Single-Channel Recordings of Three K+-Selective Currents in Cultured Chick Ciliary Ganglion Neurons

The Journal of Neurosctence July 1986, 6(7): 2106-2116

Phyllis I. Gardner Department of Pharmacology, University College London, and Department of Medicine, Stanford University Medical Center, Stanford, California 94305

Multiple distinct K+-selective channels may contribute to action for a multiplicity of outward K+ currents includesidentification potential repolarization and afterpotential generation in chick of complex gating kinetics and separationof individual current ciliary neurons. The channel types are difficult to distinguish components by pharmacologicalmanipulations. Characteriza- by traditional voltage-clamp methods, primarily because of tion of the individual componentsof outward K+ current is of coactivation during depolarization. I have used the extracellular fundamental interest, since K+ conductancesappear to be in- patch-clamp technique to resolve single-channel K+ currents in volved in such physiological processesas resting potential de- cultured chick ciliary ganglion (CC) neurons. Three unit cur- termination, action potential repolarization, afterpotential gen- rents selective for K+ ions were observed. The channels varied eration, and control of repetitive firing. Moreover, K+ channels with respect to unit conductance, sensitivity to CaZ+ ions and may be an important site for neurotransmitter modulation of voltage, and steady-state gating parameters. The first channel, neuronal excitability (for review, seeHartzell, 1981). However, GK,, was characterized by a unit conductance of 14 pica-Sie- the identification and characterization of the individual com- mens (pS) under physiological recording conditions, gating that ponents of outward K+ current by voltage-clamp analysis has was relatively independent of membrane potential and intracel- been difficult, primarily becauseall componentscan be coac- lular Ca*+ ions, and single-component open-time distributions tivated during depolarization. The extracellular patch-clamp with time constants of approximately 9 msec. The second chan- technique (Hamill et al., 1981) enablesthe resolution of aggre- nel, GK,, was characterized by a unit conductanceof 64 pS gate outward K+ currents into unitary channel currents. The under physiological recording conditions and gating that was individual K+ channelpopulation can then becharacterized with affected by membranepotential but was not dependenton the respectto conductance, ionic selectivity, steady-stategating ki- activity of intracellular Ca*+ ions. Open-time distributions in- netics, and sensitivity to Ca*+and voltage. dicated 2 open states,with open-timeconstants of 0.09 (61%) This paper reports experiments using the patch-clamp tech- and 0.35 (39%) msec,at +40 mV membranepotential. The nique to study K+ channelsin dissociatedchick ciliary para- third channel, GI(cn*+, was identified in isolated patch record- sympathetic ganglion neurons maintained in primary culture. ings in which the concentration of internal Ca*+ was lo-’ M or Previous conventional microelectrodestudies (Bader et al., 1982) greater, which wasan absoluteprerequisite for channelopening. have suggestedthat at least 2 K+ conductancescontribute to G&,*+ was characterized by a unit conductanceof 193 pS in action potential repolarization and to hyperpolarizing inhibitory symmetrical 0.15 M KC1 solutions, an open-state probability afterpotentials in chick ciliary neurons in culture. These K+ that was a function not only of [Ca2+jebut also of membrane conductances,or as-yet-unidentified ones,appear to be the site potential, and single-componentopen-time distribution with a of action mediating the hyperpolarizing postsynaptic responses time constant of 1.11 msecat - 10 mV patch potential. These of muscarinic ACh (Hartzell et al., 1977)and enkephalins(Ka- results suggestthe presenceof at least 3 distinct K+ channel tayama and Nishi, 1984),2 transmittersthat have beenlocalized populationsin the membraneof cultured chick CG neurons. by immunohistochemical study to preganglionicnerve termi- nals in the chick ciliary ganglion (Erichsen et al., 1982). In the Voltage-clamp records provide evidence that several types of present study, 3 distinct K+ channel populations are identified outward K+ current can flow across the soma membrane of and briefly characterized by single-channelrecordings. invertebrate (Connor and Stevens, 1971; Meech and Standen, 1975; Neher, 1971; Thompson, 1977) and vertebrate neurons Materials and Methods (Adams et al., 1982; Barrett and Barrett, 1976; Barrett et al., Cell preparation 1980;Belluzzi et al., 1985;Dubois, 1981; Galvan and Sedlmeir, 1984;Kostyuk et al., 1981; Krylov and Makovsky, 1978;McAfee Chick CG cells were dissociated from 8- to IO-d-old chicks and grown and Yarowsky, 1979). Supporting evidence from these studies in tissue culture by the methods of Nishi and Berg (1981). Cells were maintained in bicarbonate/CO,-buffered Eagle’s minimal essential medium (MEM), containing a raised KC1 concentation of 25 mM, 10% calf Received Oct. 31, 1985; revised Jan. 15, 1986; accepted Jan. 17, 1986. serum, 2% chick eye extract (Nishi and Berg, 198 l), 120 &ml ben- I extend my thanks to Dr. David Colquhoun for his excellent guidance and zylpenicillin, 200 &ml streptomycin, and 100 &ml ampicillin, in a instruction during my tenure in his laboratory. I also thank Chris Magnus for his humidified atmosphere of 5% CO, in air at 37°C for 5-6 d before expert instruction on tissue culture techniques and Joan Rose1 for her skilled experiments. assistance. in manuscript preparation. This work was supported initially by a fellowship from the Muscular Dystrophy Experimental conditions Association of America, and subsequently by a grant-in-aid from the American Heart Association, with funds contributed in part by the Santa Clara County During experiments, the growth medium was replaced by HEPES-buff- Chapter of the AHA, California Affiliate, and in part by the AHA, Florida Affiliate. ered Ringer’s solution with the following composition (in mM): NaCl. Correspondence should be addressed to Phyllis I. Gardner, M.D., Cardiovas- 134; KCC 5.4; MgSO,.7H,O, 0.8; NaH,PO,, 1;l; CaCl,,‘ 1.8; NiHCO,; cular Research Center, 29 1, Department of Medicine, Stanford University Medical 5; glucose, 5.5; HEPES, 10 (adjusted to pH 7.2-7.3 by NaOH). In records Center, Stanford, CA 94305 with isolated patches, a high K+ solution, consisting of (in mM): KCl, Copyright 0 1986 Society for Neuroscience 0270-6474/86/072 106- 11$02.00/O 150; CaCl,, 1.O; MgCl,, 1.O; HEPES, 10 (pH 7.2-7.3, adjusted by KOH),

