity. To further

Page 110 AHP, BK- and SK-channel references investigate the role of the linker, analogues of dequalinium have been synthesized, in which the alkyl chain has been replaced by CH2XCH2 where X is a rigid or semirigid group containing aromatic rings. The compounds have been tested for blockade of the slow after-hyperpolarization on rat sympathetic neurons. The most potent compounds have X = phenanthryl, fluorenyl, cis-stilbene, and C6H4(CH2)nC6H4, where n = 0-4. The conformational preferences of the compounds were investigated using the XED/COSMIC molecular modeling system. Although there is some dependence of the potency of the analogue on the conformational properties of the linker (X), overall, X groups having substantial structural differences are tolerated. It seems that X provides a support for the two quinolinium groups and does not interact with the channel directly. The intramolecular separation between the quinolinium rings, which is provided by rigid groups X, is not critical for activity; this may be attributed to the residual conformational mobility of the heterocycles and to the extensive delocalization of the positive charge. These two factors may permit favorable contacts between the quinolinium groups and the channel over a range of intramolecular separations.

•Campos Rosa, J., Galanakis, D., Ganellin, C.R., Dunn, P.M. & Jenkinson, D.H. (1998). Bis-quinolinium cyclophanes: 6,10-diaza-3(1,3),8(1,4)-dibenzena-1,5(1,4)- diquinolinacyclodecaphane (UCL 1684), the first nanomolar, non-peptidic blocker of the apamin-sensitive Ca2+-activated K+ channel. Journal of Medicinal Chemistry 41: 2-5. Small conductance Ca2+-activated K+ (SKCa) channels comprise a widely distributed but relatively little studied class of K+ channels. Selective blockade of SKCa channels may find application in the therapy of myotonic muscular dystrophy, gastrointestinal dysmotilities (Dunn, P. M.; Jenkinson, D. H., unpublished results), disorders of memory, narcolepsy, and abuse. Furthermore, SKCa channel blockers will be invaluable tools for further investigations into the role of this K+ channel subtype in physiological and pathophysiological processes. Blockers of SKCa channels include natural peptidic toxins such as apamin, leiurotoxin I, PO5, and Ts K, as well as nonpeptidic compounds such as the neuromuscular blockers tubocurarine, pancuronium, and atracurium. Furthermore, the antiseptic compound, dequalinium, has been shown to be a relatively potent and selective SKCa channel blocker and has constituted a lead for the development of several series of novel SKCa channel blockers. We have recently reported the design of novel bis-quinolinium cyclophanes of the general structure 2 (L = group having

Page 111 AHP, BK- and SK-channel references two aromatic rings, Chart 1) which are submicromolar blockers of the SKCa channel. In addition, we have shown that one of them, 2 [UCL 1530, L = methylenebis(benzene- 1,4-diyl)], shows selectivity for neuronal over hepatocyte SKCa channels, which adds to the functional evidence for the existence of SKCa channel isoforms in different tissues. Such selective compounds should prove to be useful pharmacological tools, especially in view of the recent cloning and expression in Xenopus oocytes of SKCa channel-forming peptides from rat and human brain.

•Campos Rosa, J., Galanakis, D., Piergentili, A., Bhandari, K., Ganellin, C.R., Dunn, P.M. & Jenkinson, D.H. (2000). Synthesis, molecular modeling, and pharmacological testing of bis-quinolinium cyclophanes: Potent, non-peptidic blockers of the apamin-sensitive Ca2+-activated K+ channel. Journal of Medicinal Chemistry 43: 420-431. The synthesis and pharmacological testing of two series of novel bis- quinolinium cyclophanes as blockers of the apamin-sensitive Ca2+-activated K(+) (SK(Ca)) channel are presented. In these cyclophanes the two 4-aminoquinolinium groups are joined at the ring N atoms (linker L) and at the exocyclic N atoms (linker A). In those cases where A and L contain two or more aromatic rings each, the activity of the compound is not critically dependent upon the nature of the linkers. When A and L each have only one benzene ring, the blocking potency changes dramatically with simple structural variations in the linkers. One of these smaller cyclophanes having A = benzene-1,4-diylbis(methylene) and L = benzene-1, 3- diylbis(methylene) (3j, 6,10-diaza-1,5(1,4)-diquinolina-3(1,3),8(1, 4)- dibenzenacyclodecaphanedium tritrifluoroacetate, UCL 1684) has an IC(50) of 3 nM and is the most potent non-peptidic SK(Ca) channel blocker described to date. Conformational analysis on the smaller cyclophanes using molecular modeling techniques suggests that the differences in the blocking potencies of the compounds may be attributable to their different conformational preferences.

•Cao, Y., Dreixler, J.C., Roizen, J.D., Roberts, M.T. & Houamed, K.M. (2001). Modulation of recombinant small-conductance Ca2+-activated K+ channels by the muscle relaxant and structurally related compounds. Journal of Pharmacology and Experimental Therapeutics 296(3): 683-689. Using the patch clamp technique we investigated the effects of the centrally acting muscle relaxant chlorzoxazone and three structurally related compounds, 1-

Page 112 AHP, BK- and SK-channel references ethyl-2-benzimidazolinone (1-EBIO), , and 1,3-dihydro-1-[2-hydroxy-5- (triflu oromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS 1619) on recombinant rat brain SK2 channels (rSK2 channels) expressed in HEK293 mammalian cells. SK channels are small conductance K(+) channels normally activated by a rise in intracellular Ca(2+) concentration; they modulate the electrical excitability in neurons and neuroendocrine cells. When applied externally, chlorzoxazone, 1-EBIO, and zoxazolamine activated rSK2 channel currents in cells dialyzed with a nominally Ca(2+)-free intracellular solution. The activation was reversible, reproducible, and depended on the chemical structure and concentration. The order of potency was 1- EBIO > chlorzoxazone > zoxazolamine. Activation of rSK2 channels by chlorzoxazone, 1-EBIO, and zoxazolamine declined at higher drug concentrations. Zoxazolamine, when applied in combination with chlorzoxazone or 1-EBIO, partially inhibited the rSK2 channel current responses, suggesting a partial-agonist mode of action. 1- EBIO failed to activate rSK2 channel currents when applied to excised inside-out membrane patches exposed to a Ca(2+)-free intracellular solution. In contrast, 1- EBIO activated rSK2 currents in a concentration-dependent manner when coapplied to the patches with a solution containing 20 nM free Ca(2+). NS 1619 did not activate rSK2 channel currents; it inhibited rSK2 channel currents activated by the other three test compounds or by high intracellular Ca(2+). We conclude that chlorzoxazone and its derivatives act through a common mechanism to modulate rSK2 channels, and SK channel modulation in the brain may partly underlie the clinical effects of chlorzoxazone.

•Carignani, C., Roncarati, R., Rimini, R. & Terstappen, G.C. (2002). Pharmacological and molecular characterisation of SK3 channels in the TE671 human medulloblastoma cell line. Brain Research 939(1-2): 11-18. The expression of the small conductance calcium-activated potassium channels SK1, SK2 and SK3 was investigated in the TE671 human medulloblastoma cell line using RT-PCR and transcripts were detected only for SK3. Immunodetection experiments confirmed this result, demonstrating the presence of the SK3 protein. This potassium channel was characterised in TE671 cells using whole-cell patch-

Page 113 AHP, BK- and SK-channel references clamp recordings. Voltage steps to -100 mV from a holding potential of 0 mV in equimolar 140 mM intra- and extracellular K(+) (K(+)(in/out)) elicited an inward current. The reversal potential of this current shifted 56.6 mV per 10-fold increase in K(+)(out) thus suggesting K(+) selectivity. This current was dependent on the concentration of Ca2+(in) with an EC(50) of 104.2 nM. A pharmacological characterisation of this current revealed that it was not blocked by 1 µM charybdotoxin (ChTX), 0.3 µM iberiotoxin (IbTX) or 10 µM clotrimazole (CLT) and only modestly inhibited (<50%) by 30 nM scyllatoxin (ScTX), 200 µM dequalinium chloride (Deq) or 300 µM d-tubocurarine (d-TC). The non-selective SK blocker d-TC blocked the current with an IC(50) of 43.2 µM while apamin blocked the current to a much greater extent (87.8% at 1 µM) with an IC(50) of 4.3 nM. Furthermore, the current was significantly increased (132.6±5.2%, n=7) by 500 µM 1-ethyl-2- benzimidazolinone (EBIO). Collectively, these data demonstrate the presence of an endogenous SK3 channel in human TE671 cells.

•Castle, N.A. (1999). Recent advances in the biology of small conductance calcium- activated potassium channels. Perspectives in Drug Discovery and Design 15/16: 131-154. Significant advances have recently been made in our understanding of the biology of small conductance calcium-activated potassium channels (SK channels). Although a long time ’Cinderella‘ to the better characterized large conductance BK channel, renewed interest in SK channels has been sparked by the recent cloning of several subtypes of this important potassium channel. The information gained from our improved understanding of the molecular nature of SK channels is already helping to elucidate the molecular basis of drug interactions, as well as to further define the physiological function and tissue distribution of these channels. Characterization of SK channels is also being aided by the availability of a wider variety of pharmacological tools including peptide toxins and potent synthetic small molecules. The contribution of SK channels to physiology and pathophysiology of CNS, skeletal and smooth muscle function clearly points to the future importance of these channels as targets for drug development.

•Dreixler, J.C., Bian, J., Cao, Y.J., Roberts, M.T., Roizen, J.D. & Houamed, K.M. (2000). Block of rat brain recombinant SK channels by tricyclic antidepressants and related compounds. European Journal of Pharmacology 401(1): 1- 7.

Page 114 AHP, BK- and SK-channel references

SK channels are small conductance, Ca(2+)-activated K(+) channels that underlie neuronal slow afterhyperpolarization and mediate spike frequency adaptation. Using the patch clamp technique, we tested the effects of eight clinically relevant psychoactive compounds structurally related to the tricyclic antidepressants, on SK2 subtype channels cloned from rat brain and functionally expressed in the human embryonic kidney cell line, HEK293. , , , , imipramine, tacrine and trifluperazine blocked SK2 channel currents with micromolar affinity. The block was reversible and concentration-dependent. The potency differed according to chemical structure. In contrast, the cognitive enhancer linopirdine was ineffective at blocking these channels. Our results point to a distinct pharmacological profile for SK channels.

•Dreixler, J.C., Jenkins, A., Cao, Y.J., Roizen, J.D. & Houamed, K.M. (2000). Patch-clamp analysis of anesthetic interactions with recombinant SK2 subtype neuronal calcium-activated potassium channels. Anesthesiology & Analgesia 90(3): 727-732. Small conductance calcium-activated potassium channels (SK) mediate spike frequency adaptation and underlie the slow afterhyperpolarization in central neurons. We tested the actions of several anesthetics on the SK2 subtype of recombinant SK channels, cloned from rat brain and functionally expressed in a mammalian cell line. Butanol, ethanol, , , and methohexital blocked recombinant SK2 channel currents, measured in the whole-cell patch clamp recording mode. The block was reversible, dose-dependent, and of variable efficacy. The inhaled anesthetics , , enflurane, halothane, isoflurane, and produced little or no block when applied at 1 minimum alveolar anesthetic concentration; varying degrees of modulation were observed at very large concentrations (10 minimum alveolar concentration). The extent of block by inhaled anesthetics did not appear to depend on concentration or membrane voltage. IMPLICATIONS: We describe differential effects of anesthetics on cloned small conductance calcium-activated potassium channels from brain that may play a role in generating the effects or side effects of anesthetics.

•Fajloun, Z., Ferrat, G., Carlier, E., M'Barek, S., Regaya, I., Fathallah, M., Rochat, H., Darbon, H., de Waard, M. & Sabatier, J.M. (2002). Synthesis, 3- D structure, and pharmacology of a reticulated chimeric peptide derived from

Page 115 AHP, BK- and SK-channel references and Tsk scorpion toxins. Biochemical & Biophysics Research Communications 291(3): 640-648. Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four bridges that acts on both Ca2+-activated (SK) and voltage-gated (Kv) K(+) channels. A 38-mer chimera of MTX, Tsk-MTX, has been synthesized by the solid-phase method. It encompasses residues from 1 to 6 of Tsk at N-terminal, and residues from 3 to 34 of MTX at C-terminal. As established by enzyme cleavage, Tsk-MTX displays half-cystine pairings of the type C1-C5, C2-C6, C3-C7 and C4-C8 which, contrary to MTX, correspond to a disulfide bridge pattern common to known scorpion toxins. The 3-D structure of Tsk-MTX, solved by (1)H NMR, demonstrates that it adopts the alpha/beta scaffold of scorpion toxins. In vivo, Tsk-MTX is lethal by intracerebroventricular injection in mice (LD(50) value of 0.2 µg/mouse). In vitro, Tsk-MTX is as potent as MTX, or Tsk, to interact with apamin-sensitive SK channels of rat brain synaptosomes (IC(50) value of 2.5 nM). It also blocks voltage-gated K(+) channels expressed in Xenopus oocytes, but is inactive on rat Kv1.3 contrary to MTX.

•Freeman, L.C., Lippold, J.J. & Mitchell, K.E. (2000). Glycosylation influences gating and pH sensitivity of I(sK). Journal of Membrane Biology 177(1): 65-79. The KvLQT1 and minK subunits that coassemble to form I(sK) channels, contain potential N-glycosylation sites. To examine the role of glycosylation in channel function, a Chinese hamster ovary cell line deficient in glycosylation (Lec-1) and its parental cell line (Pro-5) were transiently transfected with human KvLQT1 (hKvLQT1) cDNA, alone and in combination with the rat (rminK) or human minK (hminK) cDNA. Functional KvLQT1 and I(sK) currents were expressed in both cell lines, although amplitudes were larger in Pro-5 than Lec-1 cells transfected with hKvLQT1 and hKvLQT1/hminK. For I(sK), but not KvLQT1, the voltage-dependence of activation was shifted to more positive voltages and the activation kinetics were slower in the Lec-1 compared to the Pro-5 cells. The effect of extracellular acidification on recombinant KvLQT1 and I(sK) currents was investigated in Pro-5 and Lec-1 cells. Changing external pH (pH(o)) from 7.4 to 6.0 significantly decreased the amplitude and increased the half-activation voltage (V(1/2)) of KvLQT1 currents in Pro-5 and Lec-1 cells. In Pro-5 cells, decreasing pH(o) reduced I(sK) amplitude without increasing V(1/2), whether rminK or hminK was coexpressed with hKvLQT. In contrast, changing pH(o) from 7.4 to 6.0 did not significantly change I(sK) amplitude in Lec-1 cells. Thus, oligosaccharides attached to the minK subunit affect

Page 116 AHP, BK- and SK-channel references not only the gating properties, but also the pH sensitivity of I(sK).

•Galanakis, D., Calder, J.A., Ganellin, C.R., Owen, C.S. & Dunn, P.M. (1995). Synthesis and quantitative structure-activity relationships of dequalinium analogues as K+ channel blockers: investigation into the role of the substituent at position 4 of the quinoline ring. Journal of Medicinal Chemistry 38(18): 3536-46. Dequalinium (4) is a potent and selective blocker of small conductance Ca2+- activated K+ channels, an important but relatively little studied class. The 4-NH2 group of dequalinium has been shown to contribute significantly to blocking potency. In this study, we have investigated further the role of the 4-NH2 group. Replacement of this group by other substituents (R4) and quantitative structure- activity relationship (QSAR) analysis on the resultant analogues have yielded a correlation between blocking potency and sigma R for R4 for seven of the compounds. The application of calculated electronic indices enabled the extension of the QSAR to compounds for which the appropriate sigma R values are not available, allowing all 13 analogues of this series to be included in the correlations. Analysis using electronic indices obtained from AM1 MO calculations on model compounds revealed that the blocking potency correlates with the partial charge on the ring N atom, ELUMO, and EHOMO. The EHOMO correlation is qualitatively inconsistent as the HOMO is not the same orbital in all compounds. The ELUMO correlation [pEMR = 1.19(± 0.21)ELUMO + 5.41(± 1.05), n = 13, r = 0.86, s = 0.274] suggests that the higher the ELUMO the more potent is the analogue. This is consistent with simple charge transfer from the channel to the blocker and may refer to other processes which are important for the strength of the drug-K+ channel interaction such as the desolvation of the compounds.

•Galanakis, D., Davis, C.A., Del Rey Herrero, B., Ganellin, C.R., Dunn, P.M. & Jenkinson, D.H. (1995). Synthesis and structure-activity relationships of dequalinium analogues as K+ channel blockers. Investigations on the role of the charged heterocycle. Journal of Medicinal Chemistry 38(4): 595-606. Small conductance Ca2+-activated K+ (SKCa) channels occur in many cells but have been relatively little studied. Dequalinium, a bis- quinolinium compound, has recently been shown to be the most potent nonpeptidic blocker of this K+ channel subtype. This paper examines the importance of the quinolinium rings for blocking activity. Analogues of dequalinium were synthesised in which one quinolinium group was removed (1 and 2) or replaced by a triethylammonium group (3). They have been

Page 117 AHP, BK- and SK-channel references assayed in vitro for their ability to block the after-hyperpolarization (mediated by the opening of SKCa channels) that follows the action potential in rat sympathetic neurones. The compound having one quinolinium and one triethylammonium group (3) showed reduced activity, and it is suggested that the stronger binding to the channel of the quinolinium relative to the triethylammonium group may be related to differences in their electrostatic potential energy maps. Two monoquaternary compounds (1 and 2) were tested, but they exhibited a different pharmacological profile that did not allow definite conclusions to be drawn concerning their potency as blockers of the SKCa channel. Replacement of both quinolinium groups by pyridinium, acridinium, isoquinolinium, or benzimidazolium reduced but did not abolish activity. These results show that compounds having a number of different heterocyclic cations are capable of blocking the SKCa channel. However, among the heterocycles studied, quinoline is optimal. Furthermore, charge delocalization seems to be important: the higher the degree of delocalization the more potent the compound.

•Galanakis, D., Ganellin, C.R., Malik, S. & Dunn, P.M. (1996). Synthesis and pharmacological testing of dequalinium analogues as blockers of the apamin-sensitive Ca2+-activated K+ channel: Variation of the length of the alkylene chain. Journal of Medicinal Chemistry 39(18): 3592-5. Dequalinium is a potent and selective blocker of the small conductance Ca2+- activated K+ (SKCa) channel in rat sympathetic neurones. Analogues of dequalinium possessing 3-6, 8, 10, and 12 methylene groups in the linking chain have been synthesized and tested for inhibition of the afterhyperpolarization in rat sympathetic neurones. The compounds having a 5-12-carbon chain showed very little variation in their activity as SKCa channel blockers. The analogues possessing four and three methylenes exhibited 3- and 8-fold lower potency, respectively, compared with dequalinium. These results are discussed in the context of possible modes of binding of the compounds to the SKCa channel.

•Galanakis, D., Davis, C.A., Ganellin, C.R. & Dunn, P.M. (1996). Synthesis and quantitative structure-activity relationship of a novel series of small conductance Ca2+-activated K+ channel blockers related to dequalinium. Journal of Medicinal Chemistry 39(2): 359-70. The synthesis, pharmacological testing, and quantitative structure- activity relationship studies of a novel series of bisquinolinium small conductance Ca2+-

Page 118 AHP, BK- and SK-channel references activated K+ channel blockers (23) related to dequalinium are described. In this series, two quinolinium rings are linked via the 4-position to an alpha, omega-diamino alkylene chain and the ring N atom is quaternized with a methyl or benzyl group. The exocyclic N atom can be replaced by O, S, or CH2 but with some loss of potency. The quinoline groups do not have to be quaternized for blocking activity, as long as they are basic enough to be protonated at the site of action. For the quaternary compounds, there is considerable steric tolerance for the group R attached to the ring N atom of the quinoline; a benzyl group gave the optimum potency in this series. Moreover, and in contrast to previously reported results for dequalinium analogues, there is no correlation of activity to previously reported results for dequalinium analogues, there is no correlation of activity with N1 charge or EHOMO. On the other hand, a good correlation was obtained between the blocking potency of the compounds and ELUMO [pEMR = 1.16(±0.26)ELUMO + 5.33(±01.29)(n = 11, r= 0.83, s = 0.243)]. It has been possible to combine this equation with the previously reported ELUMO correlation for a series of dequalinium analogues to include all the compounds of both series [pEMR = 1.17(±0.15)ELUMO +5.33(±0.76)(n =24, r = 0.85, s = 0.249)]. A possible physical meaning for the ELUMO correlation based upon the principle of maximum hardness is discussed.

•Galanakis, D., Ganellin, C.R., Malik, S., Dunn, P.M. & Jenkinson, D.H. (1996). On the concept of a bivalent pharmacophore for SKCa channel blockers: synthesis, pharmacological testing, and radioligand binding studies on mono-, bis-, and tris- quinolinium compounds. Arch Pharmacologie (Weinheim) 329(12): 524-528. The dissociation equilibrium constants (Kd values) of dequalinium (2) and the monoquinolinium compounds 1a and 1b have been determined from competition equilibrium radioligand binding with [125I]apamin on rat brain synaptic plasma membranes (SPMs). Dequalinium binds to the channel with 2 orders of magnitude higher affinity than 1a or 1b, suggesting that both quinolinium groups are needed for potent and selective SKCa channel blockade. The trisquinolinium compound 3 (1,1'-[5- [4-(4- aminoquinolinium-1-yl)but-1-yl]non-4-en-1,9-diyl]-bis-(4- aminoquinolinium)) has been synthesized and tested for inhibition of the afterhyperpolarization of rat sympathetic neurones and on the binding assay. Compound 3 shows approximately one order of magnitude higher potency than 2, being the most potent non-peptidic SKCa channel blocker reported so far (Kd approximately 30 nM). The higher affinity of 3 compared with 2 may be due to direct binding of the third quinolinium group to the channel or may arise from a reduction of the unfavorable entropy of binding via an

Page 119 AHP, BK- and SK-channel references increase of the "local concentration" of quinolinium groups.

•Grunnet, M., Jensen, B.S., Olesen, S.P. & Klaerke, D.A. (2001). Apamin interacts with all subtypes of cloned small-conductance Ca2+-activated K+ channels. Pflugers Arch 441(4): 544-550. The purpose of the present study was to examine how apamin interacts with the three cloned subtypes of small-conductance Ca2+-activated K+ channels (hSK1, rSK2 and rSK3). Expression of the SK channel subtypes in Xenopus laevis oocytes resulted in large outward currents (0.5-5 microA) after direct injection of Ca2+. In all three cases the Ca2+-activated K+ currents could be totally inhibited by 500 nM apamin. Dose-response curves revealed a subtype-specific affinity for the apamin- induced inhibition with IC50 values of 704 pM and 196 nM (biphasic) for hSK1, 27 pM for rSK2 and 4 nM for rSK3. Consistent with these results, membranes prepared from oocytes expressing the SK channel subtypes bound 125I-labelled apamin with distinct dissociation constants (Kd values) of approx. 390 pM for hSK1, 4 pM for rSK2 and 11 pM for rSK3. These results show that apamin binds to and blocks all three subtypes of cloned SK channels, and the distinct values for IC50 and Kd suggest that apamin may be useful for determining the expression pattern of SK channel subtypes in native tissue.

•Grunnet, M., Jespersen, T., Angelo, K., Frokjaer-Jensen, C., Klaerke, D.A., Olesen, S.P. & Jensen, B.S. (2001). Pharmacological modulation of SK3 channels. Neuropharmacology 40(7): 879-887. Small-conductance, calcium-activated K+ channels (SK channels) are voltage- insensitive channels that have been identified molecularly within the last few years. As SK channels play a fundamental role in most excitable cells and participate in afterhyperpolarization (AHP) and spike-frequency adaptation, pharmacological modulation of SK channels may be of significant clinical importance. Here we report the functional expression of SK3 in HEK293 and demonstrate a broad pharmacological profile for these channels. Brain slice studies commonly employ 4- aminopyridine (4-AP) to block voltage-dependent K+ channels or a methyl derivative of bicuculline, a blocker of gamma-aminobutyric acid (GABA)-gated Cl- channels, in order to investigate the role of various synapses in specialized neural networks. However, in this study both 4-AP and bicuculline are shown to inhibit SK3 channels (IC50 values of 512 µM and 6 µM, respectively) at concentrations lower than those used for brain slice recordings. , a potent neuroprotective drug with anti-

Page 120 AHP, BK- and SK-channel references ischemic, anticonvulsant and sedative effects currently used in the treatment of amyotrophic lateral sclerosis, activates SK3 channels at concentrations of 3 µM and above. Amitriptyline, a tricyclic antidepressive widely used clinically, inhibits SK3 channels with an IC50 of 39.1 ± 10 µM (n=6).

•Hirschberg, B., Maylie, J., Adelman, J.P. & Marrion, N.V. (1998). Gating of recombinant small-conductance Ca-activated K+ channels by calcium. Journal of General Physiology 111(4): 565-81. Small-conductance Ca-activated K+ channels play an important role in modulating excitability in many cell types. These channels are activated by submicromolar concentrations of intracellular Ca2+, but little is known about the gating kinetics upon activation by Ca2+. In this study, single channel currents were recorded from Xenopus oocytes expressing the apamin-sensitive clone rSK2. Channel activity was detectable in 0.2 µM Ca2+ and was maximal above 2 µM Ca2+. Analysis of stationary currents revealed two open times and three closed times, with only the longest closed time being Ca dependent, decreasing with increasing Ca2+ concentrations. In addition, elevated Ca2+ concentrations resulted in a larger percentage of long openings and short closures. Membrane voltage did not have significant effects on either open or closed times. The open probability was approximately 0.6 in 1 µM free Ca2+. A lower open probability of approximately 0.05 in 1 µM Ca2+ was also observed, and channels switched spontaneously between behaviors. The occurrence of these switches and the amount of time channels spent displaying high open probability behavior was Ca2+ dependent. The two behaviors shared many features including the open times and the short and intermediate closed times, but the low open probability behavior was characterized by a different, long Ca2+-dependent closed time in the range of hundreds of milliseconds to seconds. Small-conductance Ca- activated K+ channel gating was modeled by a gating scheme consisting of four closed and two open states. This model yielded a close representation of the single channel data and predicted a macroscopic activation time course similar to that observed upon fast application of Ca2+ to excised inside-out patches.

•Ishii, T.M., Maylie, J. & Adelman, J.P. (1997). Determinants of apamin and d- tubocurarine block in SK potassium channels. Journal of Biological Chemistry 272(37): 23195-200. Small conductance calcium-activated potassium channels show a distinct

Page 121 AHP, BK- and SK-channel references pharmacology. Some, but not all, are blocked by the peptide toxin apamin, and apamin- sensitive channels are also blocked by d- tubocurarine. Cloned SK channels (small conductance calcium-activated potassium channel) recapitulate these properties. We have investigated the structural basis for these differences and found that two amino acid residues on either side of the deep pore are the primary determinants of sensitivity to apamin and differential block by d- tubocurarine. Therefore, the pharmacology of SK channels compared with other potassium channels correlates with structural differences in the outer pore region. However, introduction of a tyrosine residue in the position analogous to that which determines sensitivity to external tetraethylammonium for voltage-gated potassium channels endows SK channels with an equivalent tetraethylammonium sensitivity, indicating that the outer vestibules of the pores are similar. The pharmacology of channels formed in oocytes coinjected with SK1 and SK2 mRNAs, or with SK1-SK2 dimer mRNA, show that SK subunits may form heteromeric channels.

•Ishii, T.M., Silvia, C., Hirschberg, B., Bond, C.T., Adelman, J.P. & Maylie, J. (1997). A human intermediate conductance calcium-activated potassium channel. Proceedings of the National Academy of Sciences USA 94(21): 11651-6. An intermediate conductance calcium-activated potassium channel, hIK1, was cloned from human pancreas. The predicted amino acid sequence is related to, but distinct from, the small conductance calcium-activated potassium channel subfamily, which is approximately 50% conserved. hIK1 mRNA was detected in peripheral tissues but not in brain. Expression of hIK1 in Xenopus oocytes gave rise to inwardly rectifying potassium currents, which were activated by submicromolar concentrations of intracellular calcium (K0.5 = 0.3 µM). Although the K0.5 for calcium was similar to that of small conductance calcium-activated potassium channels, the slope factor derived from the Hill equation was significantly reduced (1.7 vs. 3. 5). Single-channel current amplitudes reflected the macroscopic inward rectification and revealed a conductance level of 39 pS in the inward direction. hIK1 currents were reversibly blocked by charybdotoxin (Ki = 2.5 nM) and clotrimazole (Ki = 24.8 nM) but were minimally affected by apamin (100 nM), iberiotoxin (50 nM), or (10 µM). These biophysical and pharmacological properties are consistent with native intermediate conductance calcium-activated potassium channels, including the erythrocyte Gardos channel.

