Intermediate Conductance Calcium-Activated Potassium Channels Modulate Summation of Parallel ﬁber Input in Cerebellar Purkinje Cells

Intermediate conductance calcium-activated potassium channels modulate summation of parallel ﬁber input in cerebellar Purkinje cells

Jordan D. T. Engbers1, Dustin Anderson1, Hadhimulya Asmara, Renata Rehak, W. Hamish Mehaffey, Shahid Hameed, Bruce E. McKay, Mirna Kruskic, Gerald W. Zamponi, and Ray W. Turner2

Hotchkiss Brain Institute, University of Calgary, Calgary, AB, Canada T2N 4N1

Edited* by William A. Catterall, University of Washington School of Medicine, Seattle, WA, and approved December 28, 2011 (received for review September 12, 2011)

Encoding sensory input requires the expression of postsynaptic channels are expressed in cerebellar Purkinje cells and act to ion channels to transform key features of afferent input to an shape the parallel fiber EPSP waveform, providing unique evi- appropriate pattern of spike output. Although Ca2+-activated K+ dence for a functional role for KCa3.1 channels in a CNS neu- channels are known to control spike frequency in central neurons, ron. Moreover, KCa3.1 channels colocalize with Cav3.2 Ca2+ Ca2+-activated K+ channels of intermediate conductance (KCa3.1) channels to allow even subthreshold parallel fiber EPSPs to ac- are believed to be restricted to peripheral neurons. We now report tivate KCa3.1 and suppress temporal summation, contributing to that cerebellar Purkinje cells express KCa3.1 channels, as evi- a high-pass filter that allows Purkinje cells to respond to parallel denced through single-cell RT-PCR, immunocytochemistry, phar- fiber input indicative of sensory input. macology, and single-channel recordings. Furthermore, KCa3.1 channels coimmunoprecipitate and interact with low voltage-acti- Results vated Cav3.2 Ca2+ channels at the nanodomain level to support Parallel Fiber EPSPs Activate a Ca2+-Dependent After-Hyperpolar- a previously undescribed transient voltage- and Ca2+-dependent ization. To examine the potential for parallel fiber EPSPs to ac- current. As a result, subthreshold parallel fiber excitatory postsyn- tivate postsynaptic currents, we stimulated parallel fiber inputs in aptic potentials (EPSPs) activate Cav3 Ca2+ influx to trigger a the presence of picrotoxin and CGP55845 to block GABAergic KCa3.1-mediated regulation of the EPSP and subsequent after-hy- transmission. The subthreshold parallel fiber-evoked EPSP was perpolarization. The Cav3-KCa3.1 complex provides powerful con- followed by a graded after-hyperpolarization (AHP) of up to trol over temporal summation of EPSPs, effectively suppressing ∼250-ms duration (Fig. S1A). To avoid any effects on presynaptic low frequencies of parallel fiber input. KCa3.1 channels thus con- transmisson during pharmacological tests, we simulated EPSPs tribute to a high-pass filter that allows Purkinje cells to respond by injecting excitatory postsynaptic current (EPSC) waveforms at preferentially to high-frequency parallel fiber bursts characteristic the soma (simEPSCs) in the presence of inhibitory and excitatory of sensory input. synaptic blockers, which produced very similar voltage responses (Fig. S1A). Some portion of this AHP can be attributed to IH (15). However, given that blocking IH substantially changes the mem- entral neurons receive an enormous number of spontane- brane time constant, all experiments were conducted in the pres- Cously active synaptic inputs, but exhibit the capacity to dif- − ence of IH from a membrane potential of 75 mV. ferentiate features of sensory input from background noise. We first tested the ability for blockers against HVA or LVA Cerebellar Purkinje cells are contacted by up to ∼150,000 par- 2+ fi Ca channels to affect the simEPSP-AHP. We found no sig- allel bers from granule cells, of which only a subset will convey nificant effect on the simEPSP-AHP by the Cav2.1 (P-type) sensory information at any given time. The activation of a pe- channel blocker ω-agatoxin IVA (AgTx; n =4,P = 0.93) (Fig. fi ripheral receptive eld is transmitted to the cerebellar cortex by 1A). Similarly, no effects were obtained upon pressure ejection fi mossy bers in the form of high-frequency spike bursts (1). The of a mixture of other HVA Ca2+ channel blockers, including resulting temporal summation of excitatory postsynaptic poten- ω-conotoxin GVIA, nifedipine, and SNX-482 (n =6,P = 0.084), tials (EPSPs) generates a similar high-frequency burst in granule or the general HVA blocker Cd2+ (n =5,P = 0.097) (Fig. 1A cells (2). Purkinje cells should then also possess the means to and Fig. S2). However, both the simEPSP rate of decay and AHP respond effectively to bursts of parallel fiber input that convey were significantly reduced by low concentrations of the putative sensory information compared with background activity. T-type channel blockers Ni2+ (n =6,P = 0.00050) and mibe- Postsynaptic membrane excitability can be controlled by acti- n P A 2+

fradil ( =8, = 0.00018) (Fig. 1 ) (16, 17). Indeed, Ni could NEUROSCIENCE + 2+ vation of K channels. There are two established types of Ca - affect several aspects of the simEPSP-AHP (Fig. S1B), but + activated K (KCa) channels in CNS neurons: small conduc- the most reliable effect was a reduction in the simEPSP rate of tance (SK, KCa2.x) and big conductance (BK, KCa1.1) (3, 4). A decay (Fig. 1A). third class of intermediate conductance (KCa3.1, SK4, IK1) KCa The relative lack of specificity of T-type channel blockers channel is thought to be expressed only in microglia and endo- leaves open the possibility that these effects were not entirely thelial cells in the CNS (3, 5, 6). KCa3.1 channels are gated by mediated through T-type channels. First, Ni2+ and mibefradil calmodulin in a similar manner to KCa2.x channels but are insensitive to block by apamin and tetraethylammonium (TEA) (6–8). Instead, the KCa3.1 α-subunit, encoded by the gene KCNN4,has Author contributions: J.D.T.E., D.A., W.H.M., B.E.M., G.W.Z., and R.W.T. designed research; specific residues that bind charybdotoxin and 1-[(2-chlorophenyl) J.D.T.E., D.A., H.A., R.R., W.H.M., S.H., B.E.M., and M.K. performed research; J.D.T.E., D.A., diphenylmethyl]-1H-pyrazole (TRAM-34) (5–7, 9, 10). H.A., and W.H.M. analyzed data; and J.D.T.E., G.W.Z., and R.W.T. wrote the paper. In cerebellar Purkinje cells, KCa1.1 and KCa2.2 channels are The authors declare no conflict of interest. activated during a spike by high voltage-activated (HVA) P-type *This Direct Submission article had a prearranged editor. 