2106 The Journal of Neuroscience Single-Channel K+ Currents in Chick Ciliary Neurons

3 PA

50 msec Figure1. Single-channel currents recorded from 8 d chick embryo ciliary ganglion cells dissociated and maintained in tissue culture. Recordings were from a “cell-attached” patch depolarized 60 mV from resting membrane potential, yielding an absolute patch potential of 0 mV. The patch pipette contained a physiological solution with 134 mM NaCl and 5.4 mM KCl. The outward current is upward.The presence of 2 step sizes indicates that 2 channel types are active in this patch.

was used in the circumstances stated in the text. In other indicated these records. The data from the idealized record were used for construc- circumstances, KC1 was replaced by equimolar amounts of NaCl. So- tion of histograms to display the distribution of current amplitudes lutions of variable ionized CW+ were obtained by use of appropriate (excluding open events too brief to reach full amplitude under recording Ca-EGTA buffers, taking 1Om’ M as the apparent dissociation constant conditions) and of open and shut durations, and to fit probability density (Marty, 198 1). Solutions with varying levels of Ca*+ were made as fol- functions by the method of maximum likelihood to the continuous lows: < 1O-9 M ICa2+] = 0.0 mM CaCl,, 2 mM M&l,, and 1.1 mM EGTA. distribution of data over the entire measured range. Multicomponent pH 7.3; estimated l& M [Ca*+] = 0.i mM Ca& 2 mM MgCl,, and 1. i exponential fits were assessed by eye. Probability of channel openings mM EGTA, pH 7.3; estimated lo-’ M [Ca2+] = 0.55 mM CaCl,, 2 mM (PO,“> equal to the ratio of the total time spent in the open state to the MgCl,, and 1.1 mM EGTA, pH 7.3; estimated 5 x lo-’ M [Ca2+] = 0.92 total length of record) and opening frequency (O,, equal to the number mM CaCl,, 2 mM MgCl,, and 1.1 mM EGTA, pH 7.3; and estimated of channel openings per second of closed time) were also determined 10m6M ICa2+1 = 1 mM Ca*+. 2 mM MeCl,. and 1.1 mM EGTA. DH 7.3. from the records. Further details on these methods are given by Solutions were buffered to pH 7.3 b; l{rnM HEPES and adju&ed by Colquhoun and Sakmann (198 1) and Colquhoun and Sigworth (1983). KOH. During experiments, plates were mounted on an aluminum holder incorporated into a microscope fitted with phase-contrast optics and Results viewed with a 20 x long-working-distance objective. Experiments were Currents were recorded from embryonic chick ciliary ganglion performed at room temperature, which ranged from 18-22°C. cells after 5-6 d in culture usinggigaseal patch-clamp recording Patch-clamp recording techniques.With single-channelrecords obtained from cell-attached patcheswith electrodescontaining 5.4 mM KCl, channel The techniques for high resistance (10-100 Gfl) pipette-membrane seal activity was infrequent at zero applied voltage (resting mem- formation, excision of membrane patches, and single ion-channel recording are as described by Hamill et al. (198 1). The recording config- brane potential, V,, 58 f 2 mV, 10 samples)and at hyperpo- urations of “cell-attached” patch, “inside-out” patch, and “outside-out” larized potentials. Depolarization of the patch usually revealed patch were employed. Patch-clamp circuitry (List EPC-7, List-Elec- at least 2 channel types mediating outward current in the cell- tronic, Pfungstadter Strasse 18-20,- 6 100 Darmstadt-Eberstadt, FRG) attached patches (Fig. 1). When the K+ concentration on the was similar to that described in Hamill et al. (198 1). Patch oioettes extracellular side of the membrane patch [K+], was raised to were fabricated from Pyrex glass with microfilament insertion (GC i 5OF, levels between 11 and 150 mM, these2 channeltypes were seen Clark Electromedical Instruments, Pangbourne, Reading, UK) on a ver- to be active at resting membranepotential, and inward current tical 2-stage pipette puller (pipette puller model 720, David Kopf In- stepswere observed at hyperpolarizing voltages. Most patches struments, Tujunga, CA) to form resistances of 1O-l 5 mQ. Patch pipettes studied contained both channel types. In addition, at very de- were coated at the tip with Sylgaard (Sylgaard 184 silicone elastomer kit, Dow Coming Corp., Midland, MI 48640) and fire-polished prior polarized voltages (80-120 mV from resting potential) a third to use. type of unitary outward current, consisting of rare brief events of very large amplitude, could infrequently be discernedin cell- Data analysis attached patchesexposed to pipettes with 5.4 mM KCl. These Patch-clamp currents were recorded on magnetic tape at tape speed 7% events rarely reachedfull amplitude at the recordingbandwidth ips on a Racal Store 4DS FM tape recorder (Racal Recorders Ltd., of 3 KHz. When the membrane patcheswere isolated in the Bra&tell, Berkshire, UK). Recordings were digitized at 0.1 msec time “inside-out” recording configuration and exposedto 1O-’ M or intervals (at tape speed 3.75 ips, yielding a 20 lcHz sampling frequency) greaterlevels of Ca*+on the intracellular membranesurface, the and stored on a PDP 1 l-34 computer (Digital Equipment Corporation, third current becamereadily apparent as single-channelevents 10 Forbes Road, Northboro, MA) after appropriate low-pass filtering of very large unit amplitude. at 3 kHz (- 3 dB point, Bessel characteristic, 48 dB octave rollotT). The Thus, 3 distinct channelsmediating outward current are sug- digitized record was used to measure the amplitude and duration of gestedby patch-clamp recordings of CG neurons.The experi- individual current pulses and the interval between single pulses by a semiautomatic procedure (Colquhoun and Sigworth, 1983). After the mental results describing these channels-referred to as GK,, measurement, a value was chosen for the minimum resolvable duration, GIL and GLc2+,, follow. Evidencethat GK,, GK,, and GLc2+, and the record was revised by concatenation of adjacent gaps and open- are all predominantly K+-selective but differ in unitary con- ings separated by intervals less than this chosen resolution. The mini- ductance, steady-stategating kinetics, and Ca2+and voltage sen- mum resolvable event was consistently between 100 and 200 psec in sitivity is presented. 2108 Gardner Vol. 6, No. 7, Jul. 1986