•Joiner, W.J., Tang, M.D., Wang, L.Y., Dworetzky, S.I., Boissard, C.G.,

Page 122 AHP, BK- and SK-channel references

Gan, L., Gribkoff, V.K. & Kaczmarek, L.K. (1998). Formation of intermediate- conductance calcium-activated potassium channels by interaction of Slack and Slo subunits. Nature Neuroscience 1(6): 462-469. Large-conductance calcium-activated potassium channels (maxi-K channels) have an essential role in the control of excitability and secretion. Only one gene Slo is known to encode maxi-K channels, which are sensitive to both membrane potential and intracellular calcium. We have isolated a potassium channel gene called Slack that is abundantly expressed in the nervous system. Slack channels rectify outwardly with a unitary conductance of about 25-65 pS and are inhibited by intracellular calcium. However, when Slack is co-expressed with Slo, channels with pharmacological properties and single-channel conductances that do not match either Slack or Slo are formed. The Slack/Slo channels have intermediate conductances of about 60-180 pS and are activated by cytoplasmic calcium. Our findings indicate that some intermediate-conductance channels in the nervous system may result from an interaction between Slack and Slo channel subunits.

•Joiner, W.J., Wang, L.Y., Tang, M.D. & Kaczmarek, L.K. (1997). hSK4, a member of a novel subfamily of calcium-activated potassium channels. Proceedings of the National Academy of Sciences USA 94(20): 11013-8. The gene for hSK4, a novel human small conductance calcium-activated potassium channel, or SK channel, has been identified and expressed in Chinese hamster ovary cells. In physiological saline hSK4 generates a conductance of approximately 12 pS, a value in close agreement with that of other cloned SK channels. Like other members of this family, the polypeptide encoded by hSK4 contains a previously unnoted leucine zipper-like domain in its C terminus of unknown function. hSK4 appears unique, however, in its very high affinity for Ca2+ (EC50 of 95 nM) and its predominant expression in nonexcitable tissues of adult animals. Together with the relatively low homology of hSK4 to other SK channel polypeptides (approximately 40% identical), these data suggest that hSK4 belongs to a novel subfamily of SK channels.

•Joiner, W.J., Khanna, R., Schlichter, L.C. & Kaczmarek, L.K. (2000). A dual role for calmodulin in both gating and assembly of SK channels. Society for Neuroscience Abstracts 30. Small conductance Ca2+-activated K+ (SK) channels regulate neuronal excitability by generating the slow afterhyperpolarization that commonly follows

Page 123 AHP, BK- and SK-channel references trains of action potentials. They are also important in non-excitable cells such as lymphocytes, where they regulate proliferation and cell volume. SK channels are believed to be gated indirectly by C-terminally-bound calmodulin subunits when the latter bind cytosolic Ca2+. Here we show that the gating selectivity of SK2, SK3, and SK4 channels for different metal ions matches that for activation of calmodulin. In addition, we show that the proximal C-terminus of SK4 not only binds calmodulin but suppresses whole-cell currents when coexpressed with wild-type channels. Coimmunoprecipitation, immunofluorescence, and surface biotinylation experiments all support the idea that this domain allows calmodulin to enhance assembly of SK subunits, possibly by providing anchor points for cross-linking by calmodulin. Together these data suggest a novel calmodulin-dependent mechanism for regulating ion channel activity and cell excitability. Supported by: NIH Grant DC01919 and MRC Grant MT-13657.

•Joiner, W.J., Khanna, R., Schlichter, L.C. & Kaczmarek, L.K. (2001). Calmodulin regulates assembly and trafficking of SK4/IK1 Ca2+-activated K+ channels. Journal of Biological Chemistry 276(41): 37980-37985. Calmodulin (CaM) regulates gating of several types of ion channels but has not been implicated in channel assembly or trafficking. For the SK4/IK1 K+ channel, CaM bound to the proximal C terminus ("Ct1 " domain) acts as the Ca2+ sensor. We now show that CaM interacting with the C terminus of SK4 also controls channel assembly and surface expression. In transfected cells, removing free CaM by overexpressing the CaM-binding domain, Ct1, redistributed full-length SK4 protein from the plasma membrane to the cytoplasm and decreased whole-cell currents. Making more CaM protein available by overexpressing the CaM gene abrogated the dominant-negative effect of Ct1 and restored both surface expression of SK4 protein and whole-cell currents. The distal C-terminal domain ("Ct2") also plays a role in assembly, but is not CaM-dependent. Co-immunoprecipitation experiments demonstrated that multimerization of SK4 subunits was enhanced by CaM and inhibited by removal of CaM, indicating that CaM regulates trafficking of SK4 by affecting the assembly of channels. Our results support a model in which CaM- dependent association of SK4 monomers at their Ct1 domains regulates channel assembly and surface expression. This appears to represent a novel mechanism for controlling ion channels, and consequently, the cellular functions that depend on them.

Page 124 AHP, BK- and SK-channel references

•Keen, J.E., Khawaled, R., Farrens, D.L., Neelands, T., Rivard, A., Bond, C.T., Janowsky, A., Fakler, B., Adelman, J.P. & Maylie, J. (1999). Domains responsible for constitutive and Ca2+-dependent interactions between calmodulin and small conductance Ca2+-activated potassium channels. Journal of Neuroscience 19(20): 8830-8. Small conductance Ca2+-activated potassium channels (SK channels) are coassembled complexes of pore-forming SK alpha subunits and calmodulin. We proposed a model for channel activation in which Ca2+ binding to calmodulin induces conformational rearrangements in calmodulin and the alpha subunits that result in channel gating. We now report fluorescence measurements that indicate conformational changes in the alpha subunit after calmodulin binding and Ca2+ binding to the alpha subunit-calmodulin complex. Two-hybrid experiments showed that the Ca2+-independent interaction of calmodulin with the alpha subunits requires only the C-terminal domain of calmodulin and is mediated by two noncontiguous subregions; the ability of the E-F hands to bind Ca2+ is not required. Although SK alpha subunits lack a consensus calmodulin-binding motif, mutagenesis experiments identified two positively charged residues required for Ca2+- independent interactions with calmodulin. Electrophysiological recordings of SK2 channels in membrane patches from oocytes coexpressing mutant revealed that channel gating is mediated by Ca2+ binding to the first and second E-F hand motifs in the N-terminal domain of calmodulin. Taken together, the results support a calmodulin- and Ca2+-calmodulin-dependent conformational change in the channel alpha subunits, in which different domains of calmodulin are responsible for Ca2+-dependent and Ca2+-independent interactions. In addition, calmodulin is associated with each alpha subunit and must bind at least one Ca2+ ion for channel gating. Based on these results, a state model for Ca2+ gating was developed that simulates alterations in SK channel Ca2+ sensitivity and cooperativity associated with mutations in CaM.

•Khawaled, R., Bruening-Wright, A., Adelman, J.P. & Maylie, J. (1999). Bicuculline block of small-conductance calcium-activated potassium channels. Pflügers Archiv 438(3): 314-21. Small-conductance calcium-activated potassium channels (SK channels) are gated solely by intracellular calcium ions and their activity is responsible for the slow afterhyperpolarization (AHP) that follows an action potential in many excitable cells. Brain slice studies commonly employ a methyl derivative of bicuculline

Page 125 AHP, BK- and SK-channel references

(bicuculline-m), a GABAA (gamma-aminobutyric acid) receptor antagonist, to diminish the tonic inhibitory influences of GABAergic synapses, or to investigate the role of these synapses in specialized neural networks. However, recent evidence suggests that bicuculline-m may not be specific for GABAA receptors and may also block the slow AHP. Therefore, the effects of bicuculline-m on cloned apamin-sensitive SK2 and apamin-insensitive SK1 channels were examined following expression in Xenopus oocytes. The results show that at concentrations employed for slice recordings, bicuculline-m potently blocks both apamin-sensitive SK2 currents and apamin- insensitive SK1 currents when applied to outside-out patches. Apamin-insensitive SK1 currents run down in excised patches. The potency of bicuculline-m block also decreases with time after patch excision. Site-directed mutagenesis that changes two residues in the outer vestibule of the SK1 pore that confers apamin sensitivity also reduces run down of the current in patches, and endows stable sensitivity to bicuculline-m indistinguishable from SK2. Therefore, the use of bicuculline-m in slice recordings may mask apamin-sensitive slow AHPs that are important determinants of neuronal excitability. In addition, bicuculline-m-insensitive slow AHPs may indicate that the underlying channels have run down.

•Köhler, M., Hirschberg, B., Bond, C.T., Kinzie, J.M., Marrion, N.V., Maylie, J. & Adelman, J.P. (1996). Small-conductance, calcium-activated potassium channels from mammalian brain. Science 273(5282): 1709-14. Members of a previously unidentified family of potassium channel subunits were cloned from rat and human brain. The messenger RNAs encoding these subunits were widely expressed in brain with distinct yet overlapping patterns, as well as in several peripheral tissues. Expression of the messenger RNAs in Xenopus oocytes resulted in calcium- activated, voltage-independent potassium channels. The channels that formed from the various subunits displayed differential sensitivity to apamin and tubocurare. The distribution, function, and pharmacology of these channels are consistent with the SK class of small-conductance, calcium-activated potassium channels, which contribute to the afterhyperpolarization in central neurons and other cell types.

•Legros, C., Bougis, P.E. & Martin-Eauclaire, M.-F. (1999). Molecular biology of scorpion toxins active on potassium channels. Perspectives in Drug Discovery and Design 15/16: 1-14. Peptidyl scorpion toxins are known to block diverse types of K+ channels with

Page 126 AHP, BK- and SK-channel references high affinity and, thus, can be used as powerful tools to study the physiological role of the ionic selectivity, and the architecture of the pore-region of this class of channels. Yet, diversity among K+ channels is large and there has been a profusion of research for new selective ligands in order to elucidate their mechanisms of action and pharmacology significance. Scorpion toxins active on K+ channels are short polypeptides of about 30 to 40 amino acid residues, cross-linked by three or four disulfide bridges. They display a high degree of primary sequence homologies. 1H Nuclear Magnetic Resonance (NMR) analysis has demonstrated that these toxins are composed of an [alpha]-helix and a two-stranded antiparallel [beta]-sheet, linked by two disulfide bridges. This structural motif is also found in the insect defensins. A 370 bp cDNA encoding the 2 (KTX2) precursor (a 37 amino acid residues peptide purified from the North African scorpion Androctonus australis and acting as a high affinity blocker of K+ channels) was obtained by PCR amplification and the organization of the KTX2 precursor depicted. This precursor is composed of a signal peptide followed by the mature toxin. The transcriptional unit and the promotor region of the gene encoding KTX2 was then amplified from the genomic DNA of Androctonus australis and its sequence determined. A single intron of 87 bp, located close to the region encoding the C-terminal part of the signal peptide, was found. Its A+T content was particularly high (up to 86%). The transcription unit of the gene was 390 bp long. Regulatory consensus sequences were identified. The genes of scorpion ’short‘ toxins active on K+ channels are organized similarly to those of the scorpion ’long‘ toxins active on Na+ channels and not like those of structurally related insect defensins, which are intronless.

•Legros, C., Oughuideni, R., Darbon, H., Rochat, H., Bougis, P.E. & Martin- Eauclaire, M.-F. (1996). Characterisation of a new peptide from Tityus serrulatus scorpion venom which is a ligand of the apamin-binding site. FEBS Letters 390: 81- 84. A new ligand (Ts kappa) of the apamin binding site on rat brain synaptosomes (K0.5 = 300 pM) was purified and characterized from the venom of Tityus serrulatus. It is a polypeptide toxin of 35 amino acid residues, with three disulfide bridges. Its cDNA was amplified from a venom gland cDNA library and the nucleotide sequence determined. A model of Ts kappa was constructed by amino acid replacement using charybdotoxin structure as determined by 1H nuclear magnetic resonance as starting model.

Page 127 AHP, BK- and SK-channel references

•McManus, O.B. (1991). Calcium-activated potassium channels: Regulation by calcium. Journal of Bioenergetics & Biomembranes 23(4): 537-60. A wide variety of calcium-activated K channels has been described and can be conveniently separated into three classes based on differences in single-channel conductance, voltage dependence of channel opening, and sensitivity to blockers. Large-conductance calcium-activated K channels typically require micromolar concentrations of calcium to open, and their sensitivity to calcium increases with membrane depolarization, suggesting that they may be involved in repolarization events. Small-conductance calcium-activated K channels are generally more sensitive to calcium at negative membrane potentials, but their sensitivity to calcium is independent of membrane potential, suggesting that they may be involved in regulating membrane properties near the resting potential. Intermediate- conductance calcium-activated K channels are a loosely defined group, where membership is determined because a channel does not fit in either of the other two groups. Within each broad group, variations in calcium sensitivity and single- channel conductance have been observed, suggesting that there may be families of closely related calcium-activated K channels. Kinetic studies of the gating of calcium-activated potassium channels have revealed some basic features of the mechanisms involved in activation of these channels by calcium, including the number of calcium ions participating in channel opening, the number of major conformations of the channels involved in the gating process, and the number of transition pathways between open and closed states. Methods of analysis have been developed that may allow identification of models that give accurate descriptions of the gating of these channels. Although such kinetic models are likely to be oversimplifications of the behavior of a large macromolecule, these models may provide some insight into the mechanisms that control the gating of the channel, and are subject to falsification by new data.

•Namba, T., Ishii, T.M., Ikeda, M., Hisano, T., Itoh, T., Hirota, K., Adelman, J.P. & Fukuda, K. (2000). Inhibition of the human intermediate conductance Ca2+-activated K+ channel, hIK1, by volatile anesthetics. European Journal of Pharmacology 395(2): 95-101. Ca2+-activated K(+) channels (K(Ca)) regulate a wide variety of cellular functions by coupling intracellular Ca2+ concentration to membrane potential. There are three major groups of K(Ca) classified by their unit conductances: large (BK), intermediate (IK), and small (SK) conductance of channels. BK channel is gated by

Page 128 AHP, BK- and SK-channel references combined influences of Ca2+ and voltage, while IK and SK channels are gated solely by Ca2+. Volatile anesthetics inhibit BK channel activity by interfering with the Ca2+ gating mechanism. However, the effects of anesthetics on IK and SK channels are unknown. Using cloned IK and SK channels, hIK1 and hSK1-3, respectively, we found that the currents of hIK1 were inhibited rapidly and reversibly by volatile anesthetics, whereas those of SK channels were not affected. The IC(50) values of the volatile anesthetics, halothane, sevoflurane, enflurane, and isoflurane for hIK1 inhibition were 0.69, 0.42, 1.01 and 1.03 mM, respectively, and were in the clinically used concentration range. In contrast to BK channel, halothane inhibition of hIK1 currents was independent of Ca2+ concentration, suggesting that Ca2+ gating mechanism is not involved. These results demonstrate that volatile anesthetics, such as halothane, enflurane, isoflurane, and sevoflurane, affect BK, IK, and SK channels in distinct ways.

•Paton, W.D.M. & Zaimis, E.J. (1949). Pharmacological actions of polymethylene bistrimethylammonium salts. British Journal of Pharmacology 4: 381-400.

•Peterson, B.Z., DeMaria, C.D., Adelman, J.P. & Yue, D.T. (1999). Calmodulin is the Ca2+ sensor for Ca2+-dependent inactivation of L-type calcium channels. Neuron 22(3): 549-558. Elevated intracellular Ca2+ triggers inactivation of L-type calcium channels, providing negative Ca2+ feedback in many cells. Ca2+ binding to the main alpha1c channel subunit has been widely proposed to initiate such Ca2+-dependent inactivation. Here, we find that overexpression of mutant, Ca2+-insensitive calmodulin (CaM) ablates Ca2+-dependent inactivation in a "dominant-negative" manner. This result demonstrates that CaM is the actual Ca2+ sensor for inactivation and suggests that CaM is constitutively tethered to the channel complex. Inactivation is likely to occur via Ca2+-dependent interaction of tethered CaM with an IQ-like motif on the carboxyl tail of alpha1c. CaM also binds to analogous IQ regions of N-, P/Q-, and R-type calcium channels, suggesting that CaM-mediated effects may be widespread in the calcium channel family.

•Reinhart, P.H. & Levitan, I.B. (1995). Kinase and phosphatase activities intimately associated with a reconstituted calcium-dependent potassium channel. Journal of Neuroscience 15: 4572-4579. Type-2 calcium-dependent potassium (KCa) channels from mammalian brain,

Page 129 AHP, BK- and SK-channel references reconstituted into planar phospholipid bilayers, are modulated by ATP or ATP analogs via an endogenous protein kinase activity intimately associated with the channel (Chung et al., 1991). We show here that the endogenous protein kinase activity is protein kinase C (PKC)-like because (1) modulation by ATP can be mimicked by exogenous PKC, and (2) the effects of ATP can be blocked by PKC(19-36), a specific peptide inhibitor of PKC. Furthermore, adding the PKC inhibitor peptide after the addition of ATP reverses the modulation produced by ATP, suggesting that there is a phosphoprotein phosphatase activity closely associated with type-2 KCa channels. Consistent with this idea is the finding that microcystin, a non-specific phosphatase inhibitor, enhances the modulation of KCa channel activity by ATP. Inhibitor-1, a specific protein inhibitor of phosphoprotein phosphatase-1, also enhances the effect of ATP, suggesting that the endogenous phosphatase activity is phosphatase-1-like. The results imply that type-2 KCa channels exist as part of a regulatory complex that includes a PKC-like protein kinase and a phosphatase-1-like phosphoprotein phosphatase, both of which participate in the modulation of channel function.

•Schonherr, R., Lober, K. & Heinemann, S.H. (2000). Inhibition of human ether a go-go potassium channels by Ca2+/calmodulin. EMBO Journal 19(13): 3263-3271. Intracellular Ca2+ inhibits voltage-gated potassium channels of the ether a go-go (EAG) family. To identify the underlying molecular mechanism, we expressed the human version hEAG1 in Xenopus oocytes. The channels lost Ca2+ sensitivity when measured in cell-free membrane patches. However, Ca2+ sensitivity could be restored by application of recombinant calmodulin (CaM). In the presence of CaM, half inhibition of hEAG1 channels was obtained in 100 nM Ca2+. Overlay assays using labelled CaM and glutathione S-transferase (GST) fusion fragments of hEAG1 demonstrated direct binding of CaM to a C-terminal domain (hEAG1 amino acids 673- 770). Point mutations within this section revealed a novel CaM-binding domain putatively forming an amphipathic helix with both sides being important for binding. The binding of CaM to hEAG1 is, in contrast to Ca2+-activated potassium channels, Ca2+ dependent, with an apparent K(D) of 480 nM. Co-expression experiments of wild-type and mutant channels revealed that the binding of one CaM molecule per channel complex is sufficient for channel inhibition.

•Schonherr, R., Gessner, G., Lober, K. & Heinemann, S.H. (2002). Functional distinction of human EAG1 and EAG2 potassium channels. FEBS Letters 514(2-3):

Page 130 AHP, BK- and SK-channel references

204-208. Human ether a go-go potassium channel 2 (hEAG2) was cloned and its properties were compared with the previously characterized isoform hEAG1. In the Xenopus oocyte expression system the time course of activation was about four times slower and the voltage required for half-maximal subunit activation was about 10 mV greater for hEAG2 channels. However, its voltage dependence was smaller and, therefore, hEAG2 channels start to open at more negative voltages than hEAG1. Coexpression of both isoforms and kinetic analysis of the resulting currents indicated that they can form heteromeric channel complexes in which the slow activation phenotype of hEAG2 is dominant. Upon expression in mammalian cells, blocked hEAG1 channels (IC(50) 1.4 µM) more potently than hEAG2 channels (IC(50) 152 µM), thus providing a useful tool for the functional distinction between hEAG1 and hEAG2 potassium channels.

•Schreiber, M. & Salkoff, L. (1997). A novel calcium-sensing domain in the BK channel. Biophysical Journal 73: 1355-1363.

•Schreiber, M., Yuan, A. & Salkoff, L. (1999). Transplantable sites confer calcium sensitivity to BK channels. Nature Neuroscience 2(5): 416-421. Both intracellular calcium and voltage activate Slo1, a high-conductance potassium channel, linking calcium with electrical excitability. Using molecular techniques, we created a calcium-insensitive variant of this channel gated by voltage alone. Calcium sensitivity was restored by adding back small portions of the carboxyl (C)-terminal 'tail' domain. Two separate regions of the tail independently conferred different degrees of calcium sensitivity; together, they restored essentially wild-type calcium dependence. These results suggest that, in the absence of calcium, the Slo1 tail inhibits voltage-dependent gating, and that calcium removes this inhibition. Slo1 may have evolved from an ancestral voltage-sensitive potassium channel represented by the core; the tail may represent the more recent addition of a calcium-dependent modulatory domain.

•Schumacher, M.A., Rivard, A.F., Bachinger, H.P. & Adelman, J.P. (2001). Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin. Nature 410: 1120-1124. Small-conductance Ca2+-activated K+ channels (SK channels) are independent of voltage and gated solely by intracellular Ca2+. These membrane channels are

Page 131 AHP, BK- and SK-channel references heteromeric complexes that comprise pore-forming alpha-subunits and the Ca2+- binding protein calmodulin (CaM). CaM binds to the SK channel through the CaM- binding domain (CaMBD), which is located in an intracellular region of the alpha- subunit immediately carboxy-terminal to the pore. Channel opening is triggered when Ca2+ binds the EF hands in the N-lobe of CaM. Here we report the 1.60 A crystal structure of the SK channel CaMBD/Ca2+/CaM complex. The CaMBD forms an elongated dimer with a CaM molecule bound at each end; each CaM wraps around three alpha-helices, two from one CaMBD subunit and one from the other. As only the CaM N-lobe has bound Ca2+, the structure provides a view of both calcium- dependent and -independent CaM/protein interactions. Together with biochemical data, the structure suggests a possible gating mechanism for the SK channel.

•Shah, M.M. & Haylett, D.G. (2000). The pharmacology of hSK1 Ca2+-activated K+ channels expressed in mammalian cell lines. British Journal of Pharmacology 129: 627-630. The pharmacology of hSK1, a small conductance calcium-activated potassium channel, was studied in mammalian cell lines (HEK293 and COS-7). In these cell types, hSK1 forms an apamin-sensitive channel with an IC(50) for apamin of 8 nM in HEK293 cells and 12 nM in COS-7 cells. The currents in HEK293 cells were also sensitive to tubocurarine (IC(50)=23 µM), dequalinium (IC(50)=0.4 µM), and the novel dequalinium analogue, UCL1848 (IC(50)=1 nM). These results are very different from the pharmacology of hSK1 channels expressed in Xenopus oocytes and suggest the properties of the channel may depend on the expression system. Our findings also raise questions about the role of SK1 channels in generating the apamin-insensitive slow afterhyperpolarization observed in central neurones.

•Shakkottai, V.G., Regaya, I., Wulff, H., Fajloun, Z., Tomita, H., Fathallah, M., Cahalan, M.D., Gargus, J.J., Sabatier, J.M. & Chandy, K.G. (2001). Design and characterization of a highly selective peptide inhibitor of the small conductance calcium-activated K+ channel, SkCa2. Journal of Biological Chemistry 276(46): 43145-43151. Apamin-sensitive small conductance calcium-activated potassium channels (SKCa1-3) mediate the slow afterhyperpolarization in neurons, but the molecular identity of the channel has not been defined because of the lack of specific inhibitors. Here we describe the structure-based design of a selective inhibitor of SKCa2. Leiurotoxin I (Lei) and PO5, peptide toxins that share the RXCQ motif,

Page 132 AHP, BK- and SK-channel references potently blocked human SKCa2 and SKCa3 but not SKCa1, whereas maurotoxin, Pi1, Tskappa, and PO1 were ineffective. Lei blocked these channels more potently than PO5 because of the presence of Ala(1), Phe(2), and Met(7). By replacing Met(7) in the RXCQ motif of Lei with the shorter, unnatural, positively charged diaminobutanoic acid (Dab), we generated Lei-Dab(7), a selective SKCa2 inhibitor (K(d) = 3.8 nm) that interacts with residues in the external vestibule of the channel. SKCa3 was rendered sensitive to Lei-Dab(7) by replacing His(521) with the corresponding SKCa2 residue (Asn(367)). Intracerebroventricular injection of Lei-Dab(7) into mice resulted in no gross central nervous system at concentrations that specifically blocked SKCa2 homotetramers. Lei-Dab(7) will be a useful tool to investigate the functional role of SKCa2 in mammalian tissues.

•Shmukler, B.E., Bond, C.T., Wilhelm, S., Bruening-Wright, A., Maylie, J., Adelman, J.P. & Alper, S.L. (2001). Structure and complex transcription pattern of the mouse SK1 K(Ca) channel gene, KCNN1. Biochimica Biophysica Acta 1518(1-2): 36-46. Small conductance calcium-gated K(+) channels (SK channels) are encoded by the three SK genes, SK1, SK2, and SK3. These channels likely contribute to slow synaptic afterhyperpolarizations of apamin-sensitive and apamin-insensitive types. SK channels are also widely expressed outside the nervous system. The mouse SK1 gene comprises at least 12 exons extending across 19.8 kb of genomic DNA. This gene encodes a complex pattern of alternatively spliced SK1 transcripts widely expressed among mouse tissues. These transcripts exhibit at least four distinct 5'- nucleotide sequence variants encoding at least two N-terminal amino acid sequences. Optional inclusion of exons 7 and 9, together with two alternate splice donor sites in exon 8, yields transcripts encoding eight variant C-terminal amino acid sequences for SK1. These include an altered putative S6 transmembrane span, modification of the C-terminal cytoplasmic domain binding site for calmodulin, and generation of two alternate predicted binding sites for PDZ domain-containing proteins. 20 of the 32 predicted mouse SK1 transcripts are expressed in brain at levels sufficient to allow consistent detection, and encode 16 SK1 polypeptide variants. Only four of these 16 polypeptides preserve the ability to bind calmodulin in a Ca2+-independent manner. Mouse SK1 also exhibits novel, strain-specific, length polymorphism of a polyglutamate repeat in the N-terminal cytoplasmic domain. The evolutionary conservation of this complex transcription pattern suggests a possible role in the tuning of SK1 channel function.

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•Soh, H. & Park, C.S. (2001). Inwardly rectifying current-voltage relationship of small-conductance Ca2+-activated K+ channels rendered by intracellular divalent cation blockade. Biophysics Journal 80(5): 2207-2215. Small conductance Ca2+-activated K+ channels (SK(Ca) channels) are a group of K+-selective ion channels activated by submicromolar concentrations of intracellular Ca2+ independent of membrane voltages. We expressed a cloned SK(Ca) channel, rSK2, in Xenopus oocytes and investigated the effects of intracellular divalent cations on the current-voltage (I-V) relationship of the channels. Both Mg2+ and Ca2+ reduced the rSK2 channel currents in voltage-dependent manners from the intracellular side and thus rectified the I-V relationship at physiological concentration ranges. The apparent affinity of Mg2+ was changed as a function of both transmembrane voltage and intracellular Ca2+ concentration. Extracellular K+ altered the voltage dependence as well as the apparent affinities of Mg2+ binding from intracellular side. Thus, the inwardly rectifying I-V relationship of SK(Ca) channels is likely due to the voltage-dependent blockade of intracellular divalent cations and that the binding site is located within the ion-conducting pathway. Therefore, intracellular Ca2+ modulates the permeation characteristics of SK(Ca) channels by altering the I-V relationship as well as activates the channel by interacting with the gating machinery, calmodulin, and SK(Ca) channels can be considered as Ca2+-activated inward rectifier K+ channels.

•Strobaek, D., Jorgensen, T.D., Christophersen, P., Ahring, P.K. & Olesen, S.P. (2000). Pharmacological characterization of small-conductance Ca2+-activated K+ channels stably expressed in HEK 293 cells. British Journal of Pharmacology 129: 991-999. Three genes encode the small-conductance Ca2+-activated K(+) channels (SK channels). We have stably expressed hSK1 and rSK2 in HEK 293 cells and addressed the pharmacology of these subtypes using whole-cell patch clamp recordings. The bee venom peptide apamin blocked hSK1 as well as rSK2 with IC(50) values of 3.3 nM and 83 pM, respectively. The pharmacological separation between the subtypes was even more prominent when applying the scorpion peptide blocker scyllatoxin, which blocked hSK1 with an IC(50) value of 80 nM and rSK2 at 287 pM. The potent small molecule blockers showed little differentiation between the channel subtypes. The bis- quinolinium cyclophane UCL 1684 blocked hSK1 with an IC(50) value of 762 pM and rSK2 at 364 pM. The antiseptic compound dequalinium chloride blocked hSK1 and

Page 134 AHP, BK- and SK-channel references rSK2 with IC(50) values of 444 nM and 162 nM, respectively. The nicotinic antagonist d-tubocurarine was found to block hSK1 and rSK2 with IC(50) values of 27 microM and 17 microM when measured at +80 mV. The inhibition by d-tubocurarine was voltage-dependent with increasing affinities at more hyperpolarized potentials. The GABA(A) receptor antagonist bicuculline methiodide also blocked hSK1 and rSK2 in a voltage-dependent manner with IC(50) values of 15 and 25 microM when measured at +80 mV. In conclusion, the pharmacological separation between SK channel subtypes expressed in mammalian cells is too small to support the notion that the apamin-insensitive afterhyperpolarization of neurones is mediated by hSK1.