2+ Ca channels (11). In contrast, low voltage-activated (LVA) 1J.D.T.E. and D.A. contributed equally to this work. 2+ Cav3 (T-type) Ca channels that are active during the sub- 2To whom correspondence should be addressed. E-mail: [email protected]. – threshold interspike interval (12 14) have not been associated This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. with any specific KCa channel. We now report that KCa3.1 1073/pnas.1115024109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1115024109 PNAS | February 14, 2012 | vol. 109 | no. 7 | 2601–2606 Downloaded by guest on October 2, 2021 the simEPSP rate of decay was reduced 41.7 ± 6.4% by charybdotoxin (ChTx; n =6,P = 0.00459) (Fig. 1B). ChTx is known to block Ca2+-activated KCa1.1 and KCa3.1 channels and specific Kv1.x channels (7, 20). KCa3.1 channels are apamin-insensitive but have specific binding sites for ChTx and the clotrimazole-related compound TRAM-34 (9, 21). TRAM-34 has been established as a selective KCa3.1 blocker with no effects on KCa1.1, KCa2.2, or a wide array of Kv channels (9, 10) or Cav3 channels (Fig. S3). A role for KCa3.1 channels in Pur- kinje cells was thus suggested when the simEPSP rate of decay was reduced 24.7 ± 3.9% by TRAM-34 (n =9,P = 0.000218) (Fig. 1B). The simEPSP also proved to be insensitive to TEA at concentrations < 25 mM (n = 13), a finding consistent with KCa3.1 channels but eliminating several other channels (KCa1.1, Kv1.1, Kv1.6, Kv2.x, Kv3.x channels) (20). Finally, we found that including the calmodulin blocker Camstatin (22) in the electrode occluded the effects of TRAM-34 (Fig. 1C)(n =7,P=0.31). These results strongly implicate KCa3.1 channels given that KCa1.1 channels are highly sensitive to TEA (20) and neither KCa1.1 nor Kv1.x channels are calmodulin-dependent, as shown for KCa3.1 (3). There was also no significant difference between the effects of Ni2+, mibefradil, ChTx, or TRAM-34 on the simEPSP rate of decay (F = 2.37, one-way ANOVA), suggesting actions on a common pathway. In fact, the effects of TRAM-34 on the simEPSP-AHP were occluded by prior Ni2+ treatment (Fig. 1D)(n =4,P=0.67). Taken together, these data reveal a postsynaptic current with a pharmacological profile that is unique to KCa3.1 channels (10, 20) and activated by Cav3-me- 2+ fl Fig. 1. Subthreshold parallel fiber EPSPs generate an AHP consistent with diated Ca in ux. activation of KCa3.1. All records were evoked using simEPSCs to test postsynaptic channel contributions, with drug effects on the rate of EPSP decay Purkinje Cells Express KCa3.1 Channels. Identifying an outward normalized to the control 5 mV simEPSP. (A and B) Representative record- current with KCa3.1-like properties was unexpected. Although ings (Left) and bar plots (Right) showing the effects of Ca2+ and K+ channel KCCN4 mRNA has been detected in the cerebellar Purkinje cell blockers. (A) The simEPSP rate of decay is not significantly affected by AgTx layer (23), KCa3.1 expression has not been reported in CNS (200 nM) or other HVA Ca2+ channel blockers (ω-conotoxin GVIA, 1 μM; ni- neurons (3). Of the three Cav3 channel isoforms expressed in fedipine, 1 μM; SNX-482, 200 nM) or Cd2+ (30 μM), but is reduced by putative Purkinje cells (13, 14, 24), Cav3.2 is preferentially blocked by 100 2+ 2+ T-type Ca channel blockers Ni (100 μM) and mibefradil (Mib,1 μM). (B) μMNi2+ (16), as found for the evoked AHP (Fig. 1A). We thus The simEPSP rate of decay is unaffected by the KCa2.x blocker apamin (100 used dual-label immunocytochemistry to detect KCa3.1 in re- nM), or KCa1.1 blockers IbTx (200 nM), TEA (5 mM), or paxilline (100 nM), but lation to Cav3.2 protein or calbindin, a cytosolic protein dis- is significantly reduced by ChTx (100 nM) and TRAM-34 (100 nM). (C) Internal dialysis of Camstatin (5 μM) occludes the effect of TRAM-34 on the simEPSP tributed throughout the soma and dendrites (Fig. 2 and Fig. S4). rate of decay. (D) Pretreatment with Ni2+ occludes the action of TRAM-34 on KCa3.1 immunolabel was detected at the soma and over re- the simEPSP rate of decay. Sample numbers are shown in brackets at the base stricted segments of primary and secondary dendritic branches of of bar graphs. Average values are mean ± SEM; **P < 0.01, ***P < 0.001. Purkinje cells, regions that were closely matched by that of Cav3.2 immunolabel (13), revealing colocalization (Fig. 2 A–F and Fig. S4). To further test for KCa3.1 expression, we per- also block R-type Ca2+ channels (18, 19). However, the lack of formed RT-PCR using primers directed against a 602-bp region effect by either SNX-482 or Cd2+ on the simEPSP rules out R- of rat KCCN4 mRNA that includes the pore region of the type channel involvement (Fig. 1A and Fig. S2). The effects of KCa3.1 channel (25). We also tested for the expression of Ni2+ on the simEPSP-AHP were also separate from those of microglial response factor-1 (MRF-1) as a specific marker for 2+ blocking IH (Fig. S1C). The effects of Ni were further main- microglia, which also express KCa3.1 channels (26). These tests tained in the presence of internal heparin (4 mg/mL) and bath- revealed the presence of KCa3.1 cDNA at the predicted product applied cyclopiazonic acid (6 μM) (n = 4, P=0.0032), indicating weight in homogenates of rat cerebellum and KCa1.1, KCa2.2, 2+ 2+ no involvement of IP3-mediated Ca release or Ca -ATPases and KCa3.1 in cytosolic extracts from single Purkinje cells (Fig. (Fig. S1D). Ryanodine also had no significant