+80

+60

+40

Figure 2A. Conductance and selectivity properties of single GK, channels. Examples of GK, outward currents recorded from a “cell-attached” patch at various depolarized patch membrane potentials. The current-recording pipette contained a physiological solution with 134 mM NaCl + 20 anld 5.4 mM KCl. The numbersat the left of each trace indicate the shift in patch potential (in mV) with respect to the cell’s resting potential (V,) of -60 mV, for example, +20 mV in- 3 PA dicates a patch potential depolarized 20 mV from V,, yielding an absolute J patch potential of -40 mV. 50 msec

Conductanceproperties of GK, The channel identified as GK, can be identified as the unitary current event of smaller amplitude in Figure 1. This channel is characterized by well-defined square-pulse events that increase in amplitude when the membrane potential of the patch is shift- ed to potentials more positive than V, (Fig. 2A). Figure 2B shows the relationship between unitary current amplitude and membrane potential in cell-attached patches with [K+], of 5.4 mM. The Z-I/ relationship is essentially linear, indicating a voltage-

3 TPA p a-.- ---4 /4.4- t ,p.-.-- _.a-- P 4 mV I : : I I : I I -60 -30 0 30 60 [K+l (mM) Figure 2B. Current-voltage relationship of single GK, channel currents obtained from 7 patches as in A. The mean amplitude of current step sizes is plotted as a function of patch potential. Each circle represents Figure 2C. Zero-current potentials as a function of [K’],. The data the cumulative results of the 7 patches. The mean L’, was approximately represent the results of 16 experiments. Each symbolrepresents the -60 mV (range, -58 to -63 mv). The line represents a linear-regres- average extrapolated zero-current potential from 2-7 experiments. These sion line fitted to the data points. The slope conductance is 14 pS, and values are plotted as a function of the logarithm of [K+], and fitted by the extrapolated zero-current, or reversal, potential is -95 mV. a straight line with a slope of 63.5 mV per IO-fold change in [K+],. The Journal of Neuroscience Single-Channel K+ Currents in Chick Ciliary Neurons 2109