•Syme, C.A., Gerlach, A.C., Singh, A.K. & Devor, D.C. (2000). Pharmacological activation of cloned intermediate- and small-conductance Ca2+-activated K+ channels. Proceedings of the National Academy of Sciences USA 93: 9200-9205. We previously characterized 1-ethyl-2-benzimidazolinone (1-EBIO), as well as the clinically useful benzoxazoles, chlorzoxazone (CZ), and zoxazolamine (ZOX), as pharmacological activators of the intermediate-conductance Ca2+-activated K(+) channel, hIK1. The mechanism of activation of hIK1, as well as the highly homologous small-conductance, Ca2+-dependent K(+) channel, rSK2, was determined following heterologous expression in Xenopus oocytes using two-electrode voltage clamp (TEVC) and excised, inside-out patch-clamp techniques. 1-EBIO, CZ, and ZOX activated both hIK1 and rSK2 in TEVC and excised inside-out patch-clamp experiments. In excised, inside-out patches, 1-EBIO and CZ induced a concentration-dependent activation of hIK1, with half-maximal (K(1/2)) values of 84 microM and 98 µM, respectively. Similarly, CZ activated rSK2 with a K(1/2) of 87 µM. In the absence of CZ, the Ca2+-dependent activation of hIK1 was best fit with a K(1/2) of 700 nM and a Hill coefficient (n) of 2.0. rSK2 was activated by Ca2+ with a K(1/2) of 700 nM and an n of 2.5. Addition of CZ had no effect on either the K(1/2) or n for Ca2+-dependent activation of either hIK1 or rSK2. Rather, CZ increased channel activity at all Ca2+ concentrations (V(max)). Event-duration analysis revealed hIK1 was minimally described by two open and three closed times. Activation by 1-EBIO had no effect on tau(o1), tau(o2), or tau(c1), whereas tau(c2) and tau(c3) were reduced from 9.0 and 92.6 ms to 5.0 and 44.1 ms, respectively. In conclusion, we define 1-EBIO, CZ, and ZOX as the first known activators of hIK1 and rSK2. Openers of IK and SK channels may be therapeutically beneficial in cystic fibrosis and vascular diseases.

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•Talukder, G. & Aldrich, R.W. (2000). Complex voltage-dependent behavior of single unliganded calcium-sensitive potassium channels. Biophysics Journal 78(2): 761-772. Study and characterization of unliganded openings is of central significance for the elucidation of gating mechanisms for allosteric ligand-gated ion channels. Unliganded openings have been reported for many channel types, but their low open probability can make it difficult to study their kinetics in detail. Because the large conductance calcium-activated potassium channel mSlo is sensitive to both intracellular calcium and to membrane potential, we have been able to obtain stable unliganded single-channel recordings of mSlo with relatively high opening probability. We have found that the single-channel gating behavior of mSlo is complex, with multiple open and closed states, even when no ligand is present. Our results rule out a Monod-Wyman-Changeux allosteric mechanism with a central voltage-dependent concerted step, and they support the existence of quaternary states with less than the full number of voltage sensors activated, as has been suggested by previous work involving measurements of gating currents.

•Terstappen, G.C., Pula, G., Carignani, C., Chen, M.X. & Roncarati, R. (2001). Pharmacological characterisation of the human small conductance calcium-activated potassium channel hSK3 reveals sensitivity to tricyclic antidepressants and antipsychotic . Neuropharmacology 40(6): 772-783. A stable CHO-K1 cell line was developed which expresses the human small conductance calcium-activated potassium channel hSK3. Immunofluorescence microscopy using an anti-SK3 antibody and radioligand binding using [(125)I]-apamin demonstrated the presence of hSK3 channel in the recombinant cell line. This cell line was utilised in a fluorescence assay using the membrane potential-sensitive dye DiBAC(4)(3) to functionally analyse and pharmacologically characterise this potassium channel. The analysis of known blockers of calcium-activated potassium channels revealed the highest potency for apamin (IC(50)=13.2 nM). This result was confirmed by direct recordings of SK3 currents using the whole-cell patch-clamp technique. Tricyclic antidepressants such as , imipramine and as well as phenothiazines such as fluphenazine, , chlorpromazine and blocked the hSK3 channel with micromolar potencies. These compounds also displaced [(125)I]-apamin binding to the hSK3 channel thus suggesting direct and competitive channel blocking activity. Since

Page 136 AHP, BK- and SK-channel references these compounds share a common three-ring molecular core structure, this feature seems to be important for channel blocking activity. The serine/threonine protein phosphatase inhibitors okadaic acid and calyculin A were able to abolish channel activation with nanomolar potencies, but did not displace [(125)I]-apamin binding. Thus, phosphorylation of hSK3 or an accessory channel subunit seems to be involved in its modulation.

•Wadsworth, J.D., Doorty, K.B. & Strong, P.N. (1994). Comparable 30-kDa apamin binding polypeptides may fulfill equivalent roles within putative subtypes of small conductance Ca2+-activated K+ channels. Journal of Biological Chemistry 269(27): 18053-18061. Apamin, a peptide from bee venom, blocks small conductance Ca2+- activated K+ channels in central synapses and peripheral tissues. Using 125I-apamin, single classes of high affinity binding sites (Kd 1-3 pM) were identified on plasma membranes from rat, rabbit, guinea pig, and bovine brain and from rabbit, guinea pig, and bovine liver. Binding was sensitive to scyllatoxin, dequalinium, gallamine, and d- tubocurarine but not to charybdotoxin, toxin I, or mast cell degranulating peptide. In contrast, saturable binding of 125I-apamin to rat liver plasma membranes was virtually undetectable, thereby providing a correlation with the ability to measure apamin-sensitive Ca2+-activated potassium currents in rabbit and guinea pig hepatocytes but not in rat hepatocytes. In agreement with membrane binding studies, homobifunctional cross-linkers identified apparently identical 33-kDa 125I- apamin binding polypeptides on brain plasma membranes from all species and analogous but distinct polypeptides on plasma membranes from rabbit, guinea pig, and bovine liver. None of these affinity-labeled polypeptides were detectable on plasma membranes from rat liver. Affinity labeling was abolished on both liver and brain membranes by apamin, scyllatoxin, dequalinium, gallamine, and d- tubocurarine. These results indicate that comparable approximately 30- kDa polypeptides may fulfill equivalent functional roles within putative subtypes of apamin-sensitive small conductance Ca2+- activated K+ channels.

•Wadsworth, J.D., Torelli, S., Doorty, K.B. & Strong, P.N. (1997). Structural diversity among subtypes of small-conductance Ca2+-activated potassium channels. Arch Biochem Biophys 346(1): 151-60. 125I-Apamin and photolabile derivatives of the toxin have been used to investigate the binding properties and subunit composition of small conductance

Page 137 AHP, BK- and SK-channel references

Ca2+-activated potassium channels (SK(Ca) channels) expressed on plasma membranes from rat brain, rabbit liver, or rat pheochromocytoma (PC12) cells. On all preparations, 125I-apamin recognized single classes of acceptor binding sites with similar high affinity (Kd approximately 3-6 pM). Gallamine, however, was found to readily discriminate between 125I-apamin acceptors present in these preparations, showing a maximal approx nine-fold difference in affinity for acceptors expressed by rabbit liver or PC12 cells. Affinity- labeling patterns revealed the expression of different hetero- oligomeric combinations of high (86 or 59 kDa) and low (33 or 30 kDa) molecular mass 125I-apamin-binding polypeptides, consistent with pharmacological differences. Alternative expression of either 86- or 59- kDa polypeptides appeared to be the most important factor influencing gallamine's affinity for SK(Ca) channel subtypes. Both high- and low- molecular-mass polypeptides are integral membrane proteins, the latter being glycosylated in a tissue-specific manner.

•Wissmann, R., Bildl, W., Neumann, H., Rivard, A.F., Klocker, N., Weitz, D., Schulte, U., Adelman, J.P., Bentrop, D. & Fakler, B. (2002). A helical region in the C terminus of small-conductance Ca2+-activated K+ channels controls assembly with apo-calmodulin. Journal of Biological Chemistry 277(6): 4558-4564. Small conductance Ca2+-activated potassium (SK) channels underlie the afterhyperpolarization that follows the action potential in many types of central neurons. SK channels are voltage-independent and gated solely by intracellular Ca2+ in the submicromolar range. This high affinity for Ca2+ results from Ca2+- independent association of the SK alpha-subunit with calmodulin (CaM), a property unique among the large family of potassium channels. Here we report the solution structure of the calmodulin binding domain (CaMBD, residues 396-487 in rat SK2) of SK channels using NMR spectroscopy. The CaMBD exhibits a helical region between residues 423-437, whereas the rest of the molecule lacks stable overall folding. Disruption of the helical domain abolishes constitutive association of CaMBD with Ca2+-free CaM, and results in SK channels that are no longer gated by Ca2+. The results show that the Ca2+-independent CaM-CaMBD interaction, which is crucial for channel function, is at least in part determined by a region different in sequence and structure from other CaM-interacting proteins.

•Xia, X.M., Fakler, B., Rivard, A., Wayman, G., Johnson-Pais, T., Keen, J.E., Ishii, T., Hirschberg, B., Bond, C.T., Lutsenko, S., Maylie, J. &

Page 138 AHP, BK- and SK-channel references

Adelman, J.P. (1998). Mechanism of calcium gating in small-conductance calcium- activated potassium channels. Nature 395(6701): 503-507. The slow afterhyperpolarization that follows an action potential is generated by the activation of small-conductance calcium-activated potassium channels (SK channels). The slow afterhyperpolarization limits the firing frequency of repetitive action potentials (spike- frequency adaptation) and is essential for normal neurotransmission. SK channels are voltage-independent and activated by submicromolar concentrations of intracellular calcium. They are high-affinity calcium sensors that transduce fluctuations in intracellular calcium concentrations into changes in membrane potential. Here we study the mechanism of calcium gating and find that SK channels are not gated by calcium binding directly to the channel alpha-subunits. Instead, the functional SK channels are heteromeric complexes with calmodulin, which is constitutively associated with the alpha-subunits in a calcium- independent manner. Our data support a model in which calcium gating of SK channels is mediated by binding of calcium to calmodulin and subsequent conformational alterations in the channel protein.

•Zerrouk, H., Mansuelle, P., Benslimane, A., Rochat, H. & Martin-Eauclaire, M.F. (1993). Characterization of a new leiurotoxin I-like scorpion toxin PO5 from Androctonus mauretanicus mauretanicus. FEBS Letters 320: 189-192. Three novel peptide inhibitors of the SKCa channels were purified to homogeneity from the venom of the scorpion Androctonus mauretanicus mauretanicus using one step of RP-HPLC and competition assays with [125I]apamin to rat brain synaptosomes. PO1, PO2 and PO5 have K0.5 of 100, 100 and 0.02 nM, respectively, for the apamin binding site. The sequence of PO5 was established and compared to that of other scorpion toxins active on K+ channels: it contains 31 residues and has a free carboxyl end. it shares sequence similarity with apamin and leiurotoxin I.

•Zhang, B.M., Kohli, V., Adachi, R., Lopez, J.A., Udden, M.M. & Sullivan, R. (2001). Calmodulin binding to the C-terminus of the small-conductance Ca2+- activated K+ channel hSK1 is affected by alternative splicing. Biochemistry 40(10): 3189-3195. We identified three splice variants of hSK1 whose C-terminal structures are determined by the independent deletion of two contiguous nucleotide sequences. The upstream sequence extends 25 bases in length, is initiated by a donor splice site within exon 8, and terminates at the end of the exon. The downstream sequence

Page 139 AHP, BK- and SK-channel references consists of nine bases that compose exon 9. When the upstream sequence (hSK1(- )(25b)) or both sequences (hSK1(-)(34b)) are deleted, truncated proteins are encoded in which the terminal 118 amino acids are absent. The binding of calmodulin to these variants is diminished, particularly in the absence of Ca2+ ions. The first 20 amino acids of the segment deleted from hSK1(-)(25b) and hSK1(-)(34b) contain a 1-8-14 Ca2+ calmodulin binding motif, and synthetic oligopeptides based on this region bind calmodulin better in the presence than absence of Ca2+ ions. When the downstream sequence (hSK1(-)(9b)) alone is deleted, only the three amino acids A452, Q453, and K454 are removed, and calmodulin binding is not reduced. On the basis of the relative abundance of mRNA encoding each of the four isoforms, the full-length variant appears to account for most hSK1 in the human hippocampus, while hSK1(- )(34b) predominates in reticulocytes, and hSK1(-)(9b) is especially abundant in human erythroleukemia cells in culture. We conclude that the binding of calmodulin by hSK1 can be modulated through alternative splicing.

BASAL GANGLIA

•Baranauskas, G., Tkatch, T. & Surmeier, D.J. (2000). BDS-I toxin reduces fast AHP in central fast spiking neurons. Society for Neuroscience Abstracts 30. Recently it was suggested that the fast delayed rectifier current encoded by Kv3.1/Kv3.2 genes could be responsible for spike repolarization in central fast spiking neurons. Nevertheless, the lack of specific channels blockers have not allowed direct testing of the hypothesis. BDS-I and BDS-II toxins were introduced as selective blockers of a high threshold A current encoded by Kv3.4 gene. Here we show that BDS-I toxin produces a voltage dependent block of the fast delayed rectifier current in central neurons. We studied three types of fast spiking neurons: globus pallidus (GP) and subthalamic nucleus (STN) cells and hippocampal interneurons. Potassium currents were recorded in acutely isolated neurons from 4- 6 weeks old rats. Under voltage clamp conditions BDS-I (2 mM) produced a suppression of the rapidly deactivating current but the block was rapidly relieved (t 20 msec at +20 mV) in all cells tested. This block could not be attributed to the presence of rapidly inactivating homomeric Kv3.4 channels since 80 msec long pre- pulse to +30 mV did not suppress a rapidly inactivating current We conclude, that BDS produces a voltage dependent block of the fast delayed rectifier current encoded mainly by Kv3.1 gene. We used BDS-I to test the contribution of the fast delayed rectifier current to spike repolarization and fAHP. In some hippocampal

Page 140 AHP, BK- and SK-channel references interneurons in the presence of Ca +2 there was very small or no BDS-I effect on fAHP. In all GP and STN neurons tested BDS-I produced spike broadening and reduced fAHP amplitude both in Ca+2 free solution and in the presence of 2 mM of Ca+2 . Supported by: NIH NINDS Grants NS 26473 and 34696

•Bennett, B.D., Callaway, J.C. & Wilson, C.J. (2001). Intrinsic membrane properties underlying spontaneous tonic firing in neostriatal cholinergic interneurons. Journal of Neuroscience 20(22): 8493-8503. Neostriatal cholinergic interneurons produce spontaneous tonic firing in the absence of synaptic input. Perforated patch recording and whole-cell recording combined with calcium imaging were used in vitro to identify the intrinsic membrane properties underlying endogenous excitability. Spontaneous firing was driven by the combined action of a sodium current and the hyperpolarization-activated cation current (I(h)), which together ensured that there was no zero current point in the subthreshold voltage range. Blockade of sodium channels or I(h) established a stable subthreshold resting membrane potential. A tetrodotoxin-sensitive region of negative slope conductance was observed between approximately -60 mV and threshold (approximately -50 mV) and the h-current was activated at all subthreshold voltages. Calcium imaging experiments revealed that there was minimal calcium influx at subthreshold membrane potentials but that action potentials produced elevations of calcium in both the soma and dendrites. Spike-triggered calcium entry shaped the falling phase of the action potential waveform and activated calcium-dependent potassium channels. Blockade of big-conductance channels caused spike broadening. Application of apamin, which blocks small- conductance channels, abolished the slow spike afterhyperpolarization (AHP) and caused a transition to burst firing. In the absence of synaptic input, a range of tonic firing patterns are observed, suggesting that the characteristic spike sequences described for tonically active cholinergic neurons (TANs) recorded in vivo are intrinsic in origin. The pivotal role of the AHP in regulating spike patterning indicates that burst firing of TANs in vivo could arise from direct or indirect modulation of the AHP without requiring phasic synaptic input.

•Bevan, M.D. & Wilson, C.J. (1999). Mechanisms underlying spontaneous oscillation and rhythmic firing in rat subthalamic neurons. Journal of Neuroscience 19: 7617-7628.

Page 141 AHP, BK- and SK-channel references

Subthalamic neurons drive basal ganglia output neurons in resting animals and relay cortical and thalamic activity to the same output neurons during movement. The first objective of this study was to determine the mechanisms underlying the spontaneous activity of subthalamic neurons in vitro and to gain insight into their resting discharge in vivo. The second objective was to determine the response of subthalamic neurons to depolarizing current injection and how intrinsic properties may shape their response to cortical and thalamic inputs during movement. Cell- attached and whole-cell recordings were made from subthalamic neurons in brain slices prepared from 3- to 4-week-old rats. The slow, rhythmic discharge of subthalamic neurons was resistant to blockade of excitatory synaptic transmission indicating that intrinsic currents underlie their spontaneous discharge. A persistent sodium current was the source of current during the depolarizing phase of the oscillation. A powerful afterhyperpolarization following each action potential was sufficient to terminate the depolarization. A long duration component of the spike afterhyperpolarization determined the period of the oscillation and was generated by an apamin-sensitive calcium-activated potassium current. Calcium entry responsible for that current was associated with action potentials. Subthalamic neurons exhibited a sigmoidal frequency-current relationship with the steeper portion starting at approximately 30-40 Hz. This property makes subthalamic neurons more sensitive to input at high firing rates associated with movement than at low rates associated with rest. We propose that the subthreshold persistent sodium current overcomes calcium activated potassium current which accumulates during high frequency firing and underlies the enhanced sensitivity to current >30 Hz.

THALAMUS

•Goaillard, J.-M., Legendre, P. & Vincent, P. (2000). Serotonin decreases the slow afterhyperpolarization in intralaminar thalamic neurones via 5-HT7 receptors activation: A study using cAMP imaging and electrophysiological recordings. Society for Neuroscience Abstracts 30. The 5-HT7 receptor is highly expressed in intralaminar thalamic nuclei but the functional role of this receptor in this region is unknown. This receptor is able to activate neural-specific types of AC in vitro (for review, P.Vanhoenacker et al. (2000), TIPS Vol 21(2), 70-77), however the molecular processes following activation of this receptor have not been studied in neurones in brain slice

Page 142 AHP, BK- and SK-channel references preparations. cAMP imaging on intralaminar thalamic slices showed that 5-HT (10 µM) induced an increase in cAMP which was not further increased by forskolin. Using patch-clamp recordings (V-Clamp and I-Clamp), we demonstrated that 5-HT at this concentration also induced a full inhibition of slow calcium-dependent potassium conductances (sKCa) responsible for spike discharge accomodation and afterhyperpolarization following a spike train. This latter effect was reproduced by intracellular injection of 8Br-cAMP (20 µM), showing that the electrophysiological effect of 5-HT was a direct consequence of the cAMP increase. Specific activation of 5-HT7 receptors by 5-CT (100 nM, 5-HT7 and 5-HT1A agonist) in the presence of pindolol (10 µM, 5-HT1A antagonist) also induced a full inhibition of sKCa. This study demonstrate that 5-HT7 receptor activation in situ can be implicated in the regulation of neuronal excitability by 5-HT.

•Goaillard, J.-M. & Vincent, P. (2002). Serotonin suppresses the slow afterhyperpolarization in rat intralaminar and midline thalamic neurones by activating 5-HT7 receptors. Journal of Physiology (London) 541(Pt. 2): 453-465. While the highest expression level of 5-HT(7) receptors in the brain is observed in intralaminar and midline thalamic neurones, the physiological role of these receptors in this structure is unknown. In vivo recordings have shown that stimulation of the serotonergic raphe nuclei can alter the response of these neurones to a nociceptive stimulus, suggesting that serotonin modulates their firing properties. Using the patch-clamp technique in rat thalamic brain slices, we demonstrate that activation of 5-HT(7) receptors can strongly modulate the excitability of intralaminar and midline thalamic neurones by inhibiting the calcium- activated potassium conductance that is responsible for the slow afterhyperpolarization (sAHP) following a spike discharge. This sAHP was inhibited after activation of the cAMP pathway, either by bath application of forskolin or intracellular perfusion with 8-bromo-cAMP. The inhibitory effect of 5-HT(7) receptors on sAHPs was blocked by the protein kinase A antagonist R(P)-cAMPS. Calcium-imaging experiments showed no change in intracellular calcium levels during the 5-HT(7) response, indicating that in these neurones, a global calcium signal was not necessary to activate the cAMP cascade. Finally, bath application of serotonin produced a strong increase in cytosolic cAMP concentration, as measured using the fluorescent probe FlCRhR, and an inhibition of the sAHP. Taken together, these results suggest that 5-HT(7) receptors are implicated in the effect of 5-HT on sAHP in intralaminar and midline thalamic neurones, an effect that is mediated by

Page 143 AHP, BK- and SK-channel references the cAMP second-messenger cascade.

MIDBRAIN AND BRAINSTEM

•Butcher, J.W., Kasparov, S. & Paton, J.F. (1999). Differential effects of apamin on neuronal excitability in the nucleus tractus solitarii of rats studied in vitro. Journal of the Autonomic Nervous System 77(2-3): 90-97. We demonstrated previously that microinjection of the calcium-dependent potassium channel antagonist, apamin, into the nucleus tractus solitarius (NTS) in vivo potentiated the baroreceptor reflex mediated bradycardia but attenuated the cardiopulmonary reflex. The latter result was surprising since, intuitively, potassium channel blockade would be expected to increase neuronal excitability leading to reflex potentiation. The aim of this in vitro study was to investigate possible neuronal mechanisms to explain our in vivo observations. Transverse brainstem slices of rat were cut at the level of area postrema and recordings were made from 36 NTS neurones in whole-cell mode. The neurones were classified into three groups, based on their response to apamin (10 nM). Each group had a similar resting membrane potential (RMP; -55 ± 1 mV; n = 36) and input resistance (404 ± 20 MΩ; n = 36). (1) In 15/36 neurones, apamin decreased the number of spikes evoked during injection of positive current by 37 ± 6%; this was associated with a concomitant fall in input resistance of 12 ± 2% (P < 0.05). Stimulation of the ipsilateral tractus solitarius evoked EPSP-IPSP complexes in nine of the 12 neurones tested; the inhibitory components were increased in amplitude, at a holding potential of -46 mV, from -1.7 ± 0.4 to -3.2 ± 0.6 mV (P < 0.01) in the presence of apamin, while the EPSPs were unaffected. All three of these effects were bicuculline (10 microM) sensitive. (2) In 8/36 neurones, apamin increased the number of spikes evoked during injection of positive current by 27 ± 8%, but affected neither RMP nor input resistance. Only one of five neurones tested demonstrated a synaptically evoked EPSP-IPSP complex. The remaining four neurones displayed a single evoked EPSP, the amplitudes of which were unaffected by apamin. (3) In the remaining neurones (13/36), apamin affected neither responsiveness to positive current injection, RMP, nor input resistance. Six of 12 neurones demonstrated synaptically evoked EPSP-IPSP complexes; at a holding potential of -46 mV, apamin increased the IPSP component from -2.6 ± 1 to -3.6 ± 0.8 mV (P < 0.05), while the EPSPs were unaffected. In conclusion, apamin can both increase and decrease NTS neuronal excitability: the former reflecting blockade of channels on the recorded

Page 144 AHP, BK- and SK-channel references neurone; the latter may possibly result from an increase in GABA release by interneurones impinging onto the recorded neurone. The possibility of a differential distribution of apamin-sensitive channels in sub-populations of NTS neurones subserving different reflexes is discussed.

•Carletti, R., Terstappen, G.C., Roncarati, R., Bunnemann, B., Merlo Pich, E. & Tacconi, S. (2001). Localization of small conductance calcium activated potassium channels (SK3) immunoreactivity in dopaminergic, noradrenergic, and serotonergic nuclei of the rat brain. Society for Neuroscience Abstracts 31(382.2): 196. Previous in situ hybridization and immunohistochemical studies have demonstrated the distinct expression pattern of SK3 channels in a restricted number of brain structures. Since some of these, such as dorsal raphe (DR), locus coeruleus (LC) and substantia nigra (SN)/ventral tegmental area (VTA) are of importance for serotonergic, noradrenergic and dopaminergic neurotransmission, double labelling immunohistochemistry experiments of SK3 and serotonin (5-HT), or SK3 and (TH), have been performed on rat brain sections to further define channel localization. Animals were intracardially perfused with 4% paraformaldehyde and 30m free floating brain sections were incubated with either polyclonal anti-SK3, anti-5HT or monoclonal anti-TH primary antibodies followed by visualization with fluorescent secondary antibodies. In accordance with previous results, SK3 immunoreactivity was observed in habenula, septum, DR, LC, SN and VTA. By means of confocal microscopy co-existence of SK3 and TH could be observed at the cellular level in SN and VTA, where immunoreactivity was localized mainly in neuronal cell body. In DR co-existence of SK3 and 5HT could be demonstrated in the dendritic field proximal to 5HT containing neurons. A similar pattern was also observed in LC where immunoreactivity was confined in the dendritic field close to the TH positive cell bodies. Our results pose the morphological basis for further studies on the role of SK3 channels in monoaminergic transmission.

•Johnson, S.W. & Seutin, V. (1997). Bicuculline methiodide potentiates NMDA- dependent burst firing in rat dopamine neurons by blocking apamin-sensitive Ca2+- activated K+ currents. Neuroscience Letters 231: 13-16. Apamin, a bee venom toxin which blocks a Ca2+-dependent K+ current, potentiates N-methyl-D-aspartate (NMDA)-induced burst firing in dopamine

Page 145 AHP, BK- and SK-channel references neurons. We now report that burst firing is also potentiated by an apamin-like effect of bicuculline methiodide (BMI) at the same concentration (30 microM) which blocks GABA(A) receptors in vitro. Using microelectrodes to record intracellularly from rat dopamine neurons in the midbrain slice, BMI reduced the apamin-sensitive afterhyperpolarization in all cells tested. BMI also mimicked apamin (100 nM) by potentiating burst firing produced by a concentration of NMDA (10 µM) which is too low to evoke burst firing when perfused alone. When recording under voltage-clamp, both BMI and apamin reduced a depolarization-activated outward current which was also sensitive to perfusate containing no-added Ca2+. Although (100 µM) and bicuculline free base (30 µM) blocked the inhibition of firing produced by the GABA(A) agonist isoguvacine (100 µM), neither had apamin-like effects. We conclude that BMI potentiates burst firing by blocking an apamin-sensitive Ca2+-activated K+ current.

•Kalume, F.K. & Callaway, J.C. (2001). Local dendritic tetrodotoxin application to nigral dopaminergic neurons decreases AHP amplitude and alters firing pattern. Society for Neuroscience Abstracts 31(827.13). In slice preparations, dopaminergic (DA) neurons fire in a regular pacemaker rhythm, but in vivo they also exhibit irregular and burst firing modes. The pacemaker is partially supported by subthreshold calcium-dependent membrane oscillations in both the soma and dendrites. Previous studies demonstrated that in the absence of spikes, smaller diameter distal processes oscillate faster than larger ones and theoretically can become asynchronous with the soma. During pacemaker firing, Na+ spikes actively propagate into the dendritic arbor adding to the Ca2+ transients that subsequently activate a Ca2+-dependent afterhyperpolarization (AHP). We used antisera to SK3 CaK channels and labeled both the soma and dendrites supporting a somato-dendritic localization of gKCa. Thus, dendritic Ca2+ entry likely contributes to the amplitude and duration of the AHP. We hypothesize that spike backpropagation helps to keep the oscillations synchronized and maintain regular pacemaker firing by synchronizing the dendritic Ca2+-dependent AHP. To test this hypothesis, Ca2+ imaging and patch clamp recording was performed on dopamine cells from brain slices of 13-18 d.o. rats. We disrupted dendritic spike propagation by focally applying tetrodotoxin (TTX) onto individual primary dendrites. Application of TTX reduced the amplitude of AHPs by as much as 20-40% without reducing the somatically recorded spike amplitude or increasing spike width.