effect on the 2G). As predicted by a reported decrease in KCa2.2 mRNA simEPSP rate of decay (n = 4, P=0.12), indicating no role for through development (27), the KCa2.2 band was detectable but ryanodine receptor-mediated Ca2+-induced Ca2+ release (Fig. relatively faint compared with KCa1.1 or KCa3.1 (Fig. 2G). The S1E). Taken together, these data suggest that the simEPSP band for KCa3.1 in Purkinje cell samples also matched that of activates Ca2+ influx through Cav3 Ca2+ channels to control an KCa3.1 in lysates of cultured endothelial cells known to express outward current with an onset early enough to affect both the KCa3.1 (Fig. 2G) (28). In contrast, MRF-1 cDNA was present EPSP rate of decay and AHP. only in cerebellar homogenate (Fig. 2G), indicating that KCa3.1 mRNA in Purkinje cell samples did not reflect microglial con- EPSP Rate of Decay Is Shaped by a K+ Current Consistent with KCa3.1 tamination. Furthermore, sequence analysis of the purified K+ Channels. We next examined the identity of K+ channels ac- KCa3.1 band in Purkinje cells confirmed that the product rep- tivated by the simEPSP. Surprisingly, the simEPSP rate of decay resented the region spanning the KCa3.1 channel pore, and in- was entirely unaffected by bath application of the KCa2.2 cluded the known binding sites for TRAM-34 and ChTx (25), but channel blocker apamin (n =6,P = 0.62) or by KCa1.1 channel not an apamin binding site (29). blockers iberiotoxin (IbTx; n =5,P = 0.57), TEA (n =5,P = Direct tests for the expression of K+ channels that were both 0.29), or paxilline (n =3,P=0.97) (Fig. 1B) (7), despite con- Ca2+-sensitive and of intermediate conductance were carried out firmed effects of each drug on spontaneous firing. In contrast, using on-cell recordings from Purkinje cell somata. On-cell patch

2602 | www.pnas.org/cgi/doi/10.1073/pnas.1115024109 Engbers et al. Downloaded by guest on October 2, 2021 Fig. 3. Purkinje cells express Ca2+-activated intermediate conductance K+ channels. (A) Spontaneous on-cell single-channel recordings from three different cells during a steady-state (5 min) pipette holding potential of 30 mV. Open (o) and closed (c) states are indicated. (B) Plot of mean single- Fig. 2. Purkinje cells express KCa3.1 channels that colocalize with Cav3.2 channel amplitudes in on-cell recordings at steady-state potentials up to +30 protein. (A–C) Dual-label immunocytochemistry for Cav3.2 (A) and KCa3.1 mV reveals a mean conductance of 36.3 pS (n = 5). (C)(Left) Long-duration (B) in a coronal section reveals protein colocalized (arrows) at the soma on-cell channel recordings (+30 mV pipette potential) before and after (asterisks) and restricted segments of dendritic branches (C). (D–F) High- perfusing the Ca2+ ionophore A23187 (2 μM) and block by TRAM-34 (100 power view of Purkinje cell dendrites in a sagittal section dual-labeled for nM) (n = 6). (Right) Bar plots show channel open probability under the con- Cav3.2 (D) and KCa3.1 protein (E), with overlay (F) illustrating colocalization ditions shown in the channel recordings (n = 6). Average values are mean ± over specific segments of dendritic branches. (G) RT-PCR reveals KCa3.1 and SEM; *P < 0.05; **P < 0.01. MRF-1 mRNA in whole cerebellum (Left), and KCa1.1, KCa2.2, and KCa3.1 but not MRF-1 in single Purkinje cell cytoplasmic extracts (Center). The KCa3.1 product in Purkinje cells matches that found in endothelial cells 2+ (Right). (Scale bars, 20 μm.) KCa3.1 channels are Ca -activated but show no intrinsic voltage dependence (7). Therefore, we used outside-out recordings from Purkinje cell somata to test the hypothesis that electrodes were filled with Hepes-buffered artificial cerebrospi- a closely associated Cav3 channel could confer a voltage de- nal fluid (aCSF) to provide a physiological level of Ca2+ outside, pendence on KCa3.1 current. Cav3 and KCa3.1 channels were along with tetrodotoxin (TTX), Cd2+, apamin, TEA, 4-AP, and isolated by perfusion of TTX, Cd2+, apamin, TEA, 4-AP, Cs+, Cs+ in both the electrode and external medium to block sodium, and synaptic blockers (Materials and Methods), and the current- HVA Ca2+, KCa2.2, KCa1.1, Kv, and HCN channels (Materials voltage relationship of Cav3 or KCa3.1 currents determined for and Methods and Fig. S2). T-type channel blockers were specif- steps from −110 mV to 0 mV. These recordings revealed a ically excluded and the availability of Cav3 channels promoted by voltage-dependent outward current evoked from a holding po- − − applying a +60 mV potential to the pipette at rest to hyperpo- tential of 110 mV that was absent at a holding potential of 40 B B larize the patch transmembrane potential. Channel conductance mV (Fig. 4 and Fig. S5 ), a characteristic property of channels was determined by applying steady-state commands (5 min) up that undergo voltage-dependent inactivation. Subtraction of records obtained at −40 mV vs. −110 mV isolated a transient to +30 mV from the resting condition to depolarize the patch to ± n within the subthreshold voltage range. Spontaneous channel outward current of up to 50 pA (24.8 6.2 pA, = 6) that was openings were readily detected in all patches, with either single or multiple channel openings per patch that changed linearly in amplitude with applied voltage (Fig. 3 A and B). Mean conductance, as determined from unit amplitude over a range of voltage steps, was 36.3 ± 0.1 pS (n = 5) (Fig. 3B), a value within the range previously reported for KCa3.1 channels (6). Impor- tantly, bath application of the Ca2+ ionophore A23187 (n =6) dramatically increased single channel activity (Fig. 3C), indicating an increase in probability for activation with Ca2+ influx. Moreover, channel openings were blocked by perfusion of NEUROSCIENCE TRAM-34 (n = 6), a lipophillic drug that acts at the cytoplasmic face (Fig. 3C) (9). These results, combined with the mRNA, immunolabel, and pharmacological profiles established in Figs. 1 and 2 provide strong evidence that KCa3.1 channels are expressed in Purkinje cells.