75 0 r

62.5 -

fn c = 50.0- L k 37.5- (L mw 5 25.0 - z 2PA 12.5 - t I1

5 msec AMPLITUDE (pAI Figure 3A. Occurrence of GK, current sublevels. Example of an out- Figure 3B. Distribution of current levels from a recording of 300 single- ward GK, current step recorded at absolute patch potential of + 20 mV current steps at the same membrane potential and [K+],. In addition to (depolarized 80 mV from V,), recorded with 5.4 mM KC1 in the pipette. the main peak with mean amplitude of 1.9 pA, there was a small cluster The current wave shape recording digitized at 10 KHz is represented of events around 1.O pA amplitude that may have represented the sub- by the dots. The waveform opens to the full current amplitude predicted level amplitude. by single-channelconductance, enters an intermediate level of current, and then returns to the main level before closure. behavior was studied in patches in which no more than one independent single-channel slope conductance of 14 pica-Sie- GK, channel was open at any one time during the period of observation in order to maximize the probability that there was mens (pS) and an extrapolated zero-current potential of -95 mV. only one channel in the membrane patch. (The presence of 2 The negative zero-current potential suggests that GK, is pre- or more channels that do not coincidentally open during the dominantly K+ selective. This is further indicated by Figure 2C, period of observation cannot be ruled out; in such a case, es- which depicts the semilogarithmical plot of zero-current poten- timates obtained would be too high for single-channel open- tial vs external K+ concentration. The straight line obtained by state probability.) Individual channels appeared as relatively long openings interrupted by rapid closings and separated by least-squares fit has a slope of 63.5 mV change in zero-current potential for a lo-fold increase in [K’],. The predicted change longer closed periods. The fraction of time spent in the open in zero-current potential for a lo-fold change in [K+],, which state P,,, (calculated as total open time over total length of record) was approximately 0.10, independent of the patch po- is derived from the Nemst equation with an average temperature of 20°C (293.2 K), is 58.2 mV. Within the limitation of exper- tential studied. Thus, no clear voltage dependence of either uni- imental error, the actual 63.5 mV change in zero-current po- tary conductance or open-state probability of GK, could be demonstrated. The voltage independence was further analyzed tential is very close to this predicted value. It is concluded that GK, is highly K+-selective. by measuring the open time of the channel and the interval In addition to the main conductance state, GK, appears to between successive openings from continuous recordings for more than 10 set at two different patch potentials. Probability adopt a substate of conductance. In approximately half the patches studied, events of one-halfto two-thirds full GK, current density histograms of the open time measured at patch poten- amplitude (Fig. 3A) were seen, both contiguous with and isolated tials depolarized 50 and 100 mV from V, are shown in Figure 4A, B. In each case, the histogram is well fitted by a single from full GK, currents. A correlation was seen between the occurrence of these events, suggesting that the small event was exponential with a time constant of 9.7 and 8.6 msec, respec- due to a substate of the fully conducting GK, channel; however, an entirely unrelated channel could not be absolutely ruled out. PA Distributions of GK, current amplitudes (Fig. 3B) often showed, in addition to the main peak, clusters of events approximately 0.5-0.6 the amplitude of the main current peak and were infrequent relative to the frequency of occurrence (calculated from the area under the curve) of the main current peak. Figure 3C shows the composite Z-V relationship obtained from 9 experiments in which “sublevels” were seen. The slope of the linear regression line fitted to the data points provides an estimate of conductance of 9 pS, which compares to a conductance of 14 pS for the fully conducting state. Similar substates of conductance have been described for other K+-selective channels, in- cluding the inward-rectifying K+ channel in ventricular cells .-0 2 (Sakmann and Trube, 1984a) and the slow-potential-sensitive I I I I K+ channels in longitudinal smooth muscle cells (Benham and -80 -60 -40 -20 0 20 40 60 80 Bolton, 1983). mV Voltage independence and steady-state kinetic Figure SC. Z-V relationship for sublevels observed in cell-attached patches with [K+], = 5.4 mM. Data from 9 different experiments are properties of the GK, channel shown. Each symbol represents the mean (*SD) sublevel amplitude The effect of steady-state voltages on GK, channel gating pa- from at least 5 observations, with the exception of data points without rameters was analyzed from single-channel records in which the SD bars (only 1 observation). The slope conductance from the line fitted holding voltage was maintained constant for 30-40 sec. Channel by linear regression to the data points was 9 pS. 2110 Gardner Vol. 6, No. 7, Jul. 1986