Page 146 AHP, BK- and SK-channel references

TTX application often slightly increased somatic spike amplitude and very slightly decreased spike width. When TTX application reduced AHP amplitude regular pacemaker firing often became irregular and occasionally dopamine cells exhibited bursty behavior. Supported by: NS36843

•Morikawa, H. & Williams, J.T. (2001). IP3-independent release of calcium mediated by mGluR in midbrain dopamine neurons. Society for Neuroscience Abstracts 31(41.3): 20. Rapid activation of mGluR in midbrain dopamine neurons induces a transient outward current mediated by small conductance Ca2+-activated K+ channels (SK channels). Previous work has shown that photolytic release of IP3 in these neurons elicits mobilization of Ca2+ from intracellular stores and subsequent activation of SK channels, consistent with the involvement of IP3 in the mGluR-mediated outward current. Here, it is shown that synaptic release of gluamate or iontophoretic application of aspartate caused a wave-like increase in [Ca2+]i, which was sensitive to MCPG (1 mM), an mGluR antagonist, and blocked by cyclopiazonic acid (10 µM), a blocker of endoplasmic reticlulum Ca2+-ATPase. The aspartate-induced outward current was not affected by intracellular dialysis with heparin (1 mg/ml), a blocker of IP3 receptors, whereas it completely eliminated the IP3-evoked outward current. Furthermore, treatment with U73122, a phospholipase C inhibitor (10 µM), had no significant effect on the aspartate-induced current, while the cross-desensitization of the aspartate-induced current by phenylephrine, an 1 adrenergic receptor agonist, was significantly attentuated. When ryanodine (10 µM) was perfused, aspartate still evoked a substantial current at a time when IP3-evoked current was completely abolished, suggesting the involvement of an IP3- and ryanodine-insensitive store in the mGluR-mediated Ca2+ release. Alternative second messenger pathways are currently under investigation. Supported by: NIDA 04523

•Ping, H.X. & Shepard, P.D. (1996). Apamin-sensitive Ca2+-activated K+ channels regulate pacemaker activity in nigral dopamine neurons. Neuroreport 7: 809-814.

•Ping, H.X. & Shepard, P.D. (1999). Blockade of SK-type Ca2+-activated K+ channels uncovers a Ca2+-dependent slow afterdepolarization in nigral dopamine neurons. J Neurophysiol 81(3): 977-984.

Page 147 AHP, BK- and SK-channel references

Sharp electrode current-clamp recording techniques were used to characterize the response of nigral dopamine (DA)-containing neurons in rat brain slices to injected current pulses applied in the presence of TTX (2 microM) and under conditions in which apamin-sensitive Ca2+-activated K+ channels were blocked. Addition of apamin (100-300 nM) to perfusion solutions containing TTX blocked the pacemaker oscillation in membrane voltage evoked by depolarizing current pulses and revealed an afterdepolarization (ADP) that appeared as a shoulder on the falling phase of the voltage response. ADP were preceded by a ramp-shaped slow depolarization and followed by an apamin-insensitive hyperpolarizing afterpotential (HAP). Although ADPs were observed in all apamin-treated cells, the duration of the response varied considerably between individual neurons and was strongly potentiated by the addition of TEA (2-3 mM). In the presence of TTX, TEA, and apamin, optimal stimulus parameters (0.1 nA, 200-ms duration at -55 to -68 mV) evoked ADP ranging from 80 to 1,020 ms in duration (355.3 ± 56.5 ms, n = 16). Both the ramp-shaped slow depolarization and the ensuing ADP were markedly voltage dependent but appeared to be mediated by separate conductance mechanisms. Thus, although bath application of nifedipine (10-30 µM) or low Ca2+, high Mg2+ Ringer blocked the ADP without affecting the ramp potential, equimolar substitution of Co2+ for Ca2+ blocked both components of the voltage response. Nominal Ca2+ Ringer containing Co2+ also blocked the HAP evoked between -55 and -68 mV. We conclude that the ADP elicited in DA neurons after blockade of apamin-sensitive Ca2+-activated K+ channels is mediated by a voltage-dependent, L-type Ca2+ channel and represents a transient form of the regenerative plateau oscillation in membrane potential previously shown to underlie apamin-induced bursting activity. These data provide further support for the notion that modulation of apamin-sensitive Ca2+- activated K+ channels in DA neurons exerts a permissive effect on the conductances that are involved in the expression of phasic activity.

•Scroggs, R.S., Cardenas, C.G., Whittaker, J.A. & Kitai, S.T. (2001). Muscarine reduces calcium-dependent electrical activity in substantia nigra dopaminergic neurons. Journal of Neurophysiology 86(6): 2966-2972. The effect of muscarine on Ca2+ dependent electrical activity was studied in dopamine (DA) neurons located in the substantia nigra pars compacta (SNc) in brain slices from young rats, using sharp electrodes. In most DA neurons tested, muscarine (50 µM) reduced the amplitude of spontaneous oscillatory potentials and evoked Ca2+-dependent potentials recorded in the presence of TTX. Muscarine also

Page 148 AHP, BK- and SK-channel references reduced the amplitude of the slow afterhyperpolarization (sAHP) following action potentials in most DA neurons. These data suggest that muscarine reduces Ca2+ entry in SNc DA neurons. The reduction of the amplitude of the sAHP by muscarine in DA neurons may facilitate bursting initiated by glutamatergic input by increasing the frequency at which DA neurons can fire. The reduction of the sAHP via activation of muscarinic receptors in vivo may provide a mechanism whereby cholinergic inputs to DA neurons from the tegmental peduncular pontine nucleus could modulate dopamine release at dopaminergic targets in the brain.

•Seutin, V., Johnson, S.W. & North, R.A. (1993). Apamin increases NMDA- induced burst-firing of rat mesencephalic dopamine neuron. Brain Research 630(1-2): 341-344. Intracellular recordings made in vitro from rat midbrain dopamine neurons showed that apamin (100 nM) did not alter the regular spontaneous firing of the neurons, but it increased the occurrence of bursts of action potentials in N-methyl- D-aspartate. Apamin appeared to facilitate burst-firing induced by NMDA because, by blocking an outward calcium-activated potassium current, it increased the depolarizing action of NMDA.

•Shepard, P.D. & Stump, D. (1999). Nifedipine blocks apamin-induced bursting activity in nigral dopamine-containing neurons. Brain Research 817(1-2): 104-109. Intrinsic sinusoidal oscillations in membrane potential, characteristic of nigral dopamine cells, are converted to plateau potentials following application of apamin, a potent antagonist of SK-type Ca2+-activated K+ channels. Blockade of these channels also changes neuronal firing pattern from a single-spike pacemaker discharge to a multiple spike bursting pattern. Nifedipine, a selective antagonist of L-type Ca2+ channels, blocks plateau potential generation; however, its effects on firing pattern have yet to be determined. In the present study, extracellular single unit recording techniques were used in conjunction with a brain slice preparation to determine whether nifedipine, in a concentration known to block plateau potential generation, also affects bursting activity. Nifedipine (30 µM) was equipotent in inhibiting the firing rate of control (51.2±10.8%) and apamin-treated (44.9±5.4%) neurons. Slow firing neurons (<2 Hz) were particularly sensitive to the inhibitory effects of the drug. Apamin-induced bursting was completely suppressed by nifedipine and accompanied by a significant increase in the regularity of firing. By contrast, pacemaker-like activity exhibited by control neurons was unaffected by

Page 149 AHP, BK- and SK-channel references the drug. These data demonstrate that the intrinsic plateau properties exhibited by DA neurons are responsible for the generation of phasic activity induced following blockade of apamin-sensitive Ca2+-activated K+ channels and provide further support for the involvement of an L-type Ca2+ conductance in mediating this type of activity.

•Tacconi, S., Carletti, R., Bunnemann, B., Plumpton, C., Merlo Pich, E. & Terstappen, G.C. (2001). Distribution of the messenger RNA for the small conductance calcium-activated potassium channel SK3 in the adult rat brain and correlation with immunoreactivity. Neuroscience 102(1): 209-215. Small conductance calcium-activated potassium channels are voltage independent potassium channels which modulate the firing patterns of neurons by activating the slow component of the afterhyperpolarization. The genes encoding a family of small conductance calcium-activated potassium channels have been cloned and up to now three known members have been described and named small conductance calcium-activated potassium channel type 1, small conductance calcium-activated potassium channel type 2 and small conductance calcium-activated potassium channel type 3; the distribution of their messenger RNA in the rat CNS has already been performed but only in a limited detail. The present study represents the first detailed analysis of small conductance calcium-activated potassium channel type 3 mRNA distribution in the adult rat brain and resulted in a strong to moderate expression of signal in medial habenular nucleus, substantia nigra compact part, suprachiasmatic nucleus, ventral tegmental area, lateral septum, dorsal raphe and locus coeruleus. Immunohistological experiments were also performed and confirmed the presence of small conductance calcium-activated potassium channel type 3 protein in medial habenular nucleus, locus coeruleus and dorsal raphe. Given the importance of dorsal raphe, locus coeruleus and substantia nigra/ventral tegmental area for serotonergic, noradrenergic and dopaminergic transmission respectively, our results pose the morphological basis for further studies on the action of small conductance calcium-activated potassium channel type 3 in serotonergic, noradrenergic and dopaminergic transmission.

•Wolfart, J., Neuhoff, H. & Franz, O.J. (2001). Differential expression of the small-conductance calcium-activated potassium channel SK3 is critical for pacemaker control in dopaminergic midbrain neurons. Journal of Neuroscience 21: 3443-3456.

Page 150 AHP, BK- and SK-channel references

The physiological activity of dopaminergic midbrain (DA) neurons is important for movement, cognition, and reward. Altered activity of DA neurons is a key finding in schizophrenia, but the cellular mechanisms have not been identified. Recently, KCNN3, a gene that encodes a member (SK3) of the small-conductance, calcium- activated potassium (SK) channels, has been proposed as a candidate gene for schizophrenia. However, the functional role of SK3 channels in DA neurons is unclear. We combined patch-clamp recordings with single-cell RT-PCR and confocal immunohistochemistry in mouse midbrain slices to study the function of molecularly defined SK channels in DA neurons. Biophysical and pharmacological analysis, single- cell mRNA, and protein expression profiling strongly suggest that SK3 channels mediate the calcium-dependent afterhyperpolarization in DA neurons. Perforated patch recordings of DA neurons in the substantia nigra (SN) demonstrated that SK3 channels dynamically control the frequency of spontaneous firing. In addition, SK3 channel activity was essential to maintain the high precision of the intrinsic pacemaker of DA SN neurons. In contrast, in the ventral tegmental area, DA neurons displayed significantly smaller SK currents and lower SK3 protein expression. In these DA neurons, SK3 channels were not involved in pacemaker control. Accordingly, they discharged in a more irregular manner compared with DA SN neurons. Thus, our study shows that differential SK3 channel expression is a critical molecular mechanism in DA neurons to control neuronal activity. This provides a cellular framework to understand the functional consequences of altered SK3 expression, a candidate disease mechanism for schizophrenia.

•Wolfart, J. & Roeper, J. (2002). Selective coupling of T-type calcium channels to SK potassium channels prevents intrinsic bursting in dopaminergic midbrain neurons. Journal of Neuroscience 22(9): 3404-3413. Dopaminergic midbrain (DA) neurons display two principal activity patterns in vivo, single-spike and burst firing, the latter coding for reward-related events. We have shown recently that the small-conductance calcium-activated potassium channel SK3 controls pacemaker frequency and precision in DA neurons of the substantia nigra (SN), and previous studies have implicated SK channels in the transition to burst firing. To identify the upstream calcium sources for SK channel activation in DA SN neurons, we studied the sensitivity of SK channel-mediated afterhyperpolarization (AHP) currents to inhibitors of different types of voltage- gated calcium channels in perforated patch-clamp recordings. Cobalt-sensitive AHP currents were not affected by L-type and P/Q-type calcium channel inhibitors and

Page 151 AHP, BK- and SK-channel references were reduced slightly (26%) by the N-type channel inhibitor omega-conotoxin-GVIA. In contrast, AHP currents were blocked substantially (85-94%) by micromolar concentrations of nickel (IC50, 33.75 microm) and (IC50, 4.83 microm), indistinguishable from the nickel and mibefradil sensitivities of T-type calcium currents (IC50 values, 33.86 and 4.59 microm, respectively). These results indicate that SK channels are activated selectively via T-type calcium channels in DA SN neurons. Consequently, SK currents displayed use-dependent inactivation with similar time constants when compared with those of T-type calcium currents and generated a transient rebound inhibition. Both SK and T-type channels were essential for the stability of spontaneous pacemaker activity, and, in some DA SN neurons, T-type channel inhibition was sufficient to induce intrinsic burst firing. The functional coupling of SK to T-type channels has important implications for the temporal integration of synaptic input and might help to understand how DA neurons switch between pacemaker and burst-firing modes in vivo.

CEREBELLUM

•Cingolani, L.A., Gymnopoulos, M., Boccaccio, A., Stocker, M. & Pedarzani, P. (2002). Developmental regulation of small-conductance Ca2+-activated K+ channel expression and function in rat Purkinje neurons. Journal of Neuroscience 22(11): 4456-4467. Calcium transients play an important role in the early and later phases of differentiation and maturation of single neurons and neuronal networks. Small- conductance calcium-activated potassium channels of the SK type modulate membrane excitability and are important determinants of the firing properties of central neurons. Increases in the intracellular calcium concentration activate SK channels, leading to a hyperpolarization of the membrane potential, which in turn reduces the calcium inflow into the cell. This feedback mechanism is ideally suited to regulate the spatiotemporal occurrence of calcium transients. However, the role of SK channels in neuronal development has not been addressed so far. We have concentrated on the ontogenesis and function of SK channels in the developing rat cerebellum, focusing particularly on Purkinje neurons. Electrophysiological recordings combined with specific pharmacological tools have revealed for the first time the presence of an afterhyperpolarizing current (I(AHP)) in immature Purkinje cells in rat cerebellar slices. The channel subunits underlying this current were identified as SK2 and localized by in situ hybridization and subunit-specific

Page 152 AHP, BK- and SK-channel references antibodies. Their expression level was shown to be high at birth and subsequently to decline during the first 3 weeks of postnatal life, both at the mRNA and protein levels. This developmental regulation was tightly correlated with the expression of I(AHP) and the prominent role of SK2 channels in shaping the spontaneous firing pattern in young, but not in adult, Purkinje neurons. These results provide the first evidence of the developmental regulation and function of SK channels in central neurons.

•Czubayko, U., Sultan, F., Thier, P. & Schwarz, C. (2001). Two types of neurons in the rat cerebellar nuclei as distinguished by membrane potentials and intracellular fillings. Journal of Neurophysiology 85(5): 2017-2029. Classically, three classes of neurons in the cerebellar nuclei (CN), defined by different projection targets and content of transmitters, have been distinguished. However, evidence for different types of neurons based on different intrinsic properties is lacking. The present study reports two types of neurons defined mainly by their intrinsic properties, as determined by whole-cell patch recordings. The majority of cells (type I, n = 63) showed cyclic burst firing whereas a small subset (type II, n = 7) did not. Burst firing was used to distinguish the two types of neurons because, as it turned out, pharmacological interference could not be used to convert the non-bursting cells to bursting ones. Some of the membrane potentials exclusively present in type I neurons, such as sodium and calcium plateau potentials, low-threshold calcium spikes, and a slow calcium-dependent afterhyperpolarization, were found to contribute to the generation of burst firing. Other membrane potentials of type I neurons were not obviously related to the generation of bursts. These were 1) the lower amplitude and width of the action potential during spontaneous activity, 2) a sequence of afterhyperpolarization-afterdepolarization- afterhyperpolarization following each spike, and 3) the high spontaneous firing rate. In contrast, type II neurons lacked slow plateau potentials and low threshold spikes. Their action potentials showed higher amplitude and width and were followed by a single deep afterhyperpolarization. Furthermore, they showed a lower firing rate at rest. In both types of neurons, a delayed inward rectification was present. Neurons filled with neurobiotin revealed that the sizes of the somata and dendritic fields of type I neurons comprised the whole range known from Golgi studies, whereas those of the few type II neurons recovered were found to be in the lowest range. In view of their size and scarcity, we propose that type II neurons may correspond to CN interneurons.

Page 153 AHP, BK- and SK-channel references

•Edgerton, J.R. & Reinhart, P.H. (2001). Complimentary contributions of small- conductance and large-conductance Ca2+-activated K+ channels to Purkinje cell firing properties. Society for Neuroscience Abstracts 31(382.4): 196. Purkinje neurons fire bursts of action potentials that constitute the sole output of the cerebellar cortex. Their activity is in part determined by intrinsic conductances such as Ca2+ and Ca2+-activated K+ (KCa) conductances. To determine the contributions made by KCa channels to Purkinje cell electrical function, we monitored the activity of Purkinje neurons in rat cerebellar slices in the presence or absence of channel blockers. We find that KCa channels contribute to three aspects of cell firing properties: the sodium spike fast AHP, the AHP following dendritic calcium spikes, and the firing frequency. Furthermore, we find that BK and SK channels play complimentary roles. The BK channel blocker iberiotoxin (IbTX) reduces the sodium spike fast AHP by 38 15% without affecting either the calcium spike AHP or the spontaneous firing rate. The SK channel blocker apamin increases the firing rate from 34±4 Hz to 172±59 Hz, but does not directly affect either the sodium spike fast AHP or the calcium spike AHP. When applied together, IbTX and apamin cause a 99 31% reduction in the sodium spike fast AHP amplitude, increase the firing rate from 28±6 Hz to 408±70 Hz, and also reduce the amplitude and duration of the calcium spike AHP. We conclude that in Purkinje neurons, BK channels activate during the falling phase of each action potential and contribute to the fast AHP, while SK channels have a more tonic activity profile that stabilizes the firing rate. Furthermore the combined effects of BK and SK channel blockers are much greater than the sum of the effects of the individual blockers. Supported by: NIH grant NS41866 to PHR and NIH grant NS41697 to JRE

HYPOTHALAMUS

•Bosch, M.A., Kelly, M.J. & Ronnekleiv, O.K. (2002). Distribution, neuronal colocalization, and 17beta-E2 modulation of small conductance calcium-activated K+ channel (SK3) mRNA in the guinea pig brain. Endocrinology 143(3): 1097-1107. Molecular cloning has revealed the existence of three distinct small conductance (SK1-3) Ca2+-activated K(+) channels. Because SK channels underlie the afterhyperpolarization (AHP) that is critical for sculpturing phasic firing in hypothalamic neurons, we investigated the distribution of these channels in the female guinea pig. Both SK1 and SK3 cDNA fragments were cloned using PCR, and

Page 154 AHP, BK- and SK-channel references ribonuclease protection assay as well as in situ hybridization analysis illustrated that the SK3 channel was the predominant subtype expressed in the guinea pig hypothalamus. Combined in situ hybridization and fluorescence immunocytochemistry revealed that SK3 mRNA was expressed in GnRH, dopamine, and vasopressin neurons, and all of these neurons exhibited an AHP current. Moreover, SK3 mRNA was found in other brain areas, including the septum, bed nucleus, amygdala, thalamus, midbrain, and hippocampus. Using quantitative ribonuclease protection assay, the rank order of SK3 mRNA expression was septum >or= midbrain > rostral thalamus >or= rostral basal hypothalamus >or= caudal thalamus >or= preoptic area >> caudal basal hypothalamus >or= hippocampus. Moreover, 17beta-E2 treatment, which reduces plasma LH during the negative feedback phase, significantly increased SK3 mRNA levels in the rostral basal hypothalamus (P < 0.05; n = 6). Therefore, these findings suggest that estrogen increases the mRNA expression of SK3 channels, which may represent a mechanism by which estrogen regulates hypothalamic neuronal excitability during negative feedback.

•Ghamari-Langroudi, M. & Bourque, C.W. (2001). Muscarinic excitation of magnocellular neurosecretory cells (MNC) of supraoptic nucleus (SON). Society for Neuroscience Abstracts 31(179.4). Previous studies have indicated that MNCs express a TEA-sensitive K conductance with activation properties resembling that of the M-current (Stern and Armstrong J Physiol 1995, 1997). We therefore examined the effects of muscarine on MNCs impaled using sharp electrodes in rat hypothalamic explants. Bath application of 40-100 M (+)-muscarine Cl caused a reversible depolarization and excitation of 12 of 14 MNCs initially current clamped at a voltage near -60 mV. Interestingly, muscarine also inhibited the slow (apamin-resistant) AHP evoked by long (1-2 s) trains of action potentials (n=6). These effects could be inhibited by prior application of 5-50 M atropine in each of 7 cells tested, suggesting the involvement of muscarinic cholinergic receptors. Voltage clamp analysis revealed that MNCs express a slowly developing outward current displaying an activation threshold around -70 mV and a reversal potential near -95 mV. This current could be blocked by application of either 0.2 mM Cd2+ or 5 mM TEA. Bath application of muscarine inhibited approximately 50% of the TEA-sensitive current. At -30 mV this represented the inhibition of about 200 pA of the steady-state outward current. These results suggest that activation of muscarinic cholinergic receptors can excite MNCs in the SON and that this effect may be due to the inhibition of a

Page 155 AHP, BK- and SK-channel references slow, TEA-sensitive K+ current. The muscarine-sensitive current may also underlie part of the slow AHP.

•Kelly, M.J., Ronnekleiv, O.K., Ibrahim, N., Lagrange, A.H. & Wagner, E.J. (2002). Estrogen modulation of K(+) channel activity in hypothalamic neurons involved in the control of the reproductive axis. Steroids 67(6): 447-456. Here we report on the progress we have made in elucidating the mechanisms through which estrogen alters synaptic responses in hypothalamic neurons. We examined the modulation by estrogen of the coupling of various receptor systems to inwardly rectifying and small conductance, Ca2+-activated K(+) (SK) channels. We used intracellular sharp-electrode and whole-cell recordings in hypothalamic slices from ovariectomized female guinea pigs. Estrogen rapidly uncouples mu-opioid receptors from G protein-gated inwardly rectifying K(+) (GIRK) channels in beta- endorphin neurons, manifest by a reduction in the potency of mu-opioid receptor agonists to hyperpolarize these cells. This effect is blocked by inhibitors of protein kinase A and protein kinase C. Estrogen also uncouples gamma-aminobutyric acid (GABA)(B) receptors from the same population of GIRK channels coupled to mu- opioid receptors. At 24 h after steroid administration, the GABA(B)/GIRK channel uncoupling observed in GABAergic neurons of the preoptic area (POA) is associated with reduced agonist efficacy. Conversely, estrogen enhances the efficacy of alpha(1)-adrenergic receptor agonists to inhibit apamin-sensitive SK currents in these POA GABAergic neurons, and does so in both a rapid and sustained fashion. Finally, we observed a direct, steroid-induced hyperpolarization of both arcuate and POA neurons, among which gonadotropin-releasing hormone (GnRH) neurons are particularly sensitive. These findings indicate a richly complex yet coordinated steroid modulation of K(+) channel activity that serves to control the excitability of hypothalamic neurons involved in regulating the reproductive axis.

•Kirkpatrick, K. & Bourque, C.W. (1996). Activity dependence and functional role of the apamin-sensitive K+ current in rat supraoptic neurones in vitro. Journal of Physiology (London) 494: 389-398. 1. Intracellular recordings were obtained from seventy-two magnocellular neurosecretory cells (MNCs) in superfused explants of rat hypothalamus. The current underlying the after-hyperpolarization (IAHP) following spike-evoked trains of action potentials was characterized using the hybrid-clamp technique. The activity-dependent requirements for the genesis of the AHP were determined. The

Page 156 AHP, BK- and SK-channel references functional role of the conductance was investigated using saturating concentrations (50-300 nM) of apamin, a selective blocker of the AHP in MNCs. 2. IAHP was reversibly abolished by the removal of extracellular Ca2+. The amplitude of IAHP varied linearly as a function of voltage and reversed at -100 ± 3 mV in 3 mM external K+. Changes in the concentration of extracellular K+ resulted in shifts of the reversal potential consistent with Nernst equation predictions for a K+- selective conductance. 3. Action potentials triggered by brief depolarizing pulses elicited an AHP during trains evoked at frequencies > 1 Hz. Onset of the AHP progressed exponentially, reaching a maximum after the first fifteen to twenty impulses. The steady-state amplitude of the AHP increased logarithmically between 1 and 20 Hz. 4. Switching to voltage clamp during periods of continuous cell activity (firing rate > 4 Hz) confirmed the presence of an apamin-sensitive Ca2(+)-dependent K+ current. 5. Application of apamin produced a threefold increase in the mean firing rate of spontaneously active cells, but was without effect when applied to silent cells (firing rate < 0.5 Hz). 6. Apamin did not affect the ability of MNCs to fire in a phasic manner but caused a dramatic increase in the mean intraburst firing rate. Moreover, inhibition of IAHP by apamin strongly attenuated spike accommodation normally seen at the onset of phasic bursts. 7. While apamin did not enhance the amplitude of depolarizing after-potentials following single spikes, post-train plateau potentials and associated after-discharges were enhanced. 8. The possible consequences of IAHP modulation are discussed in the context of the regulation of firing rate and pattern in MNCs.

•Teruyama, R. & Armstrong, W.E. (2000). Changes in the electrical properties of oxytocin neurons during pregnancy and lactation. Society for Neuroscience Abstracts 30. Oxytocin (OT) neurons of the supraoptic nucleus are known to undergo a remarkable morphological and physiological adaptation in response to changes in hormonal demand which occur during lactation. An important issue is whether these changes are programmed during late pregnancy, or whether they occur as a function of suckling. Previously (Stern & Armstrong, J. Neurosci. 16:4861, 1996) we found changes in spike width and the afterhyperpolarizing potential (AHP) suggesting an increased efficiency in the ability of spikes to evoke the AHP during lactation. The present experiment was done to determine whether these changes occur first during pregnancy. Sharp electrode intracellular recordings were obtained from electrophysiologically identified OT-like neurons in hypothalamic explants from

Page 157 AHP, BK- and SK-channel references virgin (random cycling), late pregnant (E19-22), and lactating (8-12 days of lactation) rats. No changes were found in input resistance and resting potential as a function of reproductive state. Action potentials were broadest during pregnancy, whereas spike height and threshold varied little across states. A prominent AHP was observed in all cells tested after an evoked discharge of spikes (400 ms pulse,+0.05- 0.35 nA). While AHP amplitude differed little across states, there was a progressive increase in the AHP amplitude per spike during pregnancy and lactation compared to virgins, with the maximal change occurring during lactation. This change was related to the fact that fewer spikes per current pulse were evoked during lactation. In conclusion, some changes are more prominent during pregnancy and therefore may be instigated by attendant steroid hormone changes, whereas others are more fully expressed after lactation. Supported by: NIH grant NS23941 (WEA).

MOTONEURONS

•Adams, P.R., Constanti, A., Brown, D.A. & Clark, R.B. (1982). Intracellular Ca2+ activates a fast voltage-sensitive K+ current in vertebrate sympathetic neurones. Nature 296: 746-749.

•Brown, D.A., Adams, P.R. & Constanti, A. (1982). Voltage-sensitive K-currents in sympathetic neurons and their modulation by neurotransmitters. Journal of the Autonomic Nervous System 6(1): 23-35. A description is given of methods for voltage clamping and for studies of the several varieties of K-currents occurring in sympathetic neurons. Conclusions were derived chiefly from experiments conducted on bullfrog lumbar paravertebral sympathetic ganglia but observations made were compared with results obtained from studies of neurons of the rat superior cervical ganglion. Discussion is given of means used for revealing and identifying these currents and for studying actions of drugs, calcium, etc. and circumstances such as membrane state on the four quite distinct time- and voltage-dependent K-currents: IK, IC, IA and IM. The latter (IM) is under direct presynaptic control and the major focus of the paper is on this current and its role. However, considerable information is given concerning the characteristic of the other three (IK, IC and IA).

•Brown, D.A., Constanti, A. & Adams, P.R. (1983). Ca-activated potassium

Page 158 AHP, BK- and SK-channel references current in vertebrate sympathetic neurons. Review. Cell Calcium 4(5-6): 407-420. Ca-activated K-currents (IC) in sympathetic neurones have been triggered by intracellular Ca-injection or by activating ICa. IC is strongly voltage-dependent, with a peak slope of 11 mV/e-fold depolarization above -50 mV. Relaxation, fluctuation and single channel analysis suggests this to result from voltage- dependent opening and closing rates. Time-constants for channel opening and closing are about 15 msec near zero mV. Single channel conductance is about 100 pS. Currents can be blocked by TEA. IC is activated very rapidly (less than or equal to 5 msec) and sometimes transiently by a depolarizing voltage-step. It is suggested that IC contributes to both spike repolarization and spike after-hyperpolarization. Spontaneous miniature ICs have also been recorded, probably activated by the release of packets of intracellular Ca.

•Callister, R.J., Keast, J.R. & Sah, P. (1997). Ca2+-activated K+ channels in rat otic ganglion cells: role of Ca2+ entry via Ca2+ channels and nicotinic receptors. Journal of Physiology (London) 500(Pt. 3): 571-582. 1. Intracellular recordings were made from neurones in the rat otic ganglion in vitro in order to investigate their morphological, physiological and synaptic properties. We took advantage of the simple structure of these cells to test for a possible role of calcium influx via nicotinic acetylcholine receptors during synaptic transmission. 2. Cells filled with biocytin comprised a homogeneous population with ovoid somata and sparse dendritic trees. Neurones had resting membrane potentials of -53 ± 0.7 mV (n = 69), input resistances of 112± 7 MΩ, and membrane time constants of 14 ±0.9 ms (n = 60). Upon depolarization, all cells fired overshooting action potentials which were followed by an apamin-sensitive after- hyperpolarization (AHP). In response to a prolonged current injection, all neurones fired tonically. 3. The repolarization phase of action potentials had a calcium component which was mediated by N-type calcium channels. Application of omega- conotoxin abolished both the repolarizing hump and the after-hyperpolarization suggesting that calcium influx via N-type channels activates SK-type calcium- activated potassium channels which underlie the AHP. 4. The majority (70%) of neurones received innervation from a single preganglionic fibre which generated a suprathreshold excitatory postsynaptic potential mediated by nicotinic acetylcholine receptors. The other 30% of neurones also had one or more subthreshold nicotinic inputs. 5. Calcium influx via synaptic nicotinic receptors contributed to the AHP current, indicating that this calcium has access to the

Page 159 AHP, BK- and SK-channel references calcium-activated potassium channels and therefore plays a role in regulating cell excitability.