Cav3 and KCa3.1 Channels Exhibit Nanodomain Coupling to Evoke Fig. 4. KCa3.1 channel activation is coupled to Cav3-mediated Ca2+ inﬂux. + a Transient Voltage-Dependent K Current. The selective reduction (A) Western blot showing coimmunoprecipitation of Cav3.2 and KCa3.1 2+ of the simEPSP rate of decay by T-type Ca channel blockers channels from cerebellar lysate. (B) Outside-out recordings from separate (Fig. 1) raised the possibility that Cav3 and KCa3.1 channels are Purkinje cell somata in response to steps from −110 mV to 0 mV. Shown are part of a physical signaling complex. We tested for an association currents calculated as the difference from those evoked at a −40 mV holding 2+ μ between Cav3.2 and KCa3.1 channels, ﬁnding that Cav3.2 chan- potential, or blocked by TRAM-34 (100 nM) or Ni (100 M). (C) Mean I-V plots for currents isolated as in B indicate a common activation in the low nels coimmunoprecipitated with KCa3.1 channels from homoge- A voltage range. (D) Mean I-V plots of TRAM-34-sensitive currents in outside- nates of rat cerebellum (Fig. 4 ). In comparison, we found no out recordings with either high EGTA or BAPTA in the electrode. All coimmunoprecipitation between KCa3.1 and Cav2.1 channels that recordings were obtained from the somata of P 18–25 Purkinje cells in 1.5 are expressed by Purkinje cells (Fig. S5A). mM external Ca2+. Average values are mean ± SEM.

Engbers et al. PNAS | February 14, 2012 | vol. 109 | no. 7 | 2603 Downloaded by guest on October 2, 2021 fast inactivating (t1/2 = 22.6 ± 1.4 ms) (Fig. 4B and Table S1). Importantly, this outward current cannot reflect activation of KCa1.1, KCa2.2, Kv1, Kv3, or Kv4 channels, as these are substantially blocked by apamin or 5 mM TEA and 4-AP (20). HVA Ca2+ currents (including R-type) are also highly effectively blocked by the 30 μMCd2+ included here (Fig. S2), leaving Cav3 channels as the predicted primary source for Ca2+ influx in these recordings. We further isolated the Ni2+ and TRAM-sensitive currents evoked by steps from −110 mV using the same conditions of HVA Ca2+ and K+ channel block. These tests showed that TRAM-34 (n =4)orNi2+ (n = 4) blocked a fast inactivating current of up to 30 pA, with mean values of 11.4 ± 2.3 pA and t1/2 inactivation of 23.6 ± 6.0 ms at −30 mV (n = 8) (Fig. 4B and Table S1). TRAM-34 and Ni2+-sensitive currents also activated in the low voltage range in a manner similar to the inactivating current isolated by membrane voltage (Fig. 4C). In support of this, current-clamp recordings indicated a clear threshold for activation of a Ni2+-sensitive AHP for membrane potentials positive to −80 mV, while voltage-ramp commands revealed Cav3 and KCa3.1 activation from membrane voltages between −80 and −90 mV (Fig. S6). Finally, to test the proximity of Cav3 to KCa3.1 channels, we used outside-out recordings to measure TRAM-34–sensitive current in the presence of either EGTA or BAPTA (10 mM) in the internal electrolyte. TRAM- 34–sensitive current was only recorded in the presence of internal EGTA (n = 5) but not BAPTA (n = 6) (Fig. 4D), indicating an interaction at the nanodomain level (30). Given that KCa3.1 channel open probability is only a function 2+ of internal Ca concentration (31), the voltage dependence of Fig. 5. The Cav3–KCa3.1 complex regulates temporal summation of parallel KCa3.1 activation must indirectly reflect the voltage dependence fiber EPSPs. (A and B) Representative recordings and plots of the baseline of Ca2+ influx through Cav3 channels. Taken together, these membrane voltage during 25-Hz trains of parallel fiber-evoked EPSPs before 2+ data indicate that an association between Cav3 and KCa3.1 and after applying Ni (A,100μM, green) or TRAM-34 (B, 100 nM, red). 2+ fl Stimulus intensity was adjusted to evoke an initial EPSP of 2 mV. (C) channels at the nanodomain level allows T-type Ca in ux to fi activate KCa3.1 as a Ca2+-dependent and transient outward Recordings and plots of baseline voltage during 25 Hz parallel ber stimulus trains in a coronal slice in the absence of picrotoxin to preserve feed-forward current in the low voltage range. inhibition. TRAM-34 (red) substantially increases temporal summation, with an additional increase upon addition of picrotoxin (50 μM, blue) to block – Cav3 KCa3.1 Interaction Controls Temporal Summation of Parallel GABAergic inhibition. Statistical significance tested for last 10 pulses of Fiber EPSP Trains. To test the hypothesis that the Cav3–KCa3.1 stimulus trains in A–C (indicated by bars). Spikes were truncated in A–C and complex could affect summation of EPSPs, we stimulated par- average values are mean ± SEM; *P < 0.05; **P < 0.01, ***P < 0.001. allel fiber inputs at 25 Hz for 2 s. Initial EPSP amplitude was 2 mV, with presynaptic facilitation causing the EPSP amplitude to reach ∼5 mV. Under control conditions, an initial summation summation during the stimulus train (Fig. 5C)(n =5,P = 0.08, of the EPSP was reduced within 250 ms to a lower, stable am- one-way ANOVA with post hoc Tukey test). These results in- plitude that remained below spike threshold for the duration of dicate that postsynaptic control of temporal summation by the the stimulus train (Fig. 