72 r +40mv

Popen = 0.12 0 freq = 11.6/s Popen = O ‘O To = 9.7 ms 0 freq’ 10 4/s ~~ = 8.6 ms

10 20 30 40 50 0 7.5 15 22.5 30 375 45 OPEN TIME (ms, OPEN TIME (ms, Figure 4A. Distributionof opentimes of GK,, plotted asprobability density.Frequency vs open-timehistogram from 300 single-channel Figure 0. Frequencyvs open-timehistogram from 370 single-channel eventsat a patchpotential of - 10 mV (depolarized50 mV from VJ. eventsat a patchpotential of +40 mV (depolarized100 mV from V,). The histogramof the channelopen times was fitted by a singleexpo- The histogramof channelopen times ar this potentialwas fitted by~a nential(curvedline) with a timeconstant of 9.7 msec.The coincidentally singleexponential (curved line) with a time constantof 8.6 msec.The determinedprobability of opening(Z’,,.; measuredas open time divided coincidentallydetermined P,,,. and O,, were0.1 and 10.4/set,respec- by total recordduration, which includedthe time segmentcontaining tively. the 300 openings)was 0.1, and the openingfrequency (0, = number of openingsper secondof closedtime) was 11.6/set, as indicated in the upperright-hand corner. Voltage dependenceand steady-state gating kinetics of GK, Steady-state mean open-channel open times and voltage de- tively, for - 10and + 40 mV. The meanchannel lifetime, channel pendence were assessedfrom records of cell-attached patches opening frequency, and fraction of time spent in the open state during maintained depolarizations for 10-l 5 set, as previously were very similar at these 2 voltages, further suggestingthat described.Probability-density histogramsof the open and closed GK, is relatively voltage-insensitive. times measuredfrom a patch depolarized 50 and 100 mV from The open-state probability of the GK, channel appeared to V, are shown in Figure 6. The open-time histogram at +40 mV be unaffectedby varying internal Ca*+concentration from < 1Om9 (Fig. 6B) consistsof 2 exponentials, a fast component with a to 1O-6M in experiments with inside-out patches. (At higher time constant of 0.09 msec (this component represents6 1% of levels of [Caz+li,the presenceof GK, was difficult to determine total area of the open-time distribution, correspondingto 475 secondaryto the presenceof large Ca2+-activatedK+ channels.) fast openings,the vast majority of which would be too short to Similarly, the fraction of time spent in the open state did not resolve with a fixed minimum resolvable open duration of 0.1 appearto changesubstantially during maintained depolariza- msec)and a slowercomponent of 0.35 msec (39% of the area). tions; that is, second-to-secondopen-state probabilities varied This suggeststhat the GK, channel may have 2 distinct open randomly without consistent changes. stateswith similar conductancesbut dissimilar lifetimes. In contrast, the open-time distribution at - 10 mV (Fig. 6A) is well Conductanceand selectivity of GK, fitted by a singleexponential with a time constant of 0.26 msec. The channelsthat are termed GK, are the unitary events of This may indicate that the presenceof 2 open statesis potential- larger amplitude seenin Figure 1. Examples of GK, currents dependent. Alternatively, the faster component may not have recordedfrom a cell-attached patch at various depolarized patch been adequately resolved at the more negative potential under potentials are seenin Figure 5A. The averageamplitude at each theserecording conditions, in which a minimum resolvableopen potential was calculated from openingsof sufficient duration to duration of 0.1 msecwas found. achieve full amplitude with a square-pulseconformation (open The degreeof voltage sensitivity could be approximated from events too rapid to be resolved,thus appearingas foreshortened the single-channelrecords in patchesin which no more than trianglesin digitized records, were not usedin mean amplitude one GK, channel was open at any one time during the period calculations).The relation betweenmean amplitude and holding of observation. In contrast to the findings with GK,, GK, ap- potential is plotted in Figure 5B. This I-Vrelation is essentially pearedto have a voltage-sensitive P,,,, which increasedwith linear, indicating voltage-independentunitary conductance.The membrane depolarization. The voltage dependenceof Popenre- linear-regressionline fitted to the data points provides an es- sidesin both longer open durations and higher opening fre- timate of single-channelconductance of 64 pS and an extrap- quencies at positive potentials. For example, Pow,,increased olated zero-current potential of - 85 mV, near the predicted K+ from 0.002 at - 10 mV (V, + 50 mV) to 0.01 at +40 mV (V, + equilibrium potential. 100 mV). Channel-openingfrequency, coincidentally, increased Relatively high K+ selectivity of GK,, which is suggestedby from 5.1 to 3 1.O/sec, while the slow-time constantof open time the negative zero-current potential under “physiological” re- increasedfrom 0.26 to 0.35 msec(Fig. 6A, B). In contrast, there cording conditions, is confirmed by experiments with variable was no evidence that Pope,,was affected by changesin Ca*+con- [K+],. The semilogarithmic plot of zero-current potential vs ex- centration from lO-9 to 1O-6M in experiments with inside-out ternal K+ concentration (Fig. 5C) provides an estimate of 55.4 patches.The presenceof GK, wasdifficult to determineat higher mV shift in zero-current potential for a 1O-fold increasein ex- levels of [Ca2+lidue to the presenceof large, Ca2+-activatedK+ tracellular K+ concentration very closeto the theoretical value channels,as describedfor GK,. As noted with GK,, there was of 58.2 mV predicted by the Nemst equation of a K+ selective no evidence of any time-dependent inactivation processduring channel at 20°C. This suggeststhat GK,, like GK,, is highly maintained depolarizations of 10-60 set; this was not explored selective for K+ ions. in detail, however. The Journal of Neuroscience Single-Channel K+ Currents in Chick Ciliary Neurons 2111

Q PA +60 100 msec Figure 5A. Conductance and selectivity properties of single GK, channels. Examples of GK, outward currents recorded from a chick CG neuron “cell-attached” patch at various depolarized patch-membrane potentials. The current-recording pipette +30 contained a physiological solution with NaCl, 134 mM, and KCl, 5.4 mM. The numbers at the left of each trace represent displacement from V,.