•Dai, Y. & Jordan, L.M. (2001). A modeling study of the relationship between the reduction of the afterhyperpolarization and hyperpolarization of voltage threshold in cat lumbar motoneurons during fictive locomotion. Society for Neuroscience Abstracts 31(625.3). Cat lumbar motoneurons displayed a reduction of the afterhyperpolarization (AHP) and a hyperpolarization of voltage threshold (Vth) during fictive locomotion. It was suggested that the reduced AHP could be due to a reduced AHP conductance (gK_AHP). Modulation of sodium conductance (gNa) was shown in a modeling study to be the most likely basis for the hyperpolarization of Vth. Here, we investigate the question of whether there is interplay between these two conductances during fictive locomotion and what could be their mixed effect on AHP. A single cell model with five-compartments was used in this study. The model included 8 active conductances in the somatic compartment, 4 in the proximal dendrite, 2 each in the axon and initial segment, and none in the distal dendrite. The Vth was used as reference to measure the AHP amplitude. Simulation results show that 1) modulation of AHP by gK_AHP and Vth by gNa was produced by two mechanisms which were not related to each other; however, increasing gNa and/or reducing gK_AHP could result in AHP reduction; 2) reduction of the AHP is dependent on the method of analysis. A constant current injection produced a larger AHP reduction than a reduced current injection, which was used to maintain a constant firing frequency, when the AHP was reduced by increasing gNa or reducing gK_AHP; 3) With constant current injection more than 40% reduction of the AHP could result from Vth hyperpolarization when Vth was hyperpolarized with an amount comparable to the experimental observation.

•Davies, P.J., Ireland, D.R., Martinez-Pinna, J. & McLachlan, E.M. (1999). Electrophysiological roles of L-type channels in different classes of guinea pig sympathetic neuron. J Neurophysiol 82(2): 818-828. The electrophysiological consequences of blocking Ca(2+) entry through L-type Ca(2+) channels have been examined in phasic (Ph), tonic (T), and long- afterhyperpolarizing (LAH) neurons of intact guinea pig sympathetic ganglia isolated in vitro. Block of Ca(2+) entry with Co(2+) or Cd(2+) depolarized T and LAH neurons, reduced action potential (AP) amplitude in Ph and LAH neurons, and increased AP

Page 160 AHP, BK- and SK-channel references half-width in Ph neurons. The afterhyperpolarization (AHP) and underlying Ca(2+)- dependent K(+) conductances (gKCa1 and gKCa2) were reduced markedly in all classes. Addition of 10 microM nifedipine increased input resistance in LAH neurons, raised AP threshold in Ph and LAH neurons, and caused a small increase in AP half-width in Ph neurons. AHP amplitude and the amplitude and decay time constant of gKCa1 were reduced by nifedipine in all classes; the slower conductance, gKCa2, which underlies the prolonged AHP in LAH neurons, was reduced by 40%. Surprisingly, AHP half- width was lengthened by nifedipine in a proportion of neurons in all classes; despite this, neuron excitability was increased during a maintained depolarization. Nifedipine's effects on AHP half-width were not mimicked by 2 mM Cs(+) or 2 mM anthracene-9-carboxylic acid, a blocker of Cl(-) channels, and it did not modify transient outward currents of the A or D types. The effects of 100 µM Ni(2+) differed from those of nifedipine. Thus in Ph neurons, Ca(2+) entry through L-type channels during a single action potential contributes to activation of K(+) conductances involved in both the AP and AHP, whereas in T and LAH neurons, it acts only on gKCa1 and gKCa2. These results differ from the results in rat superior cervical ganglion neurons, in which L-type channels are selectively coupled to BK channels, and in hippocampal neurons, in which L-type channels are selectively coupled to SK channels. We conclude that the sources of Ca(2+) for activating the various Ca(2+)-activated K(+) conductances are distinct in different types of neuron.

•Davies, P.J., Ireland, D.R. & McLachlan, E.M. (1996). Sources of Ca2+ for different Ca2+-activated K+ conductances in neurones of the rat superior cervical ganglion. Journal of Physiology (London) 495(Pt 2): 353-66. 1. The role of various Ca(2+)-activated K+ conductances were investigated using intracellular recording and single-electrode voltage clamp in neurones of superior cervical ganglia isolated in vitro from young adult rats. 2. Following replacement of Ca2+ with Co2+ (2 mM) or the addition of Cd2+ (100 µM), action potential amplitude and half- width either increased or decreased (in different cells), but both the after-hyperpolarization (AHP) and the outward tail current following a suprathreshold voltage step were markedly attenuated (by about 75%). 3. Addition of charybdotoxin (60 nM) or nifedipine (10 µM) increased action potential half-width (by about 25%) but had no significant effect on the AHP or tail current. 4. Addition of apamin (100 nM) or omega-conotoxin GVIA (100 nM) reduced the AHP and tail current (by about 60%) but did not significantly affect the action

Page 161 AHP, BK- and SK-channel references potential. A prolonged apamin-resistant component of the AHP present in 50% of neurones was blocked by ryanodine (20 µM). 5. Omega-Conotoxin MVIIC (150 nM) and omega agatoxin IVA (200 nM) had no significant effects on the action potential half-width or the AHP. 6. None of the Ca2+ channel blockers affected the prolonged ryanodine-sensitive component of the AHP and tail current. 7. We conclude that, in rat sympathetic neurones, Ca2+ entry via L-type channels selectively activates large conductance Ca(2+)-activated K+ channels (BK type) contributing to action potential repolarization, whereas Ca2+ entry via N-type channels selectively activates small conductance Ca(2+)-activated K+ channels (SK type) contributing to the AHP. Ca2+ entry via R-type Ca2+ channels prolongs the AHP by activating Ca2+ release from intracellular stores.

•Dunn, P.M. (1994). Dequalinium, a selective blocker of the slow afterhyperpolarization in rat sympathetic neurones in culture. European Journal of Pharmacology 252(2): 189-94. The actions of dequalinium have been investigated in cultured rat sympathetic neurones. It produced a rapid and reversible inhibition of the slow apamin-sensitive component of the afterhyperpolarization (AHP) which follows a single action potential in these cells. The IC50 for this effect was 1.1 microM and in voltage clamp experiments, 1 µM dequalinium produced 45% inhibition of the underlying current IAHP. When the small conductance Ca2+-activated K+ channels were blocked by 20 nM apamin the slow component of the AHP was abolished, and dequalinium (10 µM) produced no further change in the residual AHP. Dequalinium (10 µM) had no effect on the voltage-activated Ca2+ current in these cells, suggesting that the inhibition of the AHP was the result of a direct interaction with the K+ channels. The A-current as well as a composite current made up of IK and IC were all unaffected by 10 µM dequalinium. However, at this concentration it did produce 18% inhibition of the M-current. These results show dequalinium to be a potent and selective non-peptide blocker of the apamin-sensitive small conductance Ca2+- activated K+ channel in rat sympathetic neurones.

•Hirst, G.D.S., Johnson, S.M. & van Helden, D.F. (1985). The slow calcium- dependent potassium current in a myenteric neurone of the guinea-pig ileum. Journal of Physiology 361: 315-337. Experiments were performed in current-clamped and voltage-clamped after- hyperpolarizing (AH) neurones of the guinea-pig myenteric plexus to examine the

Page 162 AHP, BK- and SK-channel references properties of the potassium conductance (gK, Ca) underlying the slow calcium- activated after-hyperpolarization (VK, Ca). The action potential plateau lengthened by the addition of tetraethylammonium chloride (TEA) to the bathing medium was compared to VK, Ca. Results were consistent with enhanced calcium entry causing an increase of VK, Ca. 4-Aminopyridine (4-AP) directly reduced VK, Ca. Voltage-clamp data of gK, Ca were well fitted by a process with a delay (approximately equal to 60 ms) followed by exponential activation (time constant approximately equal to 300 ms) and inactivation (time constant approximately equal to 2 s). The presence of a small, much slower inactivating process was noted. Values for time constants were similar to those reported by Morita, North & Tokimasa (1982) and North & Tokimasa (1983) where gK, Ca was measured during VK, Ca subsequent to action potential stimulation. The relation between gK, Ca (or the calcium-activated potassium current IK, Ca) and estimated calcium influx resulting from short- duration calcium currents elicited at various voltages was compared. Both the integral of the calcium current and gK, Ca showed a similar dependence on the depolarizations used to elicit IK, Ca except there was a positive shift of about 4 mV for the gK, Ca curve. This shift was attributed to a requirement for calcium ions to prime the gK, Ca mechanism. An inward ion charge movement of about 8 pC was required before significant activation of gK, Ca occurred. After the 'priming' condition had been established, the sensitivity of gK, Ca to inward calcium current measured at the resting potential was about 500 pS/pC of inward charge. Large calcium entry obtained by prolonged calcium currents caused saturation of the peak amplitude of IK, Ca and initiated currents with slower time to peak amplitude and longer duration. Increasing the calcium concentration of the external solution provided proportionally larger IK, Ca currents before saturation. The saturation amplitude of IK, Ca (namely gK, Ca) was relatively unaffected.

•Hosseini, R., Benton, D.C.H., Dunn, P.M., Jeninson, D.H. & Moss, G.W.J. (2001). SK3 is an important component of K+ channels mediating the afterhyperpolarization in cultured rat SCG neurones. Journal of Physiology 535(323-334). 1. Our aim was to identify the small-conductance Ca2+-activated K(+) channel(s) (SK) underlying the apamin-sensitive afterhyperpolarization (AHP) in rat superior cervical ganglion (SCG) neurones. 2. Degenerate oligonucleotide primers designed to the putative calmodulin-binding domain conserved in all mammalian SK channel sequences were employed to detect SK DNA in a cDNA library from rat

Page 163 AHP, BK- and SK-channel references

SCG. Only a single band, corresponding to a fragment of the rSK3 gene, was amplified. 3. Northern blot analysis employing a PCR-generated rSK3 fragment showed the presence of mRNA coding for SK3 in SCG as well in other rat peripheral tissues including adrenal gland and liver. 4. The same rSK3 fragment enabled the isolation of a full-length rSK3 cDNA from the library. Its sequence was closely similar to, but not identical with, that of the previously reported rSK3 gene. 5. Expression of the rSK3 gene in mammalian cell lines (CHO, HEK cells) caused the appearance of a K(+) conductance with SK channel properties. 6. The application of selective SK blocking agents (including apamin, scyllatoxin and newer non-peptidic compounds) showed these homomeric SK3 channels to have essentially the same pharmacological characteristics as the SCG afterhyperpolarization, but to differ from those of homomeric SK1 and SK2 channels. 7. Immunohistochemistry using a rSK3 antipeptide antibody revealed the presence of SK3 protein in the cell bodies and processes of cultured SCG neurones. 8. Taken together, these results identify SK3 as a major component of the SK channels responsible for the afterhyperpolarization of cultured rat SCG neurones.

•Jobling, P., Mclachlan, E.M. & Sah, P. (1993). Calcium induced calcium release is involved in the afterhyperpolarization in one class of guinea pig sympathetic neurone. Journal of the Autonomic Nervous System 42(3): 251-257. The mechanisms underlying two potassium conductances which are activated by Ca2+ influx during the action potential in sympathetic prevertebral neurones of guinea pigs have been investigated pharmacologically. One Ca-activated K+ conductance, which is present in all mammalian sympathetic postganglionic neurones, is maximal after the action potential and decays exponentially with a time constant of about 130 ms; this conductance was inhibited by apamin (50-100 nM) consistent with the involvement of SK channels. A second Ca-activated K+ conductance with much slower kinetics is present in a large subpopulation of coeliac neurones. This conductance was resistant to apamin but markedly inhibited by application of ryanodine (5-20 µM), suggesting that Ca2+ influx during the action potential triggers release of Ca2+ from intracellular stores which in turn activates a different class of K+ channel. Noradrenaline (100 µoM) depressed the second K+ conductance selectively.

•Kaufmann, W.A., Sailer, C.A., Jacobson, D.A., Knopp, S.J., Bond, C.T., Adelman, J.P., Bissonnette, J.M. & Knaus, H.G. (2001). Developmental

Page 164 AHP, BK- and SK-channel references expression of a calcium-activated potassium channel, SK3, and the effects on medullary respiratory neurons in mice. Society for Neuroscience Abstracts 31(382.1). This study was undertaken to determine the developmental timetable for SK3 expression and its correlation with respiratory motoneuron activity. Expression of SK3 has been studied by immunohistochemistry using sequence specific rabbit antisera directed against SK3 protein. From embryonic day 19 to adulthood, a progressive increase of SK3 immunoreactivity was observed. The overall regional distribution pattern remained identical, however, age-dependent changes in SK3 channel localization were detected. In contrast, SK2 expression was only seen in very distinct regions of the brainstem and largely remained unchanged during development. The distribution of [125I]apamin binding supported the immunohistochemical data with low levels of toxin binding detected up to postnatal day 10 (P10) followed by a marked increase at P15. In parallel, respiratory activity was examined in a body plethysmograph. Mice studied at P4 and P7 showed a robust increase in tidal volume when exposed to 5% CO2 (170 + 24 and 175 + 20% of control, respectively). Mice targeted for the conditional repression of SK3 examined at P7 showed no difference compared to wild-type. This is in marked contrast to studies at P13-15 where repression of SK3 augments the response to CO2 (FASEB J 15:A152, 2001). These results suggest a maturation dependent function of SK3 channels. Supported by: Austrian Research Foundation grant P14954-PHA; NIH and Human Frontiers of Science Program

•Koh, D.S., Reid, G. & Vogel, W. (1994). Effect of the flavoid phloretin on Ca2+- activated K+ channels in myelinated nerve fibres of Xenopus laevis. Neuroscience Letters 165(1-2): 167-170. The effects of the phloretin on K+ channels in amphibian myelinated nerve were studied by patch clamping. The open probability of Ca2+-activated K+ channels was greatly increased by external phloretin (10-200 µM) due to a shift of the membrane potential for half-maximal activation, E50, of -63.9 mV (80 µM phloretin). Open times were prolonged and closed times shortened. Channel activation by phloretin developed slowly (tau on = 33.4 s) and its washout was even slower (tau off,1 = 4.7 and tau off,2 = 183.2 s). In contrast, submillimolar phloretin blocked two delayed rectifier K+ channels (I and F) whereas the gating of the ATP- sensitive and the flickering K+ channel were unaffected. Phloretin may directly

Page 165 AHP, BK- and SK-channel references affect the voltage sensor of K+ channels.

•Kulik, A., Brockhaus, J., Pedarzani, P. & Ballanyi, K. (2002). Chemical anoxia activates ATP-sensitive and blocks Ca2+-dependent K+ channels in rat dorsal vagal neurons in situ. Neuroscience 110(3): 541-554. The contribution of subclasses of K(+) channels to the response of mammalian neurons to anoxia is not yet clear. We investigated the role of ATP-sensitive (K(ATP)) and Ca2+-activated K(+) currents (small conductance, SK, big conductance, BK) in mediating the effects of chemical anoxia by cyanide, as determined by electrophysiological analysis and fluorometric Ca2+ measurements in dorsal vagal neurons of rat brainstem slices. The cyanide-evoked persistent outward current was abolished by the K(ATP) channel blocker , but not changed by the SK and BK channel blockers apamin or tetraethylammonium. The K(+) channel blockers also revealed that ongoing activation of K(ATP) and SK channels counteracts a tonic, spike-related rise in intracellular Ca2+ ([Ca2+](i)) under normoxic conditions, but did not modify the rise of [Ca2+](i) associated with the cyanide-induced outward current. Cyanide depressed the SK channel-mediated afterhyperpolarizing current without changing the depolarization-induced [Ca2+](i) transient, but did not affect spike duration that is determined by BK channels. The afterhyperpolarizing current and the concomitant [Ca2+](i) rise were abolished by Ca2+-free superfusate that changed neither the cyanide-induced outward current nor the associated [Ca2+](i) increase. Intracellular BAPTA for Ca2+ chelation blocked the afterhyperpolarizing current and the accompanying [Ca2+](i) increase, but had no effect on the cyanide- induced outward current although the associated [Ca2+](i) increase was noticeably attenuated. Reproducing the cyanide-evoked [Ca2+](i) transient with the Ca2+ pump blocker cyclopiazonic acid did not evoke an outward current.Our results show that anoxia mediates a persistent hyperpolarization due to activation of K(ATP) channels, blocks SK channels and has no effect on BK channels, and that the anoxic rise of [Ca2+](i) does not interfere with the activity of these K(+) channels.

•Lancaster, B. & Pennefather, P. (1987). Potassium currents evoked by brief depolarizations in bull-frog sympathetic ganglion cells. Journal of Physiology 387: 519-548. 1. Sympathetic neurones of the bull-frog Rana catesbeiana were subjected to a two-electrode voltage-clamp technique in order to investigate the K+ currents which can be elicited by action potentials or similar brief depolarizations. 2. Four

Page 166 AHP, BK- and SK-channel references separate K+ currents were observed (IC, IK, IAHP and IM). These could be separated on the basis of voltage sensitivity, Ca2+ dependence and deactivation kinetics. 3. Two of these currents, which were clearly activated by an action potential, were Ca2+ dependent. A voltage- and TEA (tetraethylammonium)-sensitive K+ current, IC, was activated within the first 1-2 ms of a depolarizing command. This current decayed on average with a time constant of 2.4 ms at -40 mV. The maximal conductance was outside the range which could be adequately voltage clamped but, as much as 2 muS could be activated by brief (2-3 ms) commands. Activation of IC during an action potential accounts for the Ca2+ dependence of the repolarization. IC did not exhibit a transient component. 4. A second Ca2+-dependent K+ current, IAHP, was also activated after as little as 1 ms depolarization but was not voltage sensitive and was much less sensitive to TEA. The current decayed with a time constant of around 150 ms at -40 mV. The maximal conductance was about 30 nS. 5. The voltage-sensitive delayed rectifying current, IK, made a contribution to the total K+ conductance of the cell similar to IC in magnitude; however, the current is not activated within the normal voltage range or time course of an action potential. The current decayed on average with a time constant of 21 ms at -40 mV. 6. IM, a muscarine- and voltage sensitive current, is not activated to any significant degree by a single action potential. The data further imply that the rate of opening of the ion channels mediating IM is less voltage sensitive than the rate of closing. 7. Large changes in the K+ reversal potential occur following depolarizing commands which evoke large K+ currents. This is attributed to K+ accumulation within a restricted extracellular space. Extracellular K+ may double or even triple during a single action potential.

•Martin-Caraballo, M. & Dryer, S.E. (2001). Activity and target-dependent regulation of Ca2+-activated K+ channels (KCa) in developing chick lumbar motoneurons. Society for Neuroscience Abstracts 31(382.15): 196. Developing neurons express particular classes of ion channels at specific developmental stages. We have examined the functional expression of large- conductance KCa channels in developing LMNs between embryonic day 6 (E6) and E13. Analyses of macroscopic fluctuations and tail currents, along with inside-out patch recordings, indicate that KCa channels of E11 LMNs have unusually fast gating kinetics. Average macroscopic KCa density was low before E8 and increased 3.3-fold by E11, with a further 1.8-fold increase occurring by E13. The density of voltage- activated Ca2+ currents did not change between E8-13. This pattern of macroscopic

Page 167 AHP, BK- and SK-channel references

KCa expression was regulated in part by interactions with target tissues. Thus, in ovo administration of D-tubocurarine, which causes an increase in axonal branching and motoneuron survival, also evoked a 1.8-fold increase in average KCa density measured at E11. Conversely, surgical ablation of target tissues at E5 caused a significant reduction in average KCa density at E11. Electrical activity also contributed to developmental regulation of LMN KCa density. A significant reduction in E11 KCa density was found after chronic in ovo treatment with the neuronal mecamylamine or the GABA receptor agonist , agents that reduce activation of LMNs in ovo. Moreover, chronic application of depolarizing concentrations of external K+ caused an increase in KCa expression, whereas chronic tetrodotoxin caused a decrease in KCa expression in LMNs developing in dissociated cell culture. Supported by: Muscular Dystrophy Association, NIH grant NS-32748, and Alberta Heritage Foundation for Medical Research

•Martinez-Pinna, J., Davies, P.J. & McLachlan, E.M. (2000). Diversity of channels involved in Ca2+ activation of K+ channels during the prolonged AHP in guinea-pig sympathetic neurons. Journal of Neurophysiology 84(3): 1346-1354. The types of Ca(2+)-dependent K(+) channel involved in the prolonged afterhyperpolarization (AHP) in a subgroup of sympathetic neurons have been investigated in guinea pig celiac ganglia in vitro. The conductance underlying the prolonged AHP (gKCa2) was reduced to a variable extent in 100 nM apamin, an antagonist of SK-type Ca(2+)-dependent K(+) channels, and by about 55% in 20 nM iberiotoxin, an antagonist of BK-type Ca(2+)-dependent K(+) channels. The reductions in gKCa2 amplitude by apamin and iberiotoxin were not additive, and a resistant component with an amplitude of nearly 50% of control remained. These data imply that, as well as apamin- and iberiotoxin-sensitive channels, other unknown Ca(2+)- dependent K(+) channels participate in gKCa2. The resistant component of gKCa2 was not abolished by 0.5-10 mM tetraethylammonium, 1 mM 4-aminopyridine, or 5 mM . We also investigated which voltage-gated channels admitted Ca(2+) for the generation of gKCa2. Blockade of Ca(2+) entry through L-type Ca(2+) channels has previously been shown to reduce gKCa2 by about 40%. Blockade of N- type Ca(2+) channels (with 100 nM omega-conotoxin GVIA) and P-type Ca(2+) channels (with 40 nM omega-agatoxin IVA) each reduced the amplitude of gKCa2 by about 35%. Thus Ca(2+) influx through multiple types of voltage-gated Ca(2+) channel can activate the intracellular mechanisms that generate gKCa2. The slow

Page 168 AHP, BK- and SK-channel references time course of gKCa2 may be explained if activation of multiple K(+) channels results from Ca(2+) influx triggering a kinetically invariant release of Ca(2+) from intracellular stores located close to the membrane.

•Middlekauff, H.R., Doering, A. & Weiss, J.N. (2001). Adenosine enhances neuroexcitability by inhibiting a slow postspike afterhyperpolarization in rabbit vagal afferent neurons. Circulation 103(9): 1325-1329. BACKGROUND: Electrophysiological mechanisms by which adenosine may activate cardiac afferent neurons are unknown. Slow afterhyperpolarizations (AHPs) follow action potentials in a subset of vagal C afferents, rendering them inexcitable. The purpose of this study was to test the hypothesis that adenosine increases vagal neuronal excitability by blocking slow AHPs and to determine the adenosine receptor subtype mediating these effects. METHODS AND RESULTS: Using the perforated patch-clamp technique, we identified cultured adult rabbit nodose ganglion cells with slow AHPs in current-clamp mode. Trains of 100 current pulses at 20% above threshold were injected, with an interspike interval of 100 ms, and the number of action potentials triggered were counted and reported as the action potential response rate. During adenosine (10 µM), slow AHPs were suppressed and action potential response rate was augmented from 3.8±0.5% at baseline to 28±7% after adenosine (P≤0.0009). The selective A(2)-adenosine receptor agonist NECA but not the A(1)-adenosine agonist CCPA replicated the adenosine effect. The selective A(2A)-adenosine antagonist ZM 241385 (10 nmol/L) but not the A(1) adenosine antagonist DPCPX (5 µM) abolished the adenosine effect. We considered two alternative hypotheses: (1) A(2)-receptor-mediated suppression of I(Ca) leading to smaller increases in intracellular Ca during stimulation, resulting in less activation of I(K(Ca)) and consequent suppression of slow AHPs, or (2) A(2)- receptor-mediated elevation of cAMP directly suppressing slow AHPs. Under voltage-clamp conditions, adenosine did not significantly inhibit I(Ca), making the latter hypothesis more likely. CONCLUSIONS: Adenosine inhibits slow AHPs in vagal afferent neurons. This effect is most likely caused by A(2A)-receptor- mediated stimulation of cAMP production.

•Nohmi, M. & Kuba, K. (1984). (+)-tubocurarine blocks Ca2+-dependent K+ channel of the bullfrog sympathetic ganglion cell. Brain Research 301: 146-148. (+)-tubocurarine [+)-Tc: 10-100 µM) reduced the duration of the afterhyperpolarization, which was induced by the activation of Ca2+-dependent K+-

Page 169 AHP, BK- and SK-channel references conductance (GK,Ca) following an action potential in the bullfrog sympathetic ganglion cell, but did not affect the maximum rates of rise and fall of Na+- and Ca2+-dependent action potentials. The amplitudes of slow rhythmic membrane hyperpolarizations produced by rhythmic rises in the GK,Ca were also decreased by (+)-Tc without a change in their intervals. Thus, (+)-Tc appears to block the Ca2+- dependent K+-channel of the bullfrog sympathetic ganglion cell.

•Pedarzani, P., Kulik, A., Muller, M., Ballanyi, K. & Stocker, M. (2000). Molecular determinants of Ca2+-dependent K+ channel function in rat dorsal vagal neurones. Journal of Physiology (London) 527: 283-290. Using in situ hybridisation histochemistry in combination with patch-clamp recordings and specific pharmacological tools, the molecular nature of the channels underlying Ca2+-dependent K+ currents was determined in dorsal vagal neurones (DVNs) of rat brainstem slices. In situ hybridisation analysis at cellular resolution revealed the presence of 'big'-conductance Ca2+- and voltage-activated K+ (BK) channel alpha-subunit mRNA, and of only one 'small'-conductance Ca2+-activated K+ (SK) channel subunit transcript, SK3, at very high levels in DVNs. By contrast, SK1 and SK2 mRNAs were below the threshold limit of detection. The SK channel- mediated after-hyperpolarising current (IAHP) was blocked by apamin with a half- maximal inhibitory concentration of approximately 2.2 nM. This is consistent with homomultimeric SK3 channels mediating IAHP in DVNs. IAHP was also blocked by scyllatoxin (20-30 nM) and curare (100-200 µM). Application of apamin (100 nM) or scyllatoxin (20 nM) invariably caused a substantial increase to 146.1 ± 10.4 and 181.8 ± 12.9 % of control, respectively, in the spontaneous firing rate of DVNs. Action potential duration was not affected by these SK channel blockers. The selective BK channel blocker iberiotoxin (50 nM) increased action potential duration by 22.5 +/- 7.3 %, as did low concentrations of tetraethylammonium (0.5 mM; 99.3 ± 16.4 %) and the Ca2+ channel blocker Cd2+ (100 µM; 49.5 ± 20.9%). BK channel blockade did not significantly affect the firing rate of DVNs. These results allow us to establish a tight correlation between the properties of cloned and native BK and SK channels, and to achieve an understanding, at the molecular level, of their role in regulating the spontaneous firing frequency and in shaping single action potentials of central neurones.

•Pennefather, P., Lancaster, B., Adams, P.R. & Nicoll, R.A. (1985). Two distinct Ca2+-dependent K+ currents in bullfrog sympathetic ganglion cells.

Page 170 AHP, BK- and SK-channel references

Proceedings of the National Academy of Sciences USA 82: 3040-3044. Healthy bullfrog sympathetic ganglion cells often show a two-component afterhyperpolarization (AHP). Both components can be reduced or abolished by adding Ca-channel blockers or by removing external Ca. Application of a single electrode "hybrid clamp"--i.e., switching from current- to voltage clamp at the peak of the AHP, reveals that the slow AHP component is generated by a small, slow, monotonically decaying outward current, which we call IAHP. IAHP is blocked by Ca- removal or by apamin and is a pure K current. It is slightly sensitive to muscarine and to tetraethylammonium ion but is much less so than muscarine-sensitive (IM) and fast Ca dependent (IC) K currents. It also can be recorded in dual-electrode voltage-clamp experiments, where it is seen as a slow, small component of the outward tail current that follows brief depolarizations to 0 mV or beyond. IC is seen as an early, fast, large component of the same tail current. Both components are blocked by Ca removal, but only the IC component is blocked by low doses of tetraethylammonium ion. Thus, bullfrog ganglion cells exhibit two quite distinct Ca- dependent K currents, which differ in size, voltage-sensitivity, kinetics, and pharmacology. These two currents also play quite separate roles in shaping the action potential.