5A). However, we found a rapid temporal Cav3–KCa3.1 complex is effective in the presence of feed- summation of EPSPs within the first five stimuli after applying forward inhibition. either Ni2+ (n =8,P = 0.00012) or TRAM-34 (n =4,P = 0.019) to block the Cav3–KCa3.1 interaction (Fig. 5 A and B). Cav3–KCa3.1 Complex Is Active During Membrane Depolarizations Measuring the baseline membrane voltage immediately pre- and Tonic Firing. Given that Cav3 channels inactivate upon de- ceding each stimulus indicated a rapid increase over the first ∼20 polarization, it is important to determine the availability of Cav3 stimuli that reached a sustained level 8–10 mV beyond initial current at physiological potentials. We thus tested if the Cav3– baseline (Fig. 5 A and B). As a result, parallel fiber-evoked KCa3.1 interaction is active at more depolarized levels of EPSPs surpassed spike threshold within 5–10 stimuli to reliably membrane potential that promote spontaneous spike firing. To generate spike output. We found no role for mGluR1 receptor measure Cav3 activation and inactivation over a full voltage activation (14) or presynaptic endocannabinoid receptors (32), range, we used whole-cell recordings from P10–P12 Purkinje as neither JNJ16259685 nor the CB1 receptor blocker AM-251 cells when the dendritic tree has not fully developed to avoid occluded the effect of Ni2+ (n = 4) during these stimuli. In space clamp errors (n = 7) (Fig. 6 A and B). The Boltzmann fits addition, the parallel fiber-evoked EPSP paired-pulse ratio was for activation and inactivation data (Fig. 6B) indicated that unaffected by either Ni2+ (n =7,P=0.95) or TRAM-34 (n =7, a Cav3 window current exists for membrane voltages up to at P=0.582) (Fig. S7 A and B), indicating that the increase in least ∼−20 mV, and thus well into the suprathreshold range (12, temporal summation did not involve presynaptic effects. 34) (Fig. 6 A and B). Temporal summation of parallel fiber EPSPs has also been We further tested the degree to which parallel fiber EPSP shown to be controlled through feed-forward inhibition (33). summation could be modified by the Cav3–KCa3.1 interaction However, when we repeated these tests in coronal slices in the during tonic firing. Bias current was applied to sustain a tonic absence of picrotoxin to preserve feed-forward inhibition, firing frequency of ∼50 Hz (49.7 ± 1.8 Hz, n = 9) before and TRAM-34 rapidly increased temporal summation of parallel fi- after drug application. Parallel fiber inputs were stimulated at ber-evoked EPSPs (Fig. 5C)(n =5,P = 0.04, one-way ANOVA 100 Hz (five pulses) to mimic frequencies of input relevant to with post hoc Tukey test). Subsequent addition of picrotoxin to sensory stimulation (1, 35). After application of TRAM-34, there block GABAergic inputs led to a further increase in temporal was an increase in both peak baseline voltage (59 ± 25.6%, n =5,

2604 | www.pnas.org/cgi/doi/10.1073/pnas.1115024109 Engbers et al. Downloaded by guest on October 2, 2021 channels, a known interaction for KCa2.x channels (38). A functional coupling between T-type Ca2+ and KCa2.x channels in select neuronal subtypes (39–41) is also reported to operate at the microdomain level compared with the nanodomain demon- strated here. A Cav3–Kv4 K+ channel complex employs K+ channel interacting protein 3 to mediate Ca2+ sensing for voltage-gated Kv4 channels (42, 43). The Cav3-KCa3.1 complex instead depends on calmodulin that has been shown to directly gate the KCa3.1 channel (3). All members of the KCa2.x and KCa3 families are known to be Ca2+- but not voltage-dependent (3). However, the close association between Cav3 and KCa3.1 channels allows KCa3.1 to acquire the properties of a low voltage-activated current, as well as the fast inactivating kinetics of Cav3 current. KCa3.1 channels also exhibit a two-to-three times greater sensitivity to internal Ca2+ than KCa2.x channels (36). These properties are consistent with the ability for a transient T-type Ca2+ influx to activate KCa3.1 with little delay at the onset of an EPSP and yet generate an AHP of up to 250-ms duration. KCa3.1 channels can thus be activated by single, low-amplitude EPSPs but also exhibit a cu- mulative activation during repetitive activity, an advantage over other K+ channels for modulating temporal summation of synaptic depolarizations. Control over temporal summation of parallel fiber EPSPs in Fig. 6. Cav3 window current straddles spike threshold in Purkinje cells and Purkinje cells has also been reported through other mechanisms. the Cav3–KCa3.1 complex affects EPSP summation under physiological con- One is a role for IH to reduce EPSP width and temporal sum- ditions. (A) Mean conductance and inactivation plots calculated for whole- mation (15). Because all tests conducted here were performed cell Cav3 current recorded from P10–12 Purkinje cells (Inset). (B) Expanded with IH intact, KCa3.1 clearly has a role distinct from IH in view of the fits for activation and inactivation curves shown in A reveal that producing the AHP and modifying temporal summation. Mo- Cav3 window current (gray) in relation to spike threshold (dashed line). (C–E) lecular layer interneurons provide a feed-forward