pended on the logarithm of [K’],, as illustrated in Figure 7C. Conductance and selectivity properties of GKc,(2+1 The slopeof the regressionline fitting the data in semilogarith- In addition to GK, and GK, channelsidentified in recordings mic coordinatesprovides an estimateof a 60.3 mV shift in zero- from cell-attached patches, a K+-selective, Ca*+- and voltage- current potential for a lo-fold increasein external K+ concen- dependent current of very large unit conductance was seenin tration. This value indicates that the current is highly selective records from inside-out patchesexposed to variable levels of for K+ ions, since it is very close to the predicted value of the internal (previously intracellular) CaZ+concentration. Figure 7A Nemst equation (58.2 mV at 20°C). In addition, it should be showsunitary outward membrane currents from a cell-free in- noted that unitary outward currents were never observedwhen side-out membrane patch in symmetrical high-K+ solutions inside-out patcheswere perfusedwith internal NaCl solutions. (KCl, 150 mM, with 1.O mM CaCl,), depolarized from absolute zero mV to 5 different positive membrane potentials. Single- channelevents characteristic of GQ,,,, were of very large unit amplitude, which depended on membrane potential. The Z-V relationship for GKeac2+,obtained from 5 inside-out patchesin symmetrical 0.15 M KC1 solutions is shown in Figure 7B. The linear-regressionline fitted to the data points provides an estimate of single-channelconductance of 193 pS. The zero-current potential was about 0 mV, as expected with identical solutions on both sidesof the membrane face. The zero-current potential of isolated patches(with constant 5.4 mM [K+]J de-

*O pA

i P/W'-, -p/e-H' &-d 10 1 -w-c *.H' , mV -100’ I I / I 1 I ; 10 20 50 100 200 -100 -50 -0 50 100 [K+lo (mM)

Figure 5B. The I-Vrelationship of GK, channel currents obtained from Figure 5C. Zero-current potentials as a function of [K+],. The data points 5 cell-attached patches. Mean amplitude of current step sizes is plotted represent the results of 13 experiments. Each symbol represents the as a function of membrane potential. The slope conductance obtained average extrapolated zero-current potential from 2-4 experiments. These from a linear-regression line fitted to the data points was 64 pS, with values are plotted as a function of the logarithm of [K+], and are fitted an extrapolated zero-current potential of - 84 mV. by a straight line with a slope of 55.4 mV per IO-fold change in [K+],. 2112 Gardner Vol. 6, No. 7, Jul. 1986

160 *40 rn”

60 P = 48 L k 36 r. : 0.26 ms (r It 5 24 z

I I I 1 I 2.5 3.0 2.5 3.0 OPEN TIME (ms) OPEN TIME (ms) Figure6B. Frequencyvs open-timehistogram from 500single-channel Figure6A. Distributionof opentimes of GK,, plotted asprobability density.Frequency vs open-timehistogram from 328 single-channel eventsat a patchpotential of +40 mV (depolarized100 mV from V,). eventsat a patchpotential of - 10 mV (depolarized50 mV from V,). The histogramof the channelopen timeswas fitted by a double-ex- The histogramof the channelopen time wasfitted by a single-expo- ponentialcurve with time constantsof 0.09 msec(representing 61% of nential curve with a time constantof 0.26 msec.The coincidentally the areaunder the curve) and 0.35 msec(39% of the area).The coin- determinedP,,. equaled0.002, and O,, equaled58/set. cidentallydetermined P,,, equaled0.0 1, and 0, equaled3 1 .O/sec.