•Protti, D.A. & Uchitel, O.D. (1997). P/Q-type calcium channels activate neighboring calcium-dependent potassium channels in mouse motor nerve terminals. Pflugers Arch 434(4): 406-412. The identity of the voltage-dependent calcium channels (VDCC), which trigger the Ca2+-gated K+ currents (IK(Ca)) in mammalian motor nerve terminals, was investigated by means of perineurial recordings. The effects of Ca2+ chelators with different binding kinetics on the activation of IK(Ca) were also examined. The calcium channel blockers of the P/Q family, omega-agatoxin IVA (omega-Aga-IVA) and funnel-web spider toxin (FTX), have been shown to exert a strong blocking effect on IK(Ca). In contrast, and omega-conotoxin GVIA (omega-CgTx) did not affect the Ca2+-activated K+ currents. The intracellular action of the fast Ca2+ buffers BAPTA and DM-BAPTA prevented the activation of the IK(Ca), while the slow Ca2+ buffer EGTA was ineffective at blocking it. These data indicate that P/Q-type VDCC mediate the Ca2+ influx which activates IK(Ca). The spatial association between Ca2+ and Ca2+-gated K+ channels is discussed, on the basis of the differential effects of the fast and slow Ca2+ chelators.

Page 171 AHP, BK- and SK-channel references

•Roncarati, R., Di Chio, M., Sava, A., Terstappen, G.C. & Fumagalli, G. (2001). Presynaptic localization of the small conductance calcium-activated potassium channel SK3 at the neuromuscular junction. Neuroscience 104(1): 253- 262. Small conductance, calcium-activated potassium channels (SK channels) are present in most neurons, in denervated muscles and in several non-excitable cell types. In excitable cells SK channels play a fundamental role in the generation of the afterhyperpolarization which follows an action potential, thereby modulating neuronal firing and regulating excitability. To date, three channel subunits (SK1-3) have been cloned from mammalian brain. Since SK3 only has been shown to be expressed in muscles upon denervation, this channel may be involved in hyperexcitability and afterhyperpolarization observed in muscle cells in the absence of the nerve. Using confocal microscopy and SK3 specific antibodies, we demonstrate that SK3 immunoreactivity is present at the rat neuromuscular junction in denervated but also in innervated muscles. In denervated muscle fibers, SK3 is localized in the extrajunctional as well as the junctional plasma membrane, where it appears to be less abundant in the acetylcholine receptor-rich domains, corresponding to the crests of the postsynaptic folds. In innervated muscles, SK3 is not detectable in the muscle fiber but is present at the neuromuscular junction and seems to be localized presynaptically in the motor nerve terminals. Axonal accumulation of SK3 immunoreactivity occurs above and below a ligature of rat sciatic nerve, indicating that the SK3 protein is transported in both directions along the axons of the motor neurons. During rat development SK3 immunoreactivity is not found at the neuromuscular junction until day 35 of postnatal development when SK3 first appears in the motor neuron terminals.These results indicate that SK3 channels are components of the presynaptic compartment in the mature neuromuscular junction, where they may play an important regulatory role in synaptic transmission.

•Sah, P. (1992). The role of calcium influx and calcium buffering in the kinetics of a Ca2+-activated K+ current in rat vagal motoneurons. Journal of Neurophysiology 68: 2237-2248. 1. Intracellular recordings were obtained from neurons of the dorsal motor nucleus of the vagus (DMV) in transverse slices of the rat medulla maintained in vitro. These neurons had a resting potential of -59.8 ± 8.7 (SD) mV. Single action potentials elicited by brief depolarizing current pulses were followed by a prolonged

Page 172 AHP, BK- and SK-channel references afterhyperpolarization (AHP). Under voltage clamp, the current underlying the AHP was found to be a calcium-activated potassium current. 2. The outward current (GkCa,1) was voltage insensitive and was not blocked by tetraethylammonium (TEA) (10 mM). Unlike the slower time course calcium-activated potassium current recorded in some other neurons, GkCa,1 was blocked by apamin (25-100 nM), indicating that SK type calcium-activated potassium channels underlie this current. 3. GkCa,1 was maximal within 10 ms of the action potential and its decay was well described by a single exponential. After a single action potential the time constant of decay of GkCa,1 was 155 ± 66 (± SD) ms. 4. Calcium influx was increased by adding TEA to the extracellular solution or by firing more than one action potential. As the calcium load was increased, both the peak amplitude and the time constant of decay of GkCa,1 increased. In cells impaled with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-filled electrodes, the time constant of decay of GkCa,1 after a single action current was 71 ± 19 ms. 5. A simple diffusion- based model that incorporates two intrinsic calcium buffers is developed that accounts for many of the properties of GkCa,1. It is concluded that the decay of GkCa,1 reflects the time course of removal of calcium that has entered the cell during the action potential.

•Sah, P. (1993). Kinetic properties of a slow apamin-insensitive Ca2+-activated K+ current in guinea pig vagal neurons. Journal of Neurophysiology 69(2): 361-366. 1. The calcium-activated outward current (gKCa,2) following an action potential was recorded from neurons in the guinea pig dorsal motor nucleus of the vagus (DMV). gKCa,2 was activated after a delay after the action current and had a distinct rising phase in contrast to the apamin sensitive calcium-activated current in rat DMV neurons (gKCa,1). 2. The time course of gKCa,2 was well described by function of the form A*[exp(t/tau 1)-exp(t/tau 2)], where tau 1 = 1.42 ± 0.05 (SE) s and tau 2 = 555 ± 24 ms. 3. Increasing calcium influx by firing multiple action currents lead to an increase in the peak amplitude of gKCa,2 with no change in its kinetics. In cells loaded with low concentrations of ethylene glycol-bis(beta- aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), gKCa,2 was smaller in amplitude but its time course was similar to that in cells not loaded with EGTA. 4. When preceded by a conditioning influx of calcium, the amplitude and time course of gKCa,2 was identical to one with no conditioning influx. These results show that, after influx of calcium, a potassium current with stereotyped amplitude and kinetics is generated. 5. These data are consistent with the idea that the source of calcium

Page 173 AHP, BK- and SK-channel references for activation of gKCa,2 is not the extracellular space.

•Sah, P. (1995). Properties of channels mediating the apamin-insensitive afterhyperpolarization in vagal motoneurons. J Neurophysiol 74(4): 1772-1776. 1. Whole cell recordings were obtained from neurons of the dorsal motor nucleus of the vagus in transverse slices of guinea pig medulla. From a holding potential of -40 mV, short depolarizing voltage steps activated two calcium- dependent potassium currents, Gk(Ca),1 and Gk(Ca),2. 2. Gk(Ca),1 was completely blocked by apamin (100 nM). Gk(Ca),2 was apamin insensitive, voltage independent, and reversed close to the potassium equilibrium potential. 3. Activation of Gk(Ca),2 was associated with an increase in current variance. The channels underlying the slow component were analyzed by stationary and nonstationary fluctuation analysis. Current variance was linearly related to mean current for small current amplitudes but clearly deviated from linearity near the peak of Gk(Ca),2. The predicted single channel conductance was 6.8 ± 2.5 (SE) pS. Probability of channel opening rose to at most 0.68. The average number of available Gk(Ca),2 channels on vagal neurons was 4,437 ± 591. 4. Power spectra were constructed from the peak current. Spectra were well fitted with a single Lorentzian with a corner frequency of 72 ± 7 Hz. The mean burst duration of the channels was 3.8 +/- 0.5 ms. These results indicate that a new type of calcium-activated channel underlies Gk(Ca),2.

•Sah, P. (1995). Different calcium channels are coupled to potassium channels with distinct physiological roles in vagal neurons. Proceedings of the Royal Society of London, Series B: Biological Sciences 260: 105-111.

•Sah, P. & McLachlan, E.M. (1991). Ca2+-activated K+ currents underlying the afterhyperpolarization in guinea pig vagal neurons: A role for Ca2+-activated Ca2+ release. Neuron 7: 257-264. We examined the possibility that Ca2+ released from intracellular stores could activate K+ currents underlying the afterhyperpolarization (AHP) in neurons. In neurons of the dorsal motor nucleus of the vagus, the current underlying the AHP had two components: a rapidly decaying component that was maximal following the action potential (GkCa,1) and a slower component that had a distinct rising phase (GkCa,2). Both components required influx of extracellular Ca2+ for their activation, and neither was blocked by extracellular TEA (10 mM). GkCa,1 was selectively blocked by apamin, whereas GkCa,2 was selectively reduced by

Page 174 AHP, BK- and SK-channel references noradrenaline. The time course of GkCa,2 was markedly temperature sensitive. GkCa,2 was selectively blocked by application of ryanodine or sodium dantrolene, or by loading cells with . These results suggest that influx of Ca2+ directly gates one class of K+ channels and leads to release of Ca2+ from intracellular stores, which activates a different class of K+ channel.

•Sah, P. & McLachlan, E.M. (1992). A slow voltage-activated potassium current in rat vagal neurons. Proceedings of the Royal Society of London, Series B: Biological Sciences 249: 71-6. Potassium currents play a key role in controlling the excitability of neurons. In this paper we describe the properties of a novel voltage-activated potassium current in neurons of the rat dorsal motor nucleus of the vagus (DMV). Intracellular recordings were made from DMV neurons in transverse slices of the medulla. Under voltage clamp, depolarization of these neurons from hyperpolarized membrane potentials (more negative than -80 mV) activated two transient outward currents. One had fast kinetics and had properties similar to A-currents. The other current had an activation threshold of around -95 mV (from a holding potential -110 mV) and inactivated with a time constant of about 3s. It had a reversal potential close to the potassium equilibrium potential. This current was not calcium dependent and was not blocked by 4-aminopyridine (5 mM), catechol (5 mM) or tetraethylammonium (20 mM). It was completely inactivated at the resting membrane potential. This current therefore represents a new type of voltage-activated potassium current. It is suggested that this current might act as a brake to repetitive firing when the neuron is depolarized from membrane potentials negative to the resting potential.

•Sah, P.A.J. & McLachlan, E.M. (1992). Potassium currents contributing to action potential repolarization and the afterhyperpolarization in rat vagal motoneurons. Journal of Neurophysiology 68: 1834-1841. 1. Intracellular recordings were made from neurons in the dorsal motor nucleus of the vagus (DMV) in transverse slices of rat medulla maintained in vitro at 30°C. Neurons had a resting potential of -59.8 ± 1.4 (SE) mV (n = 39) and input resistance of 293 ± 23 M omega (n = 44). 2. Depolarization elicited overshooting action potentials that were blocked by tetrodotoxin (TTX; 1 microM). In the presence of TTX, two types of action potentials having low and high thresholds could be elicited. The action potentials were blocked by cobalt (2 mM) indicating

Page 175 AHP, BK- and SK-channel references they were mediated by calcium currents. 3. Under voltage clamp, depolarization of the cell from membrane potentials negative of the resting potential activated a transient potassium current. This current was selectively blocked by 4- aminopyridine (4-AP) (5 mM) and catechol (5 mM) indicating that it is an A-type current. This current inactivated with a time constant of 420 ms and recovered from inactivation with a time constant of 26 ms. 4. When calcium currents were blocked by cadmium or cobalt, the rate of action potential repolarization was slower. In the presence of tetraethylammonium (TEA; 200-400 µM) or charybdotoxin (CTX; 30 nM) a small "hump" appeared on the repolarizing phase of the action potential that was abolished by addition of cadmium. These results indicate that a calcium- activated potassium current (IC) contributes to action potential repolarization. 5. Actions potentials elicited from hyperpolarized membrane potentials repolarized faster than those elicited from resting membrane potential. This effect could be blocked by catechol, indicating that voltage-dependent potassium currents (IA) can also contribute to action-potential repolarization. In the presence of catechol and calcium channel blockers, action potentials still had a significant early afterhyperpolarization suggesting that another calcium independent outward current is also active during repolarization. This fast afterhyperpolarizations (AHP) was not blocked by TEA. 6. Action potentials were followed by prolonged AHPs, which had two phases. The initial part of the AHP was blocked by apamin (100 nM) indicating that it results from activation of SK type calcium-activated potassium channels. The slow phase was selectively blocked by catechol suggesting that it is due to activation of IA. 7. It is concluded that a TTX-sensitive sodium current and two calcium currents contribute to the action potential in rat DMV neurons. At least three different currents contribute to action-potential repolarization: IC, IA, and a third unidentified calcium-insensitive outward current.(ABSTRACT TRUNCATED AT 400 WORDS)

•Sah, P. & McLachlan, E.M. (1993). Differences in electrophysiological properties between neurones of the dorsal motor nucleus of the vagus in rat and guinea pig. Journal of the Autonomic Nervous System 42: 89-98. We have examined the electrophysiological properties of neurones in the dorsal motor nucleus of the vagus (DMV) in rats and guinea pigs in transverse medullary slices maintained in vitro. There were only minor differences in the morphology of the neurones between the species, and their passive electrical properties were very similar. However, action potentials in guinea pig neurones had

Page 176 AHP, BK- and SK-channel references larger amplitudes and longer half-widths than did those in rat neurones. In both species, action potentials were followed by prolonged afterhyperpolarisations (AHPs). In the majority of guinea pig neurones, two calcium-activated potassium currents underlying the AHP could be separated into an early apamin-sensitive component and a late apamin-insensitive component. In rat neurones, the current underlying the AHP was briefer and entirely apamin-sensitive. In response to a step of depolarising current, neurones in the guinea pig only discharged once or twice and then ceased firing. In rat neurones, this manoeuvre produced repetitive firing. An inward rectifier was larger in neurones of the guinea pig than in those in the rat. The effects of 5-hydroxytryptamine and noradrenaline also differed between neurones of each species. We conclude that, despite many similarities of size and electrical properties, DMV neurones in the two species differ in terms of several voltage- and calcium-dependent conductances which determine their active electrical behaviour.

•Stauffer, E.K., Hornby, T.G., Reinking, R.M. & Stuart, D.G. (2001). Does AHP predict a spinal neuron's peak firing rate? Society for Neuroscience Abstracts 31(625.10). It is widely held that a spinal motoneuron's (MN's) peak firing rate is predicted by its AHP during a threshold (Irh) action potential (AP). We have reported some turtle MN and interneuron (IN) results to the contrary, however (J. Physiol. [Paris], 93:3-16, 1999). Here, we probe further by presenting the AHP of three 10-cell groups of turtle spinal neurons at Irh, and at the extremes of the cells' stimulus current (I)-firing frequency (f) relation under control conditions (J. Comp. Neurol. 400:544-570, 1998). The groups included high- and low-threshold MNs (MNht, like cat FF MNs; MNlt, like cat S MNs) and INs. In the table, each Irh value is the average for a single AP-AHP measurement/cell, whereas the Imin,fmin and Imax,fmax values are based on all the AP-AHPs of each cell in each 10-cell group in the second s of the 2-s stimulus trains used to generate the I-f relation. Note that the MNlt group has a higher mean fmax than the MNht one, and a longer AHP1/2-decay time at both Irh and Imin,fmin. Similar results were obtained for the AHP's integrated profile (area). Note also that the INs have a very high mean fmax, and a substantial AHP. The issues raised by these results are fundamental, and open. Supported by: NIH-NS 25077.

ADRENAL CHROMAFFIN CELLS

Page 177 AHP, BK- and SK-channel references

•Artalejo, A.R., Garcia, A.G. & Neher, E. (1993). Small-conductance Ca2+- activated K+ channels in bovine chromaffin cells. Pflugers Arch 423(1-2): 97-103. Simultaneous whole-cell patch-clamp and fura-2 fluorescence [Ca2+]i measurements were used to characterize Ca2+-activated K+ currents in cultured bovine chromaffin cells. Extracellular application of histamine (10 µM) induced a rise of [Ca2+]i concomitantly with an outward current at holding potentials positive to -80 mV. The activation of the current reflected an increase in conductance, which did not depend on membrane potential in the range -80 mV to -40 mV. Increasing the extracellular K+ concentration to 20 mM at the holding potential of -78 mV was associated with inwardly directed currents during the [Ca2+]i elevations induced either by histamine (10 µM) or short voltage-clamp depolarizations. The current reversal potential was close to the K+ equilibrium potential, being a function of external K+ concentration. Current fluctuation analysis suggested a unit conductance of 3-5 pS for the channel that underlies this K+ current. The current could be blocked by apamin (1 µM). Whole-cell current- clamp recordings showed that histamine (10 µM) application caused a transient hyperpolarization, which evolved in parallel with the [Ca2+]i changes. It is proposed that a small-conductance Ca2+-activated K+ channel is present in the membrane of bovine chromaffin cells and may be involved in regulating catecholamine secretion by the adrenal glands of various species.

•Chen, J.-Q., Galanakis, D., Ganellin, C.R., Dunn, P.M. & Jenkinson, D.H. (2000). Bis-quinolinium cyclophanes: 8,14-diaza-1,7(1,4)- diquinolinacyclotetradecaphane (UCL 1848), a highly potent and selective, non- peptidic blocker of the apamin-sensitive Ca2+-activated K+ channel. Journal of Medical Chemistry 43: 3478-3481. Small conductance Ca2+-activated K+ (SKCa) channels comprise an important subclass of K+channels. They occur in many types of cells, both excitable and inexcitable, and have a variety of physiological roles. Functional, binding, and structural data have suggested the existence of subtypes of the SKCa channel. In accordance with these observations, three SKCa channel subunits have been identified by DNA cloning: namely SK1, SK2, and SK3. Though apamin, a peptidic toxin from bee venom, potently and selectively blocks SKCa channels and has been invaluable in their study, there is considerable interest in the discovery of nonpeptidic blockers of the SKCa channel. Such compounds, in

Page 178 AHP, BK- and SK-channel references addition to being useful pharmacological tools, may have important therapeutic applications. For example, blockade of SKCa channels gives rise to an increase in gastrointestinal motility, and SKCa channels are involved in the endothelium- dependent hyperpolarizing factor (EDHF)-mediated relaxation of blood vessels. Repetitive muscle contraction in myotonic muscular dystrophy results from the aberrant expression of SKCa channels and can be prevented by intramuscular injection of apamin. In the central nervous system, a slow after-hyperpolarization (AHP) mediated by SKCa channels is responsible for the rhythmic firing of subthalamic neurons which are important for the control of movement. SKCa channels are also thought to play a part in memory and learning, sleep disorders, and the effects of chronic ethanol intoxication. Dequalinium has been shown to be a relatively potent and selective SKCa channel blocker. We have undertaken a systematic exploration of the stereoelectronic requirements for SKCa channel blockade in several series of dequalinium analogues and have developed bis-quinolinium cyclophanes of the general type 1, some of which are potent blockers of the SKCa channel. In particular, compound 1a (UCL 1684) is as effective as apamin in inhibiting the Ca2+-activated K+ currents in cultured rat superior cervical neurons and rat chromaffin cells.

•Dunn, P.M. (1999). UCL 1684: A potent blocker of Ca2+-activated K+ channels in rat adrenal chromaffin cells in culture. European Journal of Pharmacology 368: 119- 123. The novel K+ channel blocker 6,10-diaza-3(1,3)8,(1,4)-dibenzena-1,5(1,4)- diquinolinacy clodecaphane (UCL 1684) has been tested for its ability to inhibit Ca2+ -activated K+ currents in cultured rat chromaffin cells. Low nanomolar concentrations of UCL 1684 produced a rapid and reversible inhibition of the slow, apamin-sensitive, tail current activated by a depolarizing voltage command. This compound also inhibited the muscarine activated outward current with an IC50 of 6 nM. These results confirm UCL 1684 to be the most potent non-peptidic blocker of the apamin-sensitive Ca2+ -activated K+ channel so far described.

•Marty, A. (1981). Ca-dependent K channels with large unitary conductance in chromaffin cell membranes. Nature 291: 497-500.

•Marty, A. (1983). Blocking of large unitary calcium-dependent potassium currents by internal sodium ions. Pflugers Arch 396(2): 179-181.

Page 179 AHP, BK- and SK-channel references

The effects of internal Na on the outflow of K ions through a certain type of Ca-dependent K channels of chromaffin cells (Marty, 1981) were studied in isolated patches. Application of 20 mM Na ions to inside-out patches (Hamill, Marty, Neher, Sakamann and Sigworth, 1981) on the cytoplasmic (called hereafter internal) side of the membrane induced short interruptions of the single channel currents. This effect was voltage dependent. In addition, internal Na ions reduced the opening probability of the channels. Outside-out patch recordings were obtained with 20 mM internal Na and varying external K concentrations. The blocking effects of internal Na ions were relieved by increasing the external K concentration.

•Marty, A. (1989). The physiological role of calcium-dependent channels. Trends in Neurosciences 12: 420-424. Calcium (Ca2+)-dependent channels may be classified in three broad categories, which are, respectively, selective for potassium ions, for chloride ions, and for monovalent cations. The usual action of Ca2+ is to increase the probability of opening of the channels, but examples of the reverse, Ca2+-induced inhibition of ion channels, have recently been found. Ca2+-dependent channels help to shape the action potentials of excitable cells as well as the synaptic currents of muscular and neuronal preparations. They are involved in several aspects of electrolyte transport including regulation of osmolarity in animal cells and of turgor in cells, electrolyte secretion in exocrine glands, fluid absorption and secretion in epithelial tissues.

•Park, Y.B. (1994). Ion selectivity and gating of small conductance Ca2+-activated K+ channels in cultured rat adrenal chromaffin cells. Journal of Physiology (London) 481(Pt 3): 555-70. 1. The ion selectivity and gating of apamin-sensitive, small conductance Ca2+- activated K+ (SK) channels were studied in cultured rat adrenal chromaffin cells using patch clamp techniques. 2. The amplitude of slow tail currents showed a bell- shaped dependence on depolarization potentials. Slow tail currents were abolished in a Ca2+-free external solution or by adding 100 µM Cd2+ to the external solution. Reversal potentials followed the predictions of the Nernst equation for a K+ electrode. 3. Slow tail currents were largely blocked by external application of apamin (dissociation constant, Kd, 4.4 nM), (+)-tubocurarine (Kd, 20 µM), and tetraethylammonium (Kd, 5.4 mM). 4. The relative permeability (PX/PK, where X may be any one of the ions listed) of SK channels was: Tl+ (1.87) > K+ (1.0) > Rb+ (0.81) >

Page 180 AHP, BK- and SK-channel references

Cs+ (0.16) > NH4+ (0.11). Na+, Li+ and methylamine were not measurably permeant (PX/PK K+ (1.0) > Rb+ (0.85) > Cs+ (0.45) approximately NH4+ (0.44). 5. With mixtures of Tl+ and K+, SK channels showed anomalous mole-fraction behaviour. 6. Ca2+ dependence of SK channel gating was studied using inside-out macropatches. The [Ca2+] required for half-maximal activation and the Hill coefficient were 0.69 µM and 1.7, respectively, and independent of membrane potentials. 7. Single-channel conductance was 13-14 pS (160 mM K+).

•Prakriya, M. & Lingle, C.J. (1999). BK channel activation by brief depolarizations requires Ca2+ influx through L- and Q-type Ca2+ channels in rat chromaffin cells. J Neurophysiol 81(5): 2267-2278. BK channel activation by brief depolarizations requires Ca2+ influx through L- and Q-type Ca2+ channels in rat chromaffin cells. Ca2+- and voltage-dependent BK-type K+ channels contribute to action potential repolarization in rat adrenal chromaffin cells. Here we characterize the Ca2+ currents expressed in these cells and identify the Ca2+ channel subtypes that gate the activation of BK channels during Ca2+ influx. Selective Ca2+ channel antagonists indicate the presence of at least four types of high-voltage-gated Ca2+ channels: L-, N-, P, and Q type. Mean amplitudes of the L-, N-, P-, and Q-type Ca2+ currents were 33, 21, 12, and 24% of the total Ca2+ current, respectively. Five-millisecond Ca2+ influx steps to 0 mV were employed to assay the contribution of Ca2+ influx through these Ca2+ channels to the activation of BK current. Blockade of L-type Ca2+ channels by 5 µM nifedipine or Q-type Ca2+ channels by 2 µM Aga IVA reduced BK current activation by 77 and 42%, respectively. In contrast, blockade of N-type Ca2+ channels by brief applications of 1-2 µM CnTC MVIIC or P-type Ca2+ channels by 50-100 nM Aga IVA reduced BK current activation by only 11 and 12%, respectively. Selective blockade of L- and Q-type Ca2+ channels also eliminated activation of BK current during action potentials, whereas almost no effects were seen by the selective blockade of N- or P-type Ca2+ channels. Finally, the L-type Ca2+ channel agonist Bay K 8644 promoted activation of BK current by brief Ca2+ influx steps by more than twofold. These data show that, despite the presence of at least four types of Ca2+ channels in rat chromaffin cells, BK channel activation in rat chromaffin cells is predominantly coupled to Ca2+ influx through L- and Q-type Ca2+ channels.

•Xie, J. & McCobb, D.P. (1998). Control of alternative splicing of potassium channels by stress hormones. Science 280: 443-446.

Page 181 AHP, BK- and SK-channel references

Many molecular mechanisms for neural adaptation to stress remain unknown. Expression of alternative splice variants of Slo, a gene encoding calcium- and voltage-activated potassium channels, was measured in rat adrenal chromaffin tissue from normal and hypophysectomized animals. Hypophysectomy triggered an abrupt decrease in the proportion of Slo transcripts containing a "STREX" exon. The decrease was prevented by adrenocorticotropic hormone injections. In Xenopus oocytes, STREX variants produced channels with functional properties associated with enhanced repetitive firing. Thus, the hormonal stress axis is likely to control the excitable properties of epinephrine-secreting cells by regulating alternative splicing of Slo messenger RNA.

SENSORY NEURONS

•Navaratnam, D.S., Bell, T.J., Tu, T.D., Cohen, E.L. & Oberholtzer, J.C. (1997). Differential distribution of Ca2+-activated K+ channel splice variants among hair cells along the tonotopic axis of the chick cochlea. Neuron 19: 1077- 1085. We have cloned from the receptor epithelium of the chick cochlea a family of alternatively spliced cDNAs derived from cslo, which encodes a Ca2+-activated K+ channel like those shown to help determine the resonant frequency of electrically tuned hair cells. Our results from PCRs using template RNAs from both tonotopically subdivided receptor epithelia and single hair cells demonstrate differential exon usage along the frequency axis of the epithelium at multiple splice sites in cslo. We also show that single hair cells express more than one splice variant at a given splice site. Since channel isoforms encoded by differentially spliced slo transcripts in other species are functionally heterogeneous, these data suggest that differential processing of slo transcripts may account, at least in part, for the systematic variation in hair-cell membrane properties along the frequency axis of electrically tuned auditory receptor epithelia.

•Smith, M.R., Nelson, A.B. & Du Lac, S. (2002). Regulation of firing response gain by calcium-dependent mechanisms in vestibular nucleus neurons. Journal of Neurophysiology 87(4): 2031-2041. Behavioral reflexes can be modified by experience via mechanisms that are largely unknown. Within the circuitry for the vestibuloocular reflex (VOR), neurons in the medial vestibular nucleus (MVN) show adaptive changes in firing rate

Page 182 AHP, BK- and SK-channel references responses that are correlated with VOR gain (the ratio of evoked eye velocity to input head velocity). Although changes in synaptic strength are typically assumed to underlie gain changes in the VOR, modulation of intrinsic ion channels that dictate firing could also play a role. Little is known, however, about how ion channel function or regulation contributes to firing responses in MVN neurons. This study examined contributions of calcium-dependent currents to firing responses in MVN neurons recorded with whole cell patch electrodes in rodent brain stem slices. Firing responses were remarkably linear over a wide range of firing rates and showed modest spike frequency adaptation. Firing response gain, the ratio of evoked firing rate to input current, was reduced by increasing extracellular calcium and increased either by lowering extracellular calcium or with antagonists to SK- and BK-type calcium-dependent potassium channels and N- and T-type calcium channels. Blockade of SK channels occluded gain increases via N-type calcium channels, while blocking BK channels occluded gain increases via presumed T-type calcium channels, indicating specific coupling of potassium channels and their calcium sources. Selective inhibition of Ca2+/calmodulin-dependent kinase II and broad-spectrum inhibition of phosphatases modulated gain via BK-dependent pathways, indicating that firing responses are tightly regulated. Modulation of firing response gain by phosphorylation provides an attractive mechanism for adaptive control of VOR gain.