inhibitory in- The effects of blocking the Cav3–KCa3.1 complex during a five-pulse train of fluence that reduces parallel fiber EPSP summation (33). This parallel fiber stimulation (100 Hz, arrows). Resting potential was depolarized function is again different, in that the Cav3–KCa3.1 complex fi ∼ fi to a level suf cient to evoke 50-Hz tonic ring, with spike threshold in- exerts a significant effect on temporal summation even in the dicated at Left. Blocking the Cav3–KCa3.1 complex with either TRAM-34 (C, fl – 2+ presence of feed-forward inhibition. The in uence of the Cav3 100 nM, red) or Ni (D, 100 μM, green) reveals substantial control over EPSP fi summation and spike frequency (E). Spikes were truncated for display in C KCa3.1 interaction is also fully functional during tonic ring, and D, and picrotoxin (50 μM) was present for all tests in C–E. All average a result attributable to the wide extent of Cav3 window current in values are mean ± SEM; *P < 0.05. the suprathreshold voltage range. The present results then suggest that the Cav3–KCa3.1 complex functions synergistically with IH and feed-forward inhibition to reduce Purkinje cell re- P = 0.025) and peak frequency during stimulation (53 ± 16.2%, sponsiveness to background granule cell activity yet permit ac- n =5,P = 0.021) (Fig. 6 C and E). Similarly, Ni2+ increased tivation by high-frequency trains of parallel fiber input. Taken peak baseline voltage (96 ± 36%, n =4,P = 0.024) and spike together, these factors underlie a high-pass filter function that frequency (114 ± 16.5%, n =4,P = 0.012) during the stimula- allows spike bursts produced by granule cells to preferentially tion (Fig. 6 D and E). Neither result could be attributed to evoke Purkinje cell spike output (2). a change in the amplitude of the evoked EPSP (Fig. S7 C and D). These results demonstrate that both Cav3 and KCa3.1 channels Materials and Methods are available at depolarized potentials supporting tonic spike Molecular Biology and Cytochemical Methods. Materials and methods related firing, and modulate temporal summation of parallel fiber inputs to coimmunoprecipitation, RT-PCR, immunocytochemistry, and use of tSA- at physiologically relevant input frequencies. 201 cells are found in SI Materials and Methods. Discussion Animals. Timed-pregnant Sprague-Dawley rats (Charles River) were main- tained according to the Canadian Council on Animal Care; male pups ≥

The present study provides unique evidence for the activation of NEUROSCIENCE + postnatal day (P) 18 were used, unless otherwise indicated. Rats were KCa3.1 K channels in a CNS neuron and its role in creating anesthetized by inhalation of isoflurane until unresponsive to tail pinch. a postsynaptic frequency filter for synaptic input. Although Tissue dissection and preparation of parasagittal (300 μm) slices from the KCa3.1 channel activation has been documented in cells of the cerebellar vermis were previously described (24). enteric and myenteric nervous systems (36, 37), several lines of evidence now support the expression of KCa3.1 channels in Solutions. Chemicals were obtained from Sigma unless otherwise indicated. Purkinje cells, including single cell RT-PCR, KCa3.1 immuno- aCSF was composed of 125 mM NaCl, 3.25 mM KCl, 1.5 mM CaCl2, 1.5 mM μ μ label, single channels with intermediate conductance, and mac- MgCl2, 25 mM NaHCO3, and 25 mM D-glucose. Picrotoxin (50 M), 10 M 6,7- ropatch recordings of KCa currents with a pharmacological dinitroquinoxolinedione (DNQX; Tocris), 25 μM DL-AP5 (Ascent Scientific), profile that is unique to KCa3.1 channels (3, 7). Moreover, and 1 μM CGP55485 (Tocris) were added to the aCSF for all recordings that 2+ did not require stimulation. DNQX, as well as nifedipine, were dissolved in Cav3.2 Ca channels colocalize and associate with KCa3.1 fi channels to provide Ca2+-dependent regulation at the nano- dimethyl sulfoxide before use ( nal DMSO concentration less than 0.1%). Apamin was first dissolved in 50 mM acetic acid before preparing domain level to control temporal summation of parallel fiber – fi stock solution. EPSPs. This unique Cav3 KCa3.1 complex differs signi cantly Bath perfused channel blockers and ionophores were applied at con- from other ion channel complexes involving T-type channels centrations of 100 nM TTX, 100 μM NiCl ,1μM mibefradil, 30 μM CdCl ,2μM – 2 2 reported in previous studies. The ability to evoke the Cav3 A23187, 100 nM apamin, 5 mM TEA, 5 mM 4-AP, 2 mM CsCl2, 100 nM TRAM- KCa3.1 interaction using postsynaptic simEPSCs indicates that 34 (Tocris) (10), 100 nM paxilline, 6 μM cyclopiazonic acid (CPA; Ascent Sci- 2+ the complex does not require Ca influx through ligand-gated entific), 10 μM ryanodine, and 1.5 μM JNJ16259685. Heparin (4 mg/mL) or