density histogramsof open time measuredat - 10 and -60 mV Dependenceof GKcon+,opening on absolutepatch potential are shown in Figure 8B, C. The open- cytoplasmic Ca2+and voltage time histogram at each potential revealed a single-exponential G&a<,+, activation was dependenton the internal CaZ+concen- distribution with a time constant of 1.11 msecat - 10 mV and tration. Ionized Ca*+concentrations were obtained by the use 0.76 msec at -60 mV. The more depolarized potential was of appropriate Ca-EGTA buffers described in Materials and characterized not only by longer mean channel open time, but Methods. There was no evidence of channel activation with also by higher openingfrequency, asindicated in the upper right- internal Ca2+concentrations of lO-8 M or less.[Ca2+], of lo-’ M hand comer (O,, = 3750hec at -10 mV, 0, = 790/set at produced small increasesin channel-openingprobability at de- -60 mV). It should be noted that several investigators (Barrett polarized voltages (P,,, = 0 at -50 mV, P,,,” = 0.3 at 0 mV, et al., 1982; Magleby and Pallotta, 1983a,b; Moczydlowski and Popen = 0.4 at +50 mv). An increaseof [Caz+lito 1O-3M produced Latorre, 1983) have reported 2-component open-time distri- a dramatic voltage-dependentincrease in P,,,, that equaled 0.5 butions for large-conductance,Ca2+- and voltage-activated K+ at approximately - 45 mV membranepotential and approached channelsin other preparations.It is possiblethat a second,faster 1.O from potentials more positive than - 10 mV at millimolar component was not resolved under current recording condi- CaZ+concentrations (Fig. 8A). The channel disappearedwhen tions. the internal membrane surfacewas reperfused with nominally Ca*+-freesolutions. Since pipette solutions containing 1.0 mh4 Discussion Ca*+were usedfor theseinside-out patches,CaZ+ ions appeared The single-channelcurrent records in the present study defini- to be ineffective in producing channelopenings from the outside tively identify 3 distinct K+ conductancesin the membraneof of the membranes. cultured chick ciliary ganglion neurons. While all 3 channels Several investigators have described long-lived but infre- appearto be highly selective for K+ ions, they differ with respect quently occurring closed states for Caz+-activated K+-selective to unitary conductance, mean channel open times, opening fre- channelsexposed to millimolar levels of internal Ca2+concen- quency, probability of opening, and sensitivity to voltage and trations (Barrett et al., 1982; Latorre et al., 1982). This hasbeen intracellular Ca2+ions. At present,there are relatively few single- suggestedto be the result of channel blockade by Ca*+ ions channel data on K+ currents in nerves with which to compare (Vergaraand Latorre, 1983).While CY+ ion blockadeof GLcz+, the results.Comparisons will thus be made to patch-clampstud- in CG neuronsmay occur at millimolar internal CaZ+concen- ies of K+ channelsin a variety of tissues. trations, no definitive evidence of this was found in the present GK, is characterized by a low unitary conductance (14 pS) study. Infrequent, nonsustainedclosures to baselineoccasion- associatedwith high selectivity. While many highly selective ally occurred,but estimatesof open-stateprobability were great- low-conductance K+ channels have been describedin the lit- er than 0.9 at positive potentials during 1O-l 5 set maintained erature (for review, see Latorre and Miller, 1983), most are depolarizations.Longer periods of depolarization may have pro- voltage-dependent,with either rectifying unitary conductances vided evidence of a decreasedP,,,, indicating channelblockade. or gating kinetics that are sensitiveto membranepotential. GK, Channel open-stateprobability was dependent on voltage as differs from thesein its apparent voltage insensitivity with re- well as internal CaZ+concentration. As illustrated in Figure 8A, spect to either conductance or gating properties. For instance, channelopen-state probability isa sigmoidalfunction of voltage 2 examplesof singleneuronal K+ channelstudies, which coupled that increasesas the channel is depolarized for any given level low conductance with high selectivity, are the delayed rectifier of internal CaZ+concentration. This finding is similar to the of squid axon (Conti and Neher, 1980; Llano and Bezanilla, results of investigators on large-conductance, Ca*+-activated 1983) and the reconstituted lobster axon delayed rectifier (Co- channelsin several preparations. As with GK,, the increasein ronado et al., 1984), both of which exhibit strongly voltage- Popenat more positive potentials residesin both higher channel- dependent gating properties. On the other hand, patch-clamp opening frequencies and longer effective channel open times. studiesof the anomalousrectifier K+ channel in tunicate eggs Steady-stategating kinetic analysisduring maintained depolar- (Fukushima, 1981, 1982),in rat myotubes (Ohmori et al., 198l), izations was performed as previously described. Probability in atrioventricular and sinoatrial nodal cells of the rabbit heart The Journal of Neuroscience Single-Channel K+ Currents in Chick Ciliary Neurons 2113

+80

+60

Figure 7A. Conductanceand selectivity propertiesof GLt2+, currents. Examplesof C&+-activatedoutward currentsrecorded from a cell-free“inside-out” membranepatch isolated from a chick ciliary neuron.The re- cordingsolutions in the bath and pi- pettewere symmetrical, with the following composition(in mM): KCl, 150:CaCl,. 1 .o: M&l,. 1.0: KHEPES. 5.0,‘pH 7:3. The &&beri at the lest of each trace indicate displacement of 10pA the patch potential from 0 mV (positive numbers indicate depolarization). The linesat the left of each trace J100 ms indicate zero-current level.

(Sakmann et al., 1983), in guinea pig ventricular cells (Kameya- channel-openingfrequency. At least 4 other non-Ca2+-activated, ma et al., 1983; Sakmann and Trube, 1984a, b, Trube et al., K+-selective channels with similar conductancesand voltage 198 l), and in bullfrog atria1 cells (Momose et al., 1983) indicate sensitivitieshave beenstudied by single-ionchannel recordings. that these highly K+-selective channels have low unitary con- Gruol (1984) describesa 100 pS channel, which exhibits an ductances that are strongly voltage-dependent, exhibiting strong increasein opening frequency and open duration on depolari- inward rectification at positive potentials. Low-conductance, zation of the membraneof cultured Purkinje neurons.Clapham highly selective K+ channels in human erythrocytes (Grygorczyk and DeFelice (1984) have describeda 62 pS K+-selectivechan- et al., 1984; Hamill, 198 1) and HeZix pomatiu neurons (Lux et nel with open-stateprobability that increasesat positive mem- al., 198 1) have been described, these 2 channels, however, show brane potentials in embryonic chick heart sarcolemma.A 50 pS both voltage- and Ca2+-sensitive gating properties. Thus, GK, K+-selectivechannel that also hasan increasingopen-state prob- appears to be somewhat novel with respect to its apparent volt- ability on depolarization has been found in the membrane of age insensitivity with respect to conductance and gating. smooth muscle cells (Benham and Bolton, 1983). The sero- GK, is a highly K+-selective channel with a moderate (64 pS) tonin-sensitive K+ channel in Aplysia sensoryneurons has an conductance. It does not require CaZ+ ions for activation, but outwardly rectifying unitary conductance of 55 pS and gating does exhibit strongly voltage-dependent gating properties, with that is moderately affected by membranepotential (Siegelbaum an open-state probability that increases with membrane depo- et al., 1982). larization, due to increases in both channel open duration and Large-conductance“maxi” K+-selective channels activated 2114 Gardner Vol. 6, No. 7, Jul. 1986