•Yuhas, W.A. & Fuchs, P.A. (1999). Apamin-sensitive, small-conductance, calcium- activated potassium channels mediate cholinergic inhibition of chick auditory hair cells. Journal of Comparative Physiology A: Sensory, Neural and Behavioral Physiology 185(5): 455-462. Acetylcholine released from efferent neurons in the cochlea causes inhibition of mechanosensory hair cells due to the activation of calcium-dependent potassium channels. Hair cells are known to have large-conductance, "BK"-type potassium channels associated with the afferent synapse, but these channels have different properties than those activated by acetylcholine. Whole-cell (tight-seal) and cell- attached patch-clamp recordings were made from short (outer) hair cells isolated from the chicken basilar papilla (cochlea equivalent). The peptides apamin and charybdotoxin were used to distinguish the calcium-activated potassium channels involved in the acetylcholine response from the BK-type channels associated with the afferent synapse. Differential toxin blockade of these potassium currents provides definitive evidence that ACh activates apamin-sensitive, "SK"-type potassium channels, but does not activate carybdotoxin-sensitive BK channels. This

Page 183 AHP, BK- and SK-channel references conclusion is supported by tentative identification of small-conductance, calcium- sensitive but voltage-insensitive potassium channels in cell-attached patches. The distinction between these channel types is important for understanding the segregation of opposing afferent and efferent synaptic activity in the hair cell, both of which depend on calcium influx. These different calcium-activated potassium channels serve as sensitive indicators for functionally significant calcium influx in the hair cell.

MUSCLE

•Auguste, P., Hugues, M., Borsotto, M., Thibault, J., Romey, G., Coppola, T. & Lazdunski, M. (1992). Characterization and partial purification from pheochromocytoma cells of an endogenous equivalent of scyllatoxin, a scorpion toxin which blocks small conductance Ca2+-activated K+ channels. Brain Research 599: 230-236. This work describes the partial purification of a heat-stable peptide which has the same properties as the scorpion toxin, scyllatoxin, a specific blocker of one class of Ca2+-activated K+ channels: (i) it competes with [125I]apamin for binding to the same site, (ii) like apamin and scyllatoxin, it blocks the after-potential hyperpolarization in skeletal muscle cells in culture, (iii) like apamin and scyllatoxin, it contracts guinea-pig taenia coli relaxed by epinephrine, (iv) it cross-reacts with antibodies raised against scyllatoxin but not with antibodies raised against apamin.

•Auguste, P., Hugues, M., Grave, B., Gesquiere, J.-C., Maes, P., Tartar, A., Romey, G., Schweitz, H. & Lazdunski, M. (1990). Leiurotoxin I (Scyllatoxin), a peptide ligand for Ca2+-activated K+ channels. Journal of Biological Chemistry 265: 4753-4759. Leiurotoxin I (scyllatoxin) is a 31-amino acid polypeptide from the venom of the scorpion Leiurus quinquestriatus hebraeus which has been previously isolated and sequenced by others. This paper reports (i) the total synthesis of this scorpion neurotoxin as well as some aspects of its structure-function relationships; (ii) the synthesis of the analog [Tyr2]leiurotoxin I (scyllatoxin) that has been monoiodinated at high specific radioactivity (2000 Ci/mmol) and has served for the characterization of the properties of 125I-[Tyr2]leiurotoxin I binding sites (Kd = 80 pM, molecular mass of 27 and 57 kDa for two polypeptides in the leiurotoxin I binding protein); (iii) the similarity of physiological actions between leiurotoxin I

Page 184 AHP, BK- and SK-channel references and apamin. Both toxins contract Taenia coli previously relaxed with epinephrine, both toxins block the after-hyperpolarization due to Ca2(+)-activated K+ channel activity in muscle cells in culture; (iv) the probable identity of binding sites for apamin and leiurotoxin I. In spite of a different chemical structure apamin competitively inhibits 125I-[Tyr2] leiurotoxin I binding and vice versa. Moreover, the peculiar effects of K+ on 125I-[Tyr2]leiurotoxin I binding are identical to those previously observed for 125I-apamin binding.

•Auguste, P., Hugues, M., Mourre, C., Moinier, D., Tartar, A. & Lazdunski, M. (1992). Scyllatoxin, a blocker of Ca2+-activated K+ channels: Structure- function relationships and brain localization of the binding sites. Biochemistry 31(3): 648-654. Chemical modifications of scyllatoxin (leiurustoxin I) have shown that two arginines in the sequence, Arg6 and Arg13, are essential both for binding to the Ca2+-activated K+ channel protein and for the functional effect of the toxin. His31 is important both for the binding activity of the toxin and for the induction of contractions on taenia coli. However, although its iodination drastically decreases the toxin activity, it does not abolish it. Chemical modification of residues or of Glu27 does not significantly alter toxin binding, but it drastically decreases potency with respect to contraction of taenia coli. The same observation has been made after chemical modification of the lysine residues. The brain distribution of scyllatoxin binding sites has been analyzed by quantitative autoradiographic analysis. It indicates that apamin (a bee venom toxin) binding sites are colocalized with scyllatoxin binding sites. The results are consonant with the presence of apamin/scyllatoxin binding sites associated with Ca2+-activated K+ channels. High- affinity binding sites for apamin can be associated with very-high-affinity (less than 70 pM), high-affinity (approximately 100-500 pM), or moderate-affinity (greater than 800 pM) binding sites for scyllatoxin.

•Banks, B.E., Brown, C., Burgess, G.M., Burnstock, G., Claret, M., Cocks, T.M. & Jenkinson, D.H. (1979). Apamin blocks certain neurotransmitter-induced increases in potassium permeability. Nature 282: 415-417.

•Blatz, A.L. & Magleby, K.L. (1984). Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat muscle. Journal of General Physiology 84(1): 1-23.

Page 185 AHP, BK- and SK-channel references

The conductance and selectivity of the Ca-activated K channel in cultured rat muscle was studied. Shifts in the reversal potential of single channel currents when various cations were substituted for Ki+ were used with the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The selectivity was Tl+ greater than K+ greater than Rb+ greater than NH4+, with permeability ratios of 1.2, 1.0, 0.67, and 0.11. Na+, Li+, and Cs+ were not measurably permeant, with permeabilities less than 0.05 that of K+. Currents with the various ions were typically less than expected on the basis of the permeability ratios, which suggests that the movement of an ion through the channel was not independent of the other ions present. For a fixed activity of Ko+ (77 mM), plots of single channel conductance vs. activity of Ki+ were described by a two-barrier model with a single saturable site. This observation, plus the finding that the permeability ratios of Rb+ and NH+4 to K+ did not change with ion concentration, is consistent with a channel that can contain a maximum of one ion at any time. The empirically determined dissociation constant for the single saturable site was 100 mM, and the maximum calculated conductance for symmetrical solutions of K+ was 640 pS. TEAi+ (tetraethylammonium ion) reduced single channel current amplitude in a voltage-dependent manner. This effect was accounted for by assuming voltage-dependent block by TEA+ (apparent dissociation constant of 60 mM at 0 mV) at a site located 26% of the distance across the membrane potential, starting at the inner side. TEAo+ was much more effective in reducing single channel currents, with an apparent dissociation constant of approximately 0.3 mM.

•Blatz, A.L. & Magleby, K.L. (1986). Single apamin-blocked Ca-activated K+ channels of small conductance in cultured rat skeletal muscle. Nature 323(6090): 718-20. Action potentials in many excitable cells are followed by a prolonged afterhyperpolarization that modulates repetitive firing. Although it is established that the afterhyperpolarization is produced by Ca-activated K+ currents, the basis of these currents is not known. The large conductance (250 pS) Ca-activated K+ channel (BK channel) is not a major contributor to the afterhyperpolarization in non-innervated skeletal muscle and some nerve cells, because apamin, a neurotoxic component of bee venom, abolishes the afterhyperpolarization but does not block BK channels, and 5 mM extracellular tetraethylammonium ion (TEA) blocks BK channels but does not reduce the afterhyperpolarization. We now report single-channel currents from small conductance (10-14 pS) Ca-activated K+ channels (SK channels)

Page 186 AHP, BK- and SK-channel references with the necessary properties to account for the afterhyperpolarization. SK channels are blocked by apamin but not by 5 mM external TEA (TEAo). They are also highly Ca-sensitive at the negative membrane potentials associated with the AHP.

•Blatz, A.L. & Magleby, K.L. (1987). Calcium-activated potassium channels. Trends in Neurosciences 10: 463-467.

•Dunn, P.M., Benton, D.C., Campos Rosa, J., Ganellin, C.R. & Jenkinson, D.H. (1996). Discrimination between subtypes of apamin-sensitive Ca2+-activated K+ channels by gallamine and a novel bis-quaternary quinolinium cyclophane, UCL 1530. British Journal of Pharmacology 117(1): 35-42. 1. Gallamine, dequalinium and a novel bis-quaternary cyclophane, UCL 1530 (8,19-diaza-3(1,4),5(1,4)-dibenzena-1 (1,4),7(1,4)-diquinolina- cyclononadecanephanedium) were tested for their ability to block actions mediated by the small conductance, apamin-sensitive Ca2+- activated K+ (SKCa) channels in rat cultured sympathetic neurones and guinea-pig isolated hepatocytes. 2. SKCa channel block was assessed in sympathetic neurones by the reduction in the slow afterhyperpolarization (AHP) that follows an action potential, and in hepatocytes by the inhibition of the SKCa mediated net loss of K+ that results from the application of angiotensin II. 3. The order of potency for inhibition of the AHP in sympathetic neurones was UCL 1530 > dequalinium > gallamine, with IC50 values of 0.08 ± 0.02, 0.60 ± 0.05 and 68.0 ± 8.4 µM respectively, giving an equi-effective molar ratio between gallamine and UCL 1530 of 850. 4. The same three compounds inhibited angiotensin II-evoked K+ loss from guinea-pig hepatocytes in the order dequalinium > UCL 1530 > gallamine, with an equi-effective molar ratio for gallamine to UCL 1530 of 5.8, 150 fold less than in sympathetic neurones. 5. Dequalinium and UCL 1530 were as effective on guinea-pig as on rat sympathetic neurones. 6. UCL 1530 at 1 µM had no effect on the voltage-activated Ca2+ current in rat sympathetic neurones, but inhibited the hyperpolarization produced by direct elevation of cytosolic Ca2+. 7. Direct activation of SKCa channels by raising cytosolic Ca2+ in hepatocytes evoked an outward current which was reduced by the three blockers, with dequalinium being the most potent. 8. These results provide evidence that the SKCa channels present in guinea-pig hepatocytes and rat cultured sympathetic neurones are different, and that this is not attributable to species variation. UCL 1530 and gallamine should be useful tools for the investigation of subtypes of apamin- sensitive K+ channels.

Page 187 AHP, BK- and SK-channel references

•Gater, P.R., Haylett, D.G. & Jenkinson, D.H. (1985). Neuromuscular blocking agents inhibit receptor-mediated increases in the potassium permeability of intestinal smooth muscle. British Journal of Pharmacology 86: 861-868. The neuromuscular blocking agents tubocurarine, atracurium and pancuronium have been tested for their ability to inhibit receptor-mediated increases in the K+ permeability of intestinal smooth muscle. All three agents, as well as the bee venom peptide apamin, reduced both the resting efflux of 86Rb and the increase in efflux caused by the application of either bradykinin (1 µM) or an alpha 1-adrenoceptor agonist, amidephrine (20 µM), to depolarized strips of guinea-pig taenia caeci. This suggested that like apamin, the neuromuscular blocking agents inhibit the Ca2+- dependent K+ permeability (PK(Ca] mechanism which in this tissue is activated by a variety of membrane receptors. The concentrations (IC50S) of atracurium, pancuronium and (+)-tubocurarine which reduced the effect of amidephrine on 86Rb efflux by 50% were 12, 37 and 67 microM respectively. Also in keeping with an ability to block PK(Ca), the neuromuscular blockers and apamin reduced the inhibition by amidephrine and bradykinin of physalaemin-mediated contractions of the taenia caeci. The IC50 values were 15, 31 and 120 µM for atracurium, tubocurarine and pancuronium respectively, and 2.3 nM for apamin. Each of the neuromuscular blockers, and apamin, increased the spontaneous contractions of the rabbit duodenum and blocked the inhibitory effect of amidephrine thereon. It is concluded that the PK(Ca) mechanism in the longitudinal smooth muscle of the intestine It is concluded that the PK(Ca) mechanism in the longitudinal smooth muscle of the intestine resembles that of hepatocytes and sympathetic ganglion cells in its susceptibility to inhibition by neuromuscular blocking agents, as well as by apamin.

•Herrera, G.M. & Nelson, M.T. (2002). Differential regulation of SK and BK channels by Ca2+ signals from Ca2+ channels and ryanodine receptors in guinea-pig urinary bladder myocytes. Journal of Physiology (London) 541(Pt. 2): 483-492. Small-conductance (SK) and large-conductance (BK) Ca(2+)-activated K(+) channels are key regulators of excitability in urinary bladder smooth muscle (UBSM) of guinea-pig. The overall goal of this study was to define how SK and BK channels respond to Ca(2+) signals from voltage-dependent Ca(2+) channels (VDCCs) in the surface membrane and from ryanodine-sensitive Ca(2+) release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) membrane. To characterize the role of SK channels in UBSM, the effects of the SK channel blocker apamin on

Page 188 AHP, BK- and SK-channel references phasic contractions were examined. Apamin caused a dose-dependent increase in the amplitude of phasic contractions over a broad concentration range (10(-10) to 10(-6) M). To determine the effects of Ca(2+) signals from VDCCs and RyRs to SK and BK channels, whole cell membrane current was measured in isolated myocytes bathed in physiological solutions. Depolarization (-70 to +10 mV for 100 ms) of isolated myocytes caused an inward Ca(2+) current (I(Ca)), followed by an outward current. The outward current was reduced in a dose-dependent manner by apamin (10(-10) to 10(-6) M), and designated I(SK). I(SK) had a mean amplitude of 53.8 ± 6.1 pA or ~1.4 pA pF(-1) at +10 mV. The amplitude of I(SK) correlated with the peak I(Ca). Blocking I(Ca) abolished I(SK). In contrast, I(SK) was insensitive to the RyR blocker ryanodine (10 µM). These data indicate that Ca(2+) signals from VDCCs, but not from RyRs, activate SK channels. BK channel currents (I(BK)) were isolated from other currents by using the BK channel blockers tetraethylammonium ions (TEA(+); 1 mM) or iberiotoxin (200 nM). Voltage steps evoked transient and steady-state I(BK) components. Transient BK currents have previously been shown to result from BK channel activation by local Ca(2+) release through RyRs ('Ca(2+) sparks'). Transient BK currents were inhibited by ryanodine (10 µM), as expected, and had a mean amplitude of 152.6 pA at +10 mV. The mean number of transient BK currents during a voltage step (range 0 to 3) correlated with I(Ca). There was a long delay (52.4 ± 2.7 ms) between activation of I(Ca) and the first transient BK current. In contrast, ryanodine did not affect the steady-state BK current (mean amplitude 135.4 pA) during the voltage step. The steady-state BK current was reduced 95 % by inhibition of VDCCs, suggesting that this process depends largely on Ca(2+) entry through VDCCs and not Ca(2+) release through RyRs. These results indicate that Ca(2+) entry through VDCCs activates both BK and SK channels, but Ca(2+) release (Ca(2+) sparks) through RyRs activates only BK channels.

•Koh, S.D., Dick, G.M. & Sanders, K.M. (1997). Small-conductance Ca2+- dependent K+ channels activated by ATP in murine colonic smooth muscle. American Journal of Physiology 273(6 Pt 1): C2010-21. The patch-clamp technique was used to determine the ionic conductances activated by ATP in murine colonic smooth muscle cells. Extracellular ATP, UTP, and 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) increased outward currents in cells with amphotericin B-perforated patches. ATP (0.5-1 mM) did not affect whole cell currents of cells dialyzed with solutions containing ethylene glycol-bis(beta- aminoethyl ether)-N,N,N',N'-tetraacetic acid. Apamin (3 x 10(-7) M) reduced the

Page 189 AHP, BK- and SK-channel references outward current activated by ATP by 32 ± 5%. Single channel recordings from cell- attached patches showed that ATP, UTP, and 2-MeS-ATP increased the open probability of small-conductance, Ca2+- dependent K+ channels with a slope conductance of 5.3 ± 0.02 pS. Caffeine (500 µM) enhanced the open probability of the small- conductance K+ channels, and ATP had no effect after caffeine. Pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS, 10(-4) M), a nonselective P2 receptor antagonist, prevented the increase in open probability caused by ATP and 2-MeS-ATP. PPADS had no effect on the response to caffeine. ATP-induced hyperpolarization in the murine colon may be mediated by P2y-induced release of Ca2+ from intracellular stores and activation of the 5.3-pS Ca2+- activated K+ channels.

•Lazdunski, M. (1983). Apamin, a neurotoxin specific for one class of Ca2+- dependent K+ channels. Cell Calcium 4: 421-428.

•Lazdunski, M., Romey, G., Schmid-Antomarchi, H., Renaud, J.P., Mourre, C., Hugues, M. & Fosset, M. (1988). The apamin-sensitive Ca2+-dependent K+ channel: Molecular properties, differentiation, involvement in muscle disease, and endogenous ligands in mammalian brain. Handbook of Experimental Pharmacology 83: 135-145.

•Neelands, T.R., Herson, P.S., Jacobson, D., Adelman, J.P. & Maylie, J. (2001). Small-conductance calcium-activated potassium currents in mouse hyperexcitable denervated skeletal muscle. Journal of Physiology (London) 536(Pt. 2): 397-407. 1. Hyperexcitability in denervated skeletal muscle is associated with the expression of SK3, a small-conductance Ca2+-activated K+ channel (SK channel). SK currents were examined in dissociated fibres from flexor digitorum brevis (FDB) muscle using the whole-cell patch clamp configuration. 2. Depolarization activated a K+-selective, apamin-sensitive and iberiotoxin-insensitive current, detected as a tail current upon repolarization, in fibres from denervated but not innervated muscle. Dialysis of the fibres with 20 mM EGTA in the patch pipette solution eliminated the tail current, consistent with this current reflecting Ca2+-activated SK channels expressed only in denervated muscle. 3. Activation of SK tail currents depended on the duration of the depolarizing pulse, consistent with a rise in intracellular Ca2+ due to release from the sarcoplasmic reticulum (SR) and influx through voltage-gated Ca2+ channels. 4. The envelope of SK tail currents was

Page 190 AHP, BK- and SK-channel references diminished by 10 microM ryanodine for all pulse durations, whereas 2 mM cobalt reduced the SK tail current for pulses greater than 80 ms, demonstrating that Ca2+ release from the SR during short pulses primarily activated SK channels. 5. In current clamp mode with the resting membrane potential set at -70 mV, denervation decreased the action potential threshold by approximately 8 mV. Application of apamin increased the action potential threshold in denervated fibres to that measured in innervated fibres, suggesting that SK channel activity modulates the apparent action potential threshold. 6. These results are consistent with a model in which SK channel activity in the T-tubules of denervated skeletal muscle causes a local increase in K+ concentration that results in hyperexcitability.

•Pallotta, B.S. (1985). N-bromoacetamide removes a calcium-dependent component of channel opening from calcium-activated potassium channels in rat skeletal muscle. Journal of General Physiology 86(3060): 601-611. Calcium-activated potassium channels from cultured rat skeletal muscle were treated with the protein-modifying reagent N-bromoacetamide (NBA) (0.3-1 mM) and studied in excised patches using patch-clamp techniques. After NBA treatment, channels opened only occasionally, and, in contrast to untreated channels, the open probability was no longer sensitive to intracellular surface calcium ions (1 nM to 100 µM). Channel activity did, however, exhibit a voltage dependence similar in direction and magnitude to that shown before NBA treatment (increasing e-fold with 19 mV depolarization). Distributions of open channel lifetimes revealed that NBA treatment virtually abolished openings of long duration, which suggests that this class of openings requires calcium sensitivity. These effects were not reversed by subsequent washing. Quantitatively similar open probability, voltage dependence, and open-interval distributions were observed in untreated channels in calcium-free medium. These results suggest that NBA removed a calcium-dependent component of channel opening, and that normal channels are able to open in the absence of significant intracellular calcium concentrations.

•Pallotta, B.S., Magleby, K.L. & Barrett, J.N. (1981). Single channel recordings of Ca2+-activated K+ currents in rat muscle cell culture. Nature 293: 471-474.

•Pribnow, D., Johnson-Pais, T., Bond, C.T., Keen, J., Johnson, R.A., Janowsky, A., Silvia, C., Thayer, M., Maylie, J. & Adelman, J.P. (1999).

Page 191 AHP, BK- and SK-channel references

Skeletal muscle and small-conductance calcium-activated potassium channels. Muscle Nerve 22(6): 742-50. Skeletal muscle becomes hyperexcitable following denervation and when cultured in the absence of nerve cells. In these circumstances, the bee venom peptide toxin apamin, a blocker of small-conductance calcium- activated potassium (SK) channels, dramatically reduces the hyperexcitability. In this report, we show that SK3 channels are expressed in denervated skeletal muscle and in L6 cells. Action potentials evoked from normal innervated rat skeletal muscle did not exhibit an afterhyperpolarization, indicating a lack of SK channel activity; very low levels of apamin binding sites, SK3 protein, or SK3 mRNA were present. However, denervation resulted in apamin-sensitive afterhyperpolarizations and increased apamin binding sites, SK3 protein, and SK3 mRNA. Cultured rat L6 myoblasts and differentiated L6 myotubes contained similar levels of SK3 mRNA, although apamin- sensitive SK currents and apamin binding sites were detected only following myotube differentiation. Therefore, different molecular mechanisms govern SK3 expression levels in denervated muscle compared with muscle cells differentiated in culture.

•Romey, G., Hugues, M., Schmid-Antomarchi, H. & Lazdunski, M. (1984). Apamin: A specific toxin to study a class of Ca2+-dependent K+ channels. Journal of Physiology (Paris) 79: 259-264. Apamin is a bee venom neurotoxin of 18 amino-acids containing two disulfide bridges. Current clamp and voltage clamp experiments have shown that externally applied apamin blocks specifically at low concentration (0.1 µM) the Ca2+-dependent slow K+ conductance which mediates the long-lasting after-hyperpolarization in neuroblastoma cells and rat muscle cells in culture. The apamin-sensitive Ca2+- dependent slow K+ conductance is voltage-dependent and tetraethylammonium (TEA) insensitive. It is distinct from the high conductance Ca2+-dependent K+ channel revealed by patch clamp experiments. Biochemical characterization of the apamin receptor in rat striated muscle, neuroblastoma cells, rat synaptosomes, smooth muscles and hepatocytes was carried out with the use of a radiolabelled monoiodo- apamin derivative (125I-apamin) of high specific radioactivity (2 000 Ci/mmol). The dissociation constant of the apamin-receptor complex is between 15 and 60 pM for all tissue preparations. The density of binding sites is very low; it varied between 1 and 40 fmol/mg of protein. Radiation inactivation analysis indicates a molecular weight for the apamin receptor of 250 000 daltons whereas affinity labelling with 125I-apamin results in covalent labelling of a single polypeptide chain with a

Page 192 AHP, BK- and SK-channel references molecular weight of about 30 000 daltons. We conclude that the apamin-sensitive Ca2+-dependent K+ channel is probably a large oligomeric structure containing one subunit of 30K daltons.

•Romey, G. & Lazdunski, M. (1984). The coexistence in rat muscle cells of two distinct classes of Ca2+-dependent K+ channels with different pharmacological properties and different physiological functions. Biochemical and Biophysical Research Communications 118: 669-674. Ca2+-dependent K+ channels responsible for the long-lasting after- hyperpolarization in rat muscle cells in culture are not those extensively studied by the patch-clamp technique. The first ones are blocked by apamin, a bee venom polypeptide, and they are unaffected by tetraethylammonium (TEA) whereas the second ones are blocked by TEA and unaffected by apamin. These two Ca2+-dependent K+ channels coexist in rat muscle cells in culture but also probably in many other cellular types.

•Romey, G., Rieger, F., Renaud, J.F., Pinçon-Raymond, M. & Lazdunski, M. (1986). The electrophysiological expresssion of Ca2+ channels and of apamin sensitive Ca2+ activated K+ channels is abolished in skeletal muscle cells from mice with muscular dysgenesis. Biochemical and Biophysical Research Communications 136: 935-940. Action potentials of myotubes in culture prepared from 18-19 day-old mouse embryos have a contractile activity and action potentials that are followed by a long lasting after hyperpolarization (AHP) which is blocked by apamin. Myotubes prepared from embryos of mice with muscular dysgenesis (mdg/mdg) did not contract and had action potentials which were never followed by AHPs. Voltage-clamp experiments have shown that Na+ channel activity was identical in mutant and control muscles and that the activity of fast and slow Ca2+ channels was nearly absent in the mutant.

•Roxburgh, C.J., Ganellin, C.R., Shiner, M.A., Benton, D.C., Dunn, P.M., Ayalew, Y. & Jenkinson, D.H. (1996). The synthesis and some pharmacological actions of the enantiomers of the K(+)-channel blocker cetiedil. Journal of Pharmacy and Pharmacology 48(8): 851-857. Cetiedil ((±)-2-cyclohexyl-2-(3-thienyl)ethanoic acid 2-(hexahydro-1 H-azepin- 1-yl) ethyl ester) possesses anti-sickling and analgesic, antispasmodic, local

Page 193 AHP, BK- and SK-channel references anaesthetic and vasodilator activities. A total synthesis and circular dichroism spectra of the enantiomers of cetiedil is described, together with a comparison of their effectiveness as blockers of the Ca2+-activated K+ permeability of rabbit erythrocytes; the contractile response of intestinal smooth muscle to acetylcholine; the Ca2+-dependent contraction of depolarized intestinal muscle; and the cell volume-sensitive K+ permeability (Kvol) of liver cells. The enantiomers did not differ substantially in their ability to block the Ca2+-activated K+ permeability of rabbit red cells or in their effectiveness as blockers of the contractile response of depolarized smooth muscle to externally applied Ca2+. There was a clear difference in the muscarinic blocking activity of the enantiomers, as assessed by inhibition of the contractile response of intestinal smooth muscle to acetylcholine; (+)-cetiedil was 7.7 ± 0.2 (s.d.) times more active than the (-) from. The enantiomers also differed in their potency as blockers of the increase in membrane conductance which occurs when liver cells swell. The concentration of (+)-cetiedil needed to reduce the conductance increase by 50% was 2.04 ± 0.54 (s.d.) µM; (-)-cetiedil was 2.6 ± 0.8 (s.d.) times less active (IC50 of 5.2 ± 1.2 µM). Differences in the biological actions of the enantiomers of cetiedil indicate that a more extensive study could be rewarding in relation to the use of the enantiomers both in therapeutics and in the study of K+ channels.

•Sansom, S.C., Stockland, J.D., Hall, D. & Williams, B. (1997). Regulation of calcium-activated potassium channels by protein phosphatase 2A. Journal of Biological Chemistry 272: 9902-9906. Vasodilating agents induce relaxation of mesangial cells, in part through cGMP-mediated activation of large calcium-activated potassium channels (BKCa). Normally quiescent in cell-attached patches, the response of BKCa to nitric oxide, atrial natriuretic peptide, and dibutyryl cGMP (Bt2cGMP) is characterized by a biphasic increase and then decrease ("rundown") in open probability. Using the patch- clamp method in conjunction with phosphatase inhibitors, we investigated whether the run-down phase was the result of dephosphorylation by an endogenous protein phosphatase. In cell-attached patches, cantharidic acid (500 nM), okadaic acid (100 nM), and calyculin A (100 nM), nondiscriminant inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A) at these concentrations, caused a significantly greater and sustained response of BKCa to Bt2cGMP. Within 2 min, the response of BKCa to the combination of cantharidic acid and Bt2cGMP was greater than the response to these agents added separately. Incubation of mesangial cells with okadaic acid for

Page 194 AHP, BK- and SK-channel references

20 min at a concentration (5 nM) specific for PP2A increased the basal open probability of BKCa and completely inhibited rundown after activation by Bt2cGMP. Incubation with calyculin A (10 nM), a more potent inhibitor of PP1, did not affect BKCa activity. In inside-out patches, Bt2cGMP plus MgATP caused a sustained activation of BKCa that was inhibited by exogenous PP2A but not PP1. It is concluded that either BKCa or a tightly associated regulator of BKCa is a common substrate for endogenous cGMP-activated protein kinase, which activates BKCa, and PP2A, which inactivates BKCa, in human mesangial cells.