Engbers et al. PNAS | February 14, 2012 | vol. 109 | no. 7 | 2605 Downloaded by guest on October 2, 2021 camstatin (5 μM) was included in the pipette electrolyte. Toxins applied lo- Electrophysiology. Whole-cell current-clamp somatic recordings were made cally were ejected from a pressure electrode containing Hepes (10 mM)- using Axoclamp amplifiers and Digidata 1322 with a DC-10 kHz band-pass buffered aCSF with 0.1% BSA as carrier medium at concentrations of 100 nM filter and pClamp software. Negative bias current of less than 1.0 nA was μ ω ChTx, 200 nM IbTx, 200 nM AgTx, 1 M -conotoxin GVIA, 200 nM SNX-482, applied during current-clamp recordings to maintain Purkinje cell resting μ 1 M nifedipine, with appropriate synaptic blockers. When applying HVA potential at ∼−75 mV and below the level of tonic firing. See SI Materials Ca2+ channel blockers, TTX was included in the bath and pressure electrode. and Methods for further details. TTX, NiCl2, CdCl2, CsCl, TEA, 4-AP, and picrotoxin were prepared daily from stock solutions and all other drugs daily from frozen aliquots. Data Analysis and Statistics. Statistical analysis was carried out in OriginPro 8. For current clamp recordings the pipette electrolyte consisted of 130 mM Unless otherwise indicated, paired-sample Student t tests were used to de- K-gluconate, 0.1 mM EGTA, 10 mM Hepes, 7 mM NaCl, 0.3 mM MgCl2,pH7.3 termine significance. The Tukey HSD post hoc comparison was used to test with KOH, providing an ECl of −76 mV and EK of −97 mV. Di-Tris-creatine phosphate (5 mM), 2 mM Tris-ATP and 0.5 mM Na-GTP were added daily significance between means following one-way or repeated-measures from frozen stock solutions. A junction potential of −10.7 mV was sub- ANOVA. Average values are expressed as mean ± SEM; *P < 0.05, **P < 0.01, tracted from current clamp recordings. Outside-out voltage-clamp record- ***P < 0.001. ings of KCa3.1 were obtained using an electrolyte of 140 mM KCl, 1 mM MgCl2, 5 mM EGTA, and 10 mM Hepes, pH 7.3 with KOH, with bath perfu- ACKNOWLEDGMENTS. We thank L. Chen and R. Winkfein for expert sion of TTX, apamin, TEA, 4-AP, CdCl2, and CsCl. Whole-cell voltage-clamp technical assistance and Dr. A. P. Braun for helpful discussions and providing recordings of Purkinje cell Cav3 current from P10–P12 animals used an KCNN4 cDNA, endothelial cell cultures, and primers for KCa1.1 and KCa2.2 RT-PCR tests. This work was supported by grants from the Canadian Insti- electrolyte consisting of 140 mM CsCl, 1 mM MgCl2, 5 mM EGTA, and 10 mM Hepes, pH 7.3 with CsOH, and bath perfusion of TTX, apamin, CdCl , TEA, 4- tutes of Health Research (CIHR) (to R.W.T. and G.W.Z.); studentships through 2 Alberta Innovates-Health Solutions (AIHS) (to W.H.M., R.R., D.A., and J.D.T.E.); AP, and CsCl. On-cell recordings of KCa3.1 single channels used an electro- T. Chen Fong awards (to J.D.T.E. and D.A.); a Killam Scholarship (to B.E.M. lyte of Hepes-buffered aCSF containing 150 mM NaCl, 3.25 mM KCl, 1.5 mM and D.A.); a Natural Sciences and Engineering Research Council award CaCl2, 1.5 mM MgCl2, 10 mM Hepes, and 20 mM D-glucose, pH 7.3 with (to R.R.); and a CIHR-Canada Graduate Scholarships award (to J.D.T.E.). NaOH, and TTX, apamin, TEA, 4-AP, CdCl2, CsCl and synaptic blockers in both R.W.T. is an AIHS Scientist and G.W.Z. is an AIHS Scientist and Canada the electrode and external medium. Research Chair.

1. Mapelli J, Gandolfi D, D’Angelo E (2010) High-pass filtering and dynamic gain regu- 23. Lein ES, et al. (2007) Genome-wide atlas of gene expression in the adult mouse brain. lation enhance vertical bursts transmission along the mossy fiber pathway of cere- Nature 445:168–176. bellum. Front. Cell. Neurosci. 4:1–13. 24. Molineux ML, et al. (2006) Specific T-type calcium channel isoforms are associated 2. D’Angelo E, De Zeeuw CI (2009) Timing and plasticity in the cerebellum: Focus on the with distinct burst phenotypes in deep cerebellar nuclear neurons. Proc Natl Acad Sci granular layer. Trends Neurosci 32:30–40. USA 103:5555–5560. 3. Pedarzani P, Stocker M (2008) Molecular and cellular basis of small—and intermedi- 25. Nguyen TV, et al. (2007) Effects of compounds that influence IK (KCNN4) channels on — ate conductance, calcium-activated potassium channel function in the brain. Cell afterhyperpolarizing potentials, and determination of IK channel sequence, in guinea – Mol Life Sci 65:3196 3217. pig enteric neurons. J Neurophysiol 97:2024–2031. 4. Berkefeld H, Fakler B, Schulte U (2010) Ca2+-activated K+ channels: From protein 26. Kaushal V, Koeberle PD, Wang Y, Schlichter LC (2007) The Ca2+-activated K+ channel complexes to function. Physiol Rev 90:1437–1459. KCNN4/KCa3.1 contributes to microglia activation and nitric oxide-dependent neu- 5. Joiner WJ, Wang LY, Tang MD, Kaczmarek LK (1997) hSK4, a member of a novel sub- rodegeneration. J Neurosci 27:234–244. family of calcium-activated potassium channels. Proc Natl Acad Sci USA 94:11013–11018. 27. Cingolani LA, Gymnopoulos M, Boccaccio A, Stocker M, Pedarzani P (2002) De- 6. Ishii TM, et al. (1997) A human intermediate conductance calcium-activated potassium velopmental regulation of small-conductance Ca2+-activated K+ channel expression channel. Proc Natl Acad Sci USA 94:11651–11656. and function in rat Purkinje neurons. J Neurosci 22:4456–4467. 7. Wei AD, et al. (2005) International Union of Pharmacology. LII. Nomenclature and mo- 28. Hannah RM, Dunn KM, Bonev AD, Nelson MT (2011) Endothelial SK(Ca) and IK(Ca) lecular relationships of calcium-activated potassium channels. Pharmacol Rev 57:463–472. 