•Vogalis, F., Furness, J.B. & Kunze, W.A. (2001). Afterhyperpolarization current in myenteric neurons of the guinea pig duodenum. Journal of Neurophysiology 85(5): 1941-1951. Whole cell patch and cell-attached recordings were obtained from neurons in intact ganglia of the myenteric plexus of the guinea pig duodenum. Two classes of neuron were identified electrophysiologically: phasically firing AH neurons that had a pronounced slow afterhyperpolarization (AHP) and tonically firing S neurons that lacked a slow AHP. We investigated the properties of the slow AHP and the underlying current (I(AHP)) to address the roles of Ca(2+) entry and Ca(2+) release in the AHP and the characteristics of the K(+) channels that are activated. AH neurons had a resting potential of -54 mV and the AHP, which followed a volley of three suprathreshold depolarizing current pulses delivered at 50 Hz through the pipette, averaged 11 mV at its peak, which occurred 0.5-1 s following the stimulus. The duration of these AHPs averaged 7 s. Under voltage-clamp conditions, I(AHP)'s were recorded at holding potentials of -50 to -65 mV, following brief depolarization of AH neurons (20-100 ms) to positive potentials (+35 to +50 mV). The null potential of the I(AHP) at its peak was -89 mV. The AHP and I(AHP) were largely blocked by omega-conotoxin GVIA (0.6-1 &mgr;M). Both events were markedly decreased by caffeine (2-5 mM) and by ryanodine (10-20 µM) added to the bathing solution. Pharmacological suppression of the I(AHP) with TEA (20 mM) or charybdotoxin (50- 100 nM) unmasked an early transient inward current at -55 mV following step depolarization that reversed at -34 mV and was inhibited by (50-100 µM). Mean-variance analysis performed on the decay of the I(AHP) revealed that the AHP K(+) channels have a mean chord conductance of ~10 pS, and there are ~4,000 per AH neuron. Spectral analysis showed that the AHP channels have a mean open dwell time of 2.8 ms. Cell-attached patch recordings from AH neurons confirmed that the channels that open following action currents have a small unitary

Page 195 AHP, BK- and SK-channel references conductance (10-17 pS) and open with a high probability (≤0.5) within the first 2 s following an action potential. These results indicate that the AHP is largely a consequence of Ca(2+) entry through omega-conotoxin GVIA-sensitive Ca(2+) channels during the action potential, Ca(2+)-triggered Ca(2+) release from caffeine- sensitive stores and the opening of Ca(2+)-sensitive small-conductance K(+) channels.

•Vogalis, F., Harvey, J.R. & Furness, J.B. (2002). TEA- and apamin-resistant K(Ca) channels in guinea-pig myenteric neurons: slow AHP channels. Journal of Physiology (London) 538(Pt. 2): 421-433. The patch-clamp technique was used to record from intact ganglia of the guinea-pig duodenum in order to characterize the K(+) channels that underlie the slow afterhyperpolarization (slow AHP) of myenteric neurons. Cell-attached patch recordings from slow AHP-generating (AH) neurons revealed an increased open probability (P(o)) of TEA-resistant K(+) channels following action potentials. The P(o) increased from < 0.06 before action potentials to 0.33 in the 2 s following action potential firing. The ensemble averaged current had a similar time course to the current underlying the slow AHP. TEA- and apamin-resistant Ca(2+)-activated K(+) (K(Ca)) channels were present in inside-out patches excised from AH neurons. The P(o) of these channels increased from < 0.03 to approximately 0.5 upon increasing cytoplasmic [Ca(2+)] from < 10 nM to either 500 nM or 10 microM. P(o) was insensitive to changes in transpatch potential. The unitary conductance of these TEA- and apamin-resistant K(Ca) channels measured approximately 60 pS under symmetric K(+) concentrations between -60 mV and +60 mV, but decreased outside this range. Under asymmetrical [K(+)], the open channel current showed outward rectification and had a limiting slope conductance of about 40 pS. Activation of the TEA- and apamin-resistant K(Ca) channels by internal Ca(2+) in excised patches was not reversed by washing out the Ca(2+)-containing solution and replacing it with nominally Ca(2+)-free physiological solution. Kinetic analysis of the single channel recordings of the TEA- and apamin-resistant K(Ca) channels was consistent with their having a single open state of about 2 ms (open dwell time distribution was fitted with a single exponential) and at least two closed states (two exponential functions were required to adequately fit the closed dwell time distribution). The Ca(2+) dependence of the activation of TEA- and apamin-resistant K(Ca) channels resides in the long-lived closed state which decreased from > 100 ms in the absence of Ca(2+) to about 7 ms in the presence of submicromolar cytoplasmic Ca(2+). The Ca(2+)-insensitive closed dwell time had a time constant of about 1 ms.

Page 196 AHP, BK- and SK-channel references

We propose that these small-to-intermediate conductance TEA- and apamin- resistant Ca(2+)-activated K(+) channels are the channels that are primarily responsible for the slow AHP in myenteric AH neurons.

OTHER CELL TYPES

•Burgess, G.M., Claret, M. & Jenkinson, D.H. (1981). Effects of quinine and apamin on the calcium-dependent potassium permeability of mammalian hepatocytes and red cells. Journal of Physiology (London) 317: 67-90. 1. K-sensitive electrodes placed in the extracellular fluid have been used to show that ATP and noradrenaline cause a rapid loss of up to 10% of the K content of isolated guinea-pig hepatocytes. 2. The hypothesis tha this response is a consequence of a rise in the K permeability of the hepatocyte membrane triggered by an increase in cytosolic Ca is supported by the finding that the divalent cation ionophore A23187 also initiated K loss, in this instance of up to 20-25% of the amount in the cells. 3. Under similar conditions A23187 caused a transient increase, followed by a larger decrease, in the 45Ca content of guinea-pig hepatocytes equilibrated with this isotope. The decrease alone was seen with ATP and noradrenaline. 4. Quinine (1 mM) and the bee venom neurotoxin apamin (10 nM) greatly reduced the effect of ATP, noradrenaline and A23187 on K content without affecting the changes in 45Ca movement. 5. Apamin (10 nM) also abolished the increase in 42K efflux which follows the application of the alpha-adrenoceptor agonist amidephrine to rabbit liver slices; the concurrent rises in 45Ca efflux and glucose release were unaffected. 6. It was concluded that quinine and apamin are able to block either the Ca-dependent K channels present in guinea-pig and rabbit liver cell membranes or the mechanism that controls them. 7. Surprisingly, rat hepatocytes took up rather than lost K when treated with the concentrations of ATP, noradrenaline or A23187 that initiated K loss from guinea-pig cells. This response was greatly reduced by ouabain. 8. Application of large concentrations of A23187 to rat hepatocytes caused K loss associated with cell death. 9. The influence of apamin (10-1000 nM) and quinine (200-1000 µM) on the Ca-dependent K permeability of red blood cells and ghosts was also studied. Apamin was without effect even when applied to both sides of the ghost membrane, whereas quinine caused inhibition, as reported by others. 10. The results suggest that Ca-dependent K channels or carriers are present in the membranes of liver cells of the guinea-pig and rabbit, but are either lacking or inactive in rat liver. The finding that apamin

Page 197 AHP, BK- and SK-channel references blocks this mechanism in hepatocytes but not in erythrocytes may mean that the channels differ in these cells.

•Castle, N.A., Haylett, D.G., Morgan, J.M. & Jenkinson, D.H. (1993). Dequalinium: A potent inhibitor of apamin-sensitive K+ channels in hepatocytes and of nicotinic responses in skeletal muscle. European Journal of Pharmacology 236: 201-207. The bisquaternary compound dequalinium has been tested for its ability to inhibit the loss of K+ which angiotensin II causes in guinea-pig hepatocytes and which occurs through apamin-sensitive Ca2+-activated K+ (SKCa) channels. Dequalinium blocked angiotensin II-evoked K+ loss with an IC50 of 1.5 ± 0.1 µM and also inhibited 125I-monoiodoapamin binding with a KI of 1.1 ± 0.1 µM. It is the most active non-peptide SKCa blocker so far described. The neuromuscular blocking agent vecuronium was also tested, and proved to be considerably less effective (IC50, 4.5 ± 0.3 µM; KI, 3.6 ± 0.5 µM). Dequalinium was also examined for its actions at nicotinic receptors in skeletal muscle and was found to be a potent, non-competitive antagonist of carbachol contractions of the frog rectus abdominis. In the frog cutaneous pectoris muscle, end-plate depolarizations induced by carbachol became smaller and more transient in the presence of dequalinium at 10 nM. However, contractions of the frog sartorius and rat diaphragm in response to nerve stimulation were inhibited only by concentrations > 1 µM. These apparently discrepant effects of dequalinium on nicotinic responses could be explained either by open channel block of slow onset or by 'stabilization' of the desensitized state of the receptor. The potency of dequalinium will make it a useful agent for the study of nicotinic receptors as well as of SKCa channels.

•Castle, N.A. & Strong, P.N. (1986). Identification of two toxins from scorpion (Leiurus quinquestriatus) venom which block distinct classes of calcium-activated potassium channel. Proceedings of the Federation of Experimental Biology 209: 117- 121. Two polypeptide toxins from scorpion (Leiurus quinquestriatus) venom which block distinct classes of calcium-activated potassium channels have been identified and partially purified. One toxin, at 50-100 ng/ml, blocks apamin-sensitive potassium fluxes in hepatocytes and inhibits [125I]monoiodoapamin binding. The other, more basic, toxin blocks apamin-insensitive potassium fluxes in erythrocytes at 200 ng/ml and, to our knowledge, is the first toxin shown to block the

Page 198 AHP, BK- and SK-channel references erythrocyte calcium-activated potassium channel with high affinity. The possible co- identity of this latter toxin with charybdotoxin is discussed.

•Desai, R., Peretz, A., Reuveny, E. & Attali, B. (2000). Molecular and functional properties of human SK2, calcium-activated potassium channels. Society for Neuroscience Abstracts 30. Previous electrophysiological studies have demonstrated the presence of apamin-sensitive, small-conductance Ca2+-activated K+ channels (SK) in human brain tissue as well as in peripheral organs including liver, skeletal and smooth muscles and leukemic Jurkat T cells. Using a combined cDNA and RT-PCR cloning strategy, we have isolated from Jurkat T cells a 2.5 kb cDNA, hSK2, encoding the human isoform of SK2 channel a subunit. Northern blot analysis reveals the presence of a 2.5 kb hSK2 transcript in Jurkat T cells however, no hSK2 mRNA could be detected in resting and activated normal human T cells. hSK2 transcripts were also expressed in various human tissues, including brain, heart, skeletal muscle, kidney and liver. The protein encoded by hSK2 is 579 aminoacid long and exhibits 97% identity with its rat protein counterpart rSK2. Similar to the native Jurkat SK currents, hSK2 produces in transfected mammalian cells a time- and voltage-independent K+ current which is inhibited by apamin and d-tubocurarine (dTC). Overexpression of the Src- family tyrosine kinase p56lck up-regulates the Jurkat SK channel activity while the calmodulin antagonist W7 inhibits the Jurkat SK current and that heterologously expressed in CHO cells. Combined cross-linking and immunoprecipitation experiments performed in Jurkat T cells and in hSK2-transfected cells indicate that [ 125I] apamin binds to high (57 kDa) and low (31 kDa) molecular weight polypeptides, suggesting that native SK2 channels consist of hetero-oligomeric complexes comprising a and b subunits, respectively.

•Grissmer, S., Nguyen, A.N. & Cahalan, M.D. (1993). Calcium-activated potassium channels in resting, calcium dependence, ion selectivity and pharmacology. Journal of General Physiology 102: 601-630. Ca2+-activated K+[K(Ca)] channels in resting and activated human peripheral blood T lymphocytes were characterized using simultaneous patch-clamp recording and fura-2 monitoring of cytosolic Ca2+ concentration, [Ca2+]i. Whole-cell experiments, using EGTA-buffered pipette solutions to raise [Ca2+]i to 1 microM, revealed a 25-fold increase in the number of conducting K(Ca) channels per cell, from an average of 20 in resting T cells to > 500 channels per cell in T cell blasts after

Page 199 AHP, BK- and SK-channel references mitogenic activation. The opening of K(Ca) channels in both whole-cell and inside-out patch experiments was highly sensitive to [Ca2+]i (Hill coefficient of 4, with a midpoint of approximately 300 nM). At optimal [Ca2+]i, the open probability of a K(Ca) channel was 0.3-0.5. K(Ca) channels showed little or no voltage dependence from -100 to 0 mV. Single-channel I-V curves were linear with a unitary conductance of 11 pS in normal Ringer and exhibited modest inward rectification with a unitary conductance of approximately 35 pS in symmetrical 160 mM K+. Permeability ratios, relative to K+, determined from reversal potential measurements were: K+ (1.0) > Rb+ (0.96) > NH4+ (0.17) > Cs+ (0.07). Slope conductance ratios were: NH4+ (1.2) > K+ (1.0) > Rb+ (0.6) > Cs+ (0.10). Extracellular Cs+ or Ba2+ each induced voltage-dependent block of K(Ca) channels, with block increasing at hyperpolarizing potentials in a manner suggesting a site of block 75% across the membrane field from the outside. K(Ca) channels were blocked by tetraethylammonium (TEA) applied externally (Kd = 40 mM), but were unaffected by 10 mM TEA applied inside by pipette perfusion. K(Ca) channels were blocked by charybdotoxin (CTX) with a half-blocking dose of 3-4 nM, but were resistant to block by noxiustoxin (NTX) at 1-100 nM. Unlike K(Ca) channels in Jurkat T cells, the K(Ca) channels of normal resting or activated T cells were not blocked by apamin. We conclude that while K(Ca) and voltage-gated K+ channels in the same cells share similarities in ion permeation, Cs+ and Ba2+ block, and sensitivity to CTX, the underlying proteins differ in structural characteristics that determine channel gating and block by NTX and TEA.

•Lang, D.G. & Ritchie, A.K. (1987). Large and small conductance calcium- activated potassium channels in the GH3 anterior pituitary cell line. Pflugers Arch 410(6): 614-22. Single Ca2+-activated K+ channels were studied in membrane patches from the GH3 anterior pituitary cell line. In excised inside-out patches exposed to symmetrical 150 mM KCl, two channel types with conductances in the ranges of 250- 300 pS and 9-14 pS were routinely observed. The activity of the large conductance channel is enhanced by internal Ca2+ and by depolarization of the patch membrane. This channel contributes to the repolarization of Ca2+ action potentials but has a Ca2+ sensitivity at-50 mV that is too low for it to contribute to the resting membrane conductance. The small conductance channel is activated by much lower concentrations of Ca2+ at -50 mV, and its open probability is not strongly voltage sensitive. In cell-attached patches from voltage-clamped cells, the small

Page 200 AHP, BK- and SK-channel references conductance channels were found to be active during slowly decaying Ca2+-activated K+ tails currents and during Ca2+-activated K+ currents stimulated by thyrotropin- releasing hormone induced elevations of cytosolic calcium. In cell-attached patches on unclamped cells, the small conductance channels were also active at negative membrane potentials when the frequency of spontaneously firing action potentials was high or during the slow afterhyperpolarization following single spontaneous action potentials of slightly prolonged duration. The small conductance channel may thus contribute to the regulation of membrane excitability.

•Logsdon, N.J., Kang, J., Togo, J.A., Christian, E.P. & Aiyar, J. (1997). A novel gene, hKCa4, encodes the calcium-activated potassium channel in human T lymphocytes. Journal of Biological Chemistry 272(52): 32723-32726. We have isolated a novel gene, hKCa4, encoding an intermediate conductance, calcium-activated potassium channel from a human lymph node library. The translated protein comprises 427 amino acids, has six transmembrane segments, S1-S6, and a pore motif between S5 and S6. hKCa4 shares 41-42% similarity at the amino acid level with three small conductance calcium-activated potassium channels cloned from brain. Northern blot analysis of primary human T lymphocytes reveals a 2.2- kilobase transcript that is highly up-regulated in activated compared with resting cells, concomitant with an increase in KCa current. hKCa4 transcript is also detected by Northern blots or by polymerase chain reaction in placenta, prostate, thymus, spleen, colon, and many cell lines of hematopoietic origin. Patch-clamp recordings of hKCa4- transfected HEK 293 cells reveal a large voltage-independent, inwardly rectifying potassium current that is blocked by externally applied tetraethylammonium (Kd = 30±7 mM), charybdotoxin (Kd = 10±1 nM), and clotrimazole (Kd = 387±34 nM), but is resistant to apamin, iberiotoxin, kaliotoxin, scyllatoxin (Kd > 1 µM), and (Kd > 100 nM). Single hKCa4 channels have a conductance of 33±2 picosiemens in symmetrical potassium solutions. The channel is activated by intracellular calcium (Kd = 270±8 nM) with a highly cooperative interaction of approximately three calcium ions per channel. These properties of the cloned channel are very similar to those reported for the native KCa channel in activated human T lymphocytes, indicating that hKCa4 encodes this channel type.

•Noonan, L.M., Morales, E., Rashid, A., Dunn, R. & Turner, R.W. (2000). KV3.3 K+ channels have multiple roles in regulating somatic and dendritic spike discharge. Society for Neuroscience Abstracts 30.

Page 201 AHP, BK- and SK-channel references

AptKv3.3 K+ channels are distributed over the soma-dendritic axis of pyramidal cells in the electrosensory lobe (ELL) of A. leptorhynchus. We examined the role of AptKv3.3 channels in regulating somatic and dendritic spikes and afterpotentials in AptKv3.3-expressing HEK cells and an in vitro ELL slice preparation. Digitized spikes used as voltage clamp commands to HEK cells revealed a potential contribution to somatic and dendritic spike repolarization, a somatic fAHP, dendritic slow AHP, and a burst AHP involved in generating oscillatory bursts in the g frequency range. Focal ejections of TEA or 4-AP in vitro (1 mM) confirmed these results by reducing spike repolarization and AHPs. Focal drug ejections that reduced dendritic spike repolarization or the somatic fAHP lowered burst threshold by enhancing a DAP at the soma. Focal ejections of either Cd2+ (200 mM) or Dendrotoxin (2 mM) had no marked affect, indicating little if any influence by BK K(Ca) channels or Kv1.x K+ channels. These results indicate that AptKv3.3 K+ channels affect multiple facets of spike discharge and afterpotentials in somatic and dendritic regions, and regulate the threshold for generating oscillatory burst discharge.

•Pallotta, B.S., Hepler, J.R., Oglesby, S.A. & Harden, T.K. (1987). A comparison of calcium-activated potassium channel currents in cell-attached and excised patches. Journal of General Physiology 89(6): 985-997. Single channel currents from Ca-activated K channels were recorded from cell-attached patches, which were then excised from 1321N1 human astrocytoma cells. Cells were depolarized with K (110 mM) so that the membrane potential was known in both patch configurations, and the Ca ionophore A23187 or ionomycin (20- 100 µM) was used to equilibrate intracellular and extracellular [Ca] (0.3 or 1 µM). Measurements of intracellular [Ca] with the fluorescent Ca indicator quin2 verified that [Ca] equilibration apparently occurred in our experiments. Under these conditions, where both membrane potential and intracellular [Ca] were known, we found that the dependence of the channel percent open time on membrane potential and [Ca] was similar in both the cell-attached and excised patch configuration for several minutes after excision. Current-voltage relations were also similar, and autocorrelation functions constructed from the single channel currents revealed no obvious change in channel gating upon patch excision. These findings suggest that the results of studies that use excised membrane patches can be extrapolated to the K-depolarized cell-attached configuration, and that the relation between [Ca] and channel activity can be used to obtain a quantitative measure of [Ca] near the

Page 202 AHP, BK- and SK-channel references membrane intracellular surface.

•Wei, Y. & Wang, W.H. (2002). Role of the cytoskeleton in mediating effect of vasopressin and herbimycin A on secretory K channels in CCD. American Journal of Physiology: Renal Physiology 282(4): F680-686. We have previously demonstrated that inhibiting protein tyrosine kinase (PTK) and stimulating protein kinase A (PKA) increase the activity of the small- conductance K (SK) channel in the cortical collecting duct (CCD) of rat kidneys (Cassola AC, Giebisch G, and Wang WH. Am J Physiol Renal Fluid Electrolyte Physiol 264: F502-F509, 1993; Wang WH, Lerea KM, Chan M, and Giebisch G. Am J Physiol Renal Physiol 278: F165-F171, 2000). In the present study, we used the patch-clamp technique to study the role of the cytoskeleton in mediating the effect of herbimycin A, an inhibitor of PTK, and vasopressin on the SK channels in the CCD. The addition of colchicine, an inhibitor of microtubule assembly, or taxol, an agent that blocks microtubule reconstruction, had no significant effect on channel activity. However, colchicine and taxol treatment completely abolished the stimulatory effect of herbimycin A on the SK channels in the CCD. Removal of the microtubule inhibitors restored the stimulatory effect of herbimycin A. In contrast, treatment of the tubules with either taxol or colchicine did not block the stimulatory effect of vasopressin on the SK channels. Moreover, the effect of herbimycin A on the SK channels was also absent in the CCDs treated with either D or . In contrast, the stimulatory effect of vasopressin was still observed in the tubules treated with phalloidin. However, cytochalasin D treatment abolished the effect of vasopressin on the SK channels. Finally, the effects of vasopressin and herbimycin A are additive because inhibiting PTK can still increase the channel activity in CCD that has been challenged by vasopressin. We conclude that an intact cytoskeleton is required for the effect on the SK channels of inhibiting PTK and that the SK channels that are activated by inhibiting PTK were differently regulated from those stimulated by vasopressin.

BEHAVIORAL EFFECTS OF BLOCKADE

•Belcadi-Abbassi, W. & Destrade, C. (1995). Post-test apamin injection suppresses a Kamin-like effect following a learning session in mice. Neuroreport 6: 1293-1296. BALB/c mice were trained in a partial acquisition session of an appetitive

Page 203 AHP, BK- and SK-channel references bar-pressing task. They then received an immediate post-acquisition i.p. injection of either saline or apamin 0.2 mg kg-1. Each group was submitted to a retention test that was delayed either 25, 85 or 180 min after initial acquisition. In saline- injected groups retention of the original training was a U-shaped function of intersession interval with a significant drop in performance (Kamin-like effect) at the 85 min time interval. In contrast, at this same time, apamin injected subjects made significantly more reinforced responses than control animals. The suppression of the Kamin-like effect by apamin could be a consequence of an acceleration of the neuronal mechanisms implicated in consolidation and long-term memory storage processes.

•Benington, J.H., Woudenberg, M.C. & Heller, H.C. (1995). Apamin, a selective SK potassium channel blocker, suppresses REM sleep without a compensatory rebound. Brain Research 692: 86-92. To determine the role of neuronal potassium conductance in rapid-eye- movement (REM)-sleep homeostasis, we have administered small doses of apamin (2-5 ng), a selective blocker of the calcium-dependent SK potassium channel, injected into the lateral ventricle in rats, and characterized the resultant effects on REM-sleep expression. Apamin produces a dose-dependent reduction in REM-sleep expression without an increase in the frequency of attempts to enter REM sleep, suggesting that accumulation of REM-sleep propensity is suppressed. The vast majority (84- 95%) of lost REM sleep is not recovered 40 h after apamin administration. These findings suggest that accumulation of REM-sleep propensity is linked to the increased neuronal potassium conductance in nonREM sleep.

•Bond, C.T., Sprengel, R., Bissonnette, J.M., Kaufmann, W.A., Pribnow, D., Neelands, T., Storck, T., Baetscher, M., Jerecic, J., Maylie, J., Knaus, H.G., Seeburg, P.H. & Adelman, J.P. (2000). Respiration and parturition affected by conditional overexpression of the Ca2+-activated K+ channel subunit, SK3. Science 289(5486): 1942-1946. In excitable cells, small-conductance Ca2+-activated potassium channels (SK channels) are responsible for the slow after-hyperpolarization that often follows an action potential. Three SK channel subunits have been molecularly characterized. The SK3 gene was targeted by homologous recombination for the insertion of a gene switch that permitted experimental regulation of SK3 expression while retaining normal SK3 promoter function. An absence of SK3 did not present overt phenotypic

Page 204 AHP, BK- and SK-channel references consequences. However, SK3 overexpression induced abnormal respiratory responses to hypoxia and compromised parturition. Both conditions were corrected by silencing the gene. The results implicate SK3 channels as potential therapeutic targets for disorders such as sleep apnea or sudden infant death syndrome and for regulating uterine contractions during labor.

•Brosh, I. & Barkai, E. (2000). Odor-learning is accompanied by reduced noradrenergic ability to enhance neuronal excitability but not to enhance paired pulse facilitation in the piriform cortex. Society for Neuroscience Abstracts 30(73.9). We have previously shown that the afterhyperpolarization (AHP) generated by a burst of action potentials and paired pulse facilitation (PPF) evoked by stimulating the intrinsic fibers, are both reduced in layer II piriform cortex pyramidal neurons following odor learning (Saar et al., EJN(10) 1518, 1998; Saar et al., J. Neuroscience (19) 8616, 1999). Here we studied learning-related modifications in noradrenaline (NE) capability to reduce AHP's amplitude and to increase PPF of excitatory intrinsic synaptic inputs onto these neurons in brain slice preparation. Rats were trained to discriminate positive cues in pairs of odors, until they demonstrated dramatic increase in acquiring the same task with unfamiliar odors (rule learning). Single cell recordings were performed three days after training completion. In the presence of NE (10mM), neurons from trained rats did not differ from neurons from pseudo trained and naive animals in the averaged amplitude of AHP evoked by generating 6 action potentials at Vm= -60mV (-4.7mV±2.1, n=7 in neurons from trained vs.-4.8mV±1.9, n=21 in neurons from pseudo trained and 4.6mV±1.6, n=12 in neurons from naive rats). Accordingly, neuronal adaptation was also similar in neurons from the different groups. In contrast to its effect on intrinsic neuronal properties, the learning-induced differences in PPF were not abolished by NE. Averaged PPF value, at inter stimulus interval of 50 ms, in neurons from trained rats (1.23±0.16, n=11) was significantly lower than in neurons from pseudo trained (1.39±0.12, n=10) and naive (1.34±0.16, n=10) animals. Our data show that odor learning is accompanied by reduction in NE capability to enhance neuronal excitability, but not to reduce synaptic release. Thus, these preliminary results raise the possibility that while post-synaptic NE-dependent process are modified by learning, pre-synaptic processes affected by this neuromodulator remain intact.

•Fournier, C., Kourrich, S., Soumireu-Mourat, B. & Mourre, C. (2001). Apamin

Page 205 AHP, BK- and SK-channel references improves reference memory but not procedural memory in rats by blocking small conductance Ca2+-activated K+ channels in an olfactory discrimination task. Behavioral Brain Research 121(1-2): 81-93. Apamin blocks SK channels responsible for long-lasting hyperpolarization following the action potential. Using an olfactory associative task, the effect of an intracerebroventricular 0.3 ng apamin injection was tested on learning and memory. Apamin did not modify the learning of the procedure side of the task or the learning of the odor-reward association. To test reference memory specifically, the rats were trained on a new odor-association problem using the same procedure (acquisition session), and they were tested for retention 24 h later. Apamin injected before or after the acquisition session improved retention of the valence of a new odor pair. Apamin injected before the retention session did not affect the retrieval of the new valence. Thus, the results indicate that the blockage of apamin-sensitive SK channels facilitate consolidation on new-odor-reward association.

•Gandolfo, G., Schweitz, H., Lazdunski, M. & Gottesmann, C. (1996). Sleep cycle disturbances induced by apamin, a selective blocker of Ca2+-activated K+ channels. Brain Research 736(1-2): 344-347. Intracerebroventricular injections of low doses of apamin, a specific blocker of a class of Ca(2+)-activated K+ channels, induced insomnia and a long-lasting suppression of deep slow sleep and paradoxical sleep. Injected animals showed a late but important rebound of paradoxical sleep. Even after the recovery of a normal sleep amount, the circadian cycle remained disturbed during all the recording duration (96 h).

•Messier, C., Mourre, C., Bontempi, B., Sif, J., Lazdunski, M. & Destrade, C. (1991). Effect of apamin, a toxin that inhibits Ca2+-dependent K+ channels, on learning and memory processes. Brain Research 551: 322-326. Apamin, a neurotoxin extracted from bee venom, specifically binds to a particular class of Ca2+-activated K+ channels which are involved in the slow afterhyperpolarization (S-AHP) that follows action potentials in many excitable cells. We tested in mice the effects of apamin on learning and memory processes. The results showed that pre-training injection of apamin accelerated the acquisition of a bar-pressing response but also increased the bar-pressing rates of the animals. This latter result suggests that apamin accelerated acquisition because it increased behavioral activity in general and the number of bar-presses in particular. Post-

Page 206 AHP, BK- and SK-channel references training apamin injection retroactively and non-contingently facilitated memory processes taking place shortly after training in a bar-pressing task. The lack of an effect of the delayed apamin injection showed that apamin did not act proactively on memory retrieval processes. These results suggest that apamin-sensitive KCa channels may contribute to memory processes.

•van der Staay, F.J., Fanelli, R.J., Blokland, A. & Schmidt, B.H. (1999). Behavioral effects of apamin, a selective inhibitor of the SK(Ca)-channel, in mice and rats. Neuroscience Biobehavioral Reviews 23(8): 1087-1110. Apamin, a highly selective and potent peptide that blocks the SK(Ca)-channels has been suggested to be a cognition enhancer. We tested apamin in the Morris water escape task, in shock motivated avoidance tasks, and in operant tasks in the Skinnerbox. We also used non-cognitive tests, such as the rat forced swimming test and -induced locomotor activity in the open field, and a test to assess the side effect profile. Mice and rats from different strains, and rats of different ages were used. The rat studies provided only weak support for the notion that apamin acts as a cognition enhancer. More convincing evidence was obtained from the mouse studies. Overt side effects of apamin were found at the dose of 0.3 mg kg(-1). This dose was close to, or even overlapped, the doses which improved cognition in mice. We conclude that apamin is a poor tool to assess the role of SK(Ca)-channels in learning and memory processes.

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