8. Joiner WJ, Khanna R, Schlichter LC, Kaczmarek LK (2001) Calmodulin regulates as- channels regulate brain parenchymal arteriolar diameter and cortical cerebral blood fl – sembly and trafficking of SK4/IK1 Ca2+-activated K+ channels. J Biol Chem 276: ow. J Cereb Blood Flow Metab 31:1175 1186. 37980–37985. 29. Ishii TM, Maylie J, Adelman JP (1997) Determinants of apamin and d-tubocurarine – 9. Wulff H, Gutman GA, Cahalan MD, Chandy KG (2001) Delineation of the clotrimazole/ block in SK potassium channels. J Biol Chem 272:23195 23200. TRAM-34 binding site on the intermediate conductance calcium-activated potassium 30. Fakler B, Adelman JP (2008) Control of K(Ca) channels by calcium nano/microdomains. channel, IKCa1. J Biol Chem 276:32040–32045. Neuron 59:873–881. 10. Wulff H, et al. (2000) Design of a potent and selective inhibitor of the intermediate- 31. Oliver D, et al. (2000) Gating of Ca2+-activated K+ channels controls fast inhibitory conductance Ca2+-activated K+ channel, IKCa1: A potential immunosuppressant. Proc synaptic transmission at auditory outer hair cells. Neuron 26:595–601. Natl Acad Sci USA 97:8151–8156. 32. Brenowitz SD, Best AR, Regehr WG (2006) Sustained elevation of dendritic calcium 11. Hosy E, Piochon C, Teuling E, Rinaldo L, Hansel C (2011) SK2 channel expression and evokes widespread endocannabinoid release and suppression of synapses onto cer- function in cerebellar Purkinje cells. J Physiol 589:3433–3440. ebellar Purkinje cells. J Neurosci 26:6841–6850. 12. Swensen AM, Bean BP (2003) Ionic mechanisms of burst firing in dissociated Purkinje 33. Mittmann W, Koch U, Häusser M (2005) Feed-forward inhibition shapes the spike neurons. J Neurosci 23:9650–9663. output of cerebellar Purkinje cells. J Physiol 563:369–378. 13. McKay BE, et al. (2006) Ca(V)3 T-type calcium channel isoforms differentially distrib- 34. Raman IM, Bean BP (1999) Ionic currents underlying spontaneous action potentials in ute to somatic and dendritic compartments in rat central neurons. Eur J Neurosci 24: isolated cerebellar Purkinje neurons. J Neurosci 19:1663–1674. 2581–2594. 35. Rancz EA, et al. (2007) High-fidelity transmission of sensory information by single 14. Hildebrand ME, et al. (2009) Functional coupling between mGluR1 and Cav3.1 T-type cerebellar mossy fibre boutons. Nature 450:1245–1248. fi calcium channels contributes to parallel ber-induced fast calcium signaling within 36. Vogalis F, Harvey JR, Neylon CB, Furness JB (2002) Regulation of K+ channels underlying – Purkinje cell dendritic spines. J Neurosci 29:9668 9682. the slow afterhyperpolarization in enteric afterhyperpolarization-generating myenteric 15. Angelo K, London M, Christensen SR, Häusser M (2007) Local and global effects of I(h) neurons: Role of calcium and phosphorylation. Clin Exp Pharmacol Physiol 29:935–943. – distribution in dendrites of mammalian neurons. J Neurosci 27:8643 8653. 37. Neylon CB, et al. (2004) Intermediate-conductance calcium-activated potassium 16. Lee JH, Gomora JC, Cribbs LL, Perez-Reyes E (1999) Nickel block of three cloned T-type channels in enteric neurones of the mouse: Pharmacological, molecular and immu- calcium channels: Low concentrations selectively block alpha1H. Biophys J 77:3034–3042. nochemical evidence for their role in mediating the slow afterhyperpolarization. J 17. McDonough SI, Bean BP (1998) Mibefradil inhibition of T-type calcium channels in Neurochem 90:1414–1422. cerebellar purkinje neurons. Mol Pharmacol 54:1080–1087. 38. Ngo-Anh TJ, et al. (2005) SK channels and NMDA receptors form a Ca2+-mediated 18. Randall AD, Tsien RW (1997) Contrasting biophysical and pharmacological properties feedback loop in dendritic spines. Nat Neurosci 8:642–649. of T-type and R-type calcium channels. Neuropharmacology 36:879–893. 39. Wolfart J, Roeper J (2002) Selective coupling of T-type calcium channels to SK po- 19. Zamponi GW, Bourinet E, Snutch TP (1996) Nickel block of a family of neuronal calcium channels: Subtype- and subunit-dependent action at multiple sites. J Membr Biol tassium channels prevents intrinsic bursting in dopaminergic midbrain neurons. J – 151:77–90. Neurosci 22:3404 3413. 20. Coetzee WA, et al. (1999) Molecular diversity of K+ channels. Ann N Y Acad Sci 868: 40. Cueni L, et al. (2008) T-type Ca2+ channels, SK2 channels and SERCAs gate sleep-re- 233–285. lated oscillations in thalamic dendrites. Nat Neurosci 11:683–692. 21. Rauer H, et al. (2000) Structure-guided transformation of charybdotoxin yields an 41. Smith MR, Nelson AB, Du Lac S (2002) Regulation of firing response gain by calcium- analog that selectively targets Ca(2+)-activated over voltage-gated K(+) channels. J dependent mechanisms in vestibular nucleus neurons. J Neurophysiol 87:2031–2042. Biol Chem 275:1201–1208. 42. Anderson D, et al. (2010) Regulation of neuronal activity by Cav3-Kv4 channel sig- 22. Slemmon JR, et al. (1996) Camstatins are peptide antagonists of calmodulin based naling complexes. Nat Neurosci 13:333–337. upon a conserved structural motif in PEP-19, neurogranin, and neuromodulin. J Biol 43. Anderson D, et al. (2010) Regulation of the KV4.2 complex by CaV3.1 calcium chan- Chem 271:15911–15917. nels. Channels (Austin) 4:163–167.

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