P(0P.w

L / 1 , I I I I II 1 300 -80 -60 -40 -20 0 20 40 60 80 100 mV Figure 8A. Channel-openingkinetics as a functionof membranevolt- -201 age.Probability of a channelbeing in the openstate as a functionof membranepotential, Po( P), at a fixed interval (previouslyintracelhtlar) Figure 7B. Closed circles indicatethe current-voltagerelationship of Ca*+concentration of 1O-’ M. Po(v) wasdetermined as the sumof open- singleGL12+, channelcurrents obtained from 5 patchesin symmetrical statedwell times divided by the total timeof the single-channelrecords 0.15 M KCl, as in A. Slopeconductance determined from the linear- at eachpotential. Recordsat all potentialswere obtainedfrom one regressionline fitted to the data points is 193 pS, and extrapolated inside-outpatch with a singleCa2+- and voltage-activated K+ channel, reversalpotential is close to 0 mV, asexpected in symmetricalsolutions. asconfirmed during maximal activation by 1Om3 M Ca2+(Marty, 1981). All experimentswere carried out in the presenceof symmetrical0.15 M KC1solutions. The potentialacross the membranepatch was calcu- latedby taking the external(previously extracellular) side of the mem- braneas reference;that is, negativepotentials indicate hyperpolariza- by micromolar concentrationsof Ca2+at the intracellular surface tion. and by membrane depolarization have been describedin a variety of preparations’(for review, seeLatorre and Miller, 1983). Other than the kinetic findings cited in the text, GJ&+), which is characterized by a very large conductance(approximately 200 kinetic analysiswithout selectivepharmacological blockade. Two pS in 0.15 M KC1 solutions)despite high selectivity for K+ ions possibleexperimental approaches,whole-cell aggregatecurrent and by an open-state probability that is a function of both in- analysisor averagedunitary current responsesto repetitive volt- ternal Ca2+concentration and membrane potential, does not age jumps, will be contaminated by overlap of channel popu- differ substantiallyfrom reports of thesechannels in other prep- lations coactivated during depolarization. arations. The major purpose of the present study was to confirm the As noted, the present study doesnot attempt to addressthe presenceof multiple K+ channel populations underlying the time dependenceof channelgating. While no definitive evidence outward current activated by depolarization of chick ciliary gan- of inactivation processeswas seenfor any of the 3 channels,as glion neurons.Each of thesechannels represents a potential site judged by a decline in open-stateprobability during maintained for modulation of cellular excitability by neurotransmitters or voltage steps,the methods employed in this study could give hormones. only an approximateexpression of time and voltage dependence due to the random behavior of single channels. The present study highlights the difficulties of interpreting non-steady-state

72 -10 mV 60 / I 1 I I 1’ 1 60 R

P open = 0.89 0 freq = 3750/S To = 1.11 ms k$ 36

0” ’ I I 20 50 100 200 500 1000 [K’]~ ImM) 1 2 3 4 5 6 Figure 7C. Zero-currentpotential as a functionof [K+],. The data in- OPEN TIME (ms) cludethe resultsfrom 4 different“inside-out” patches, which have the indicatedK+ concentrations in the pipetteand face an internalsolution with K+ concentrationof 5.4 mM. The valuesof zero-currentpotential, Figure 8B. Frequencyvs open-timehistogram from 300single G&(,+, determinedby linear-regressionanalysis from the current-voltagere- channelevents at an isolatedpatch potential of - 10mV. Thehistogram lationships,are plotted as a function of the logarithmof [K+],. The of channelopen times was fitted by a singleexponential (curved line) experimentalpoints are fitted by a straightline with a slopeof 60.3 mV with a time constantof 1.11msec. The coincidentallydetermined P,,, per IO-foldchange in [K’],. equaled0.89, and 0, equaled3750/set of closedtime. The Journal of Neuroscience Single-Channel K+ Currents in Chick Ciliary Neurons 2115

180 Fukushima, Y. (1982) Blocking kinetics of the anomalous potassium -60 mV rectifier of tunicate egg studied by single channel recording. J. Physiol. R (Lond.). 331: 311-331. ‘50 t\ Galvan, M., and C. Sedlmeir (1984) Outward currents in voltage- clamped rat sympathetic neurones. J. Physiol. (Lond.). 356: 115-l 33. P ope” = 0.33 Gruol, D. L. (1984) Single-channel analysis of voltage-sensitive K+ 0 channels in cultured Purkinje neurons. Biophys. J. 45: 33-55. freq =790/s Grygorczyk, R., W. Schwarz, and H. Passow (1984) (X+-activated To = 0.76 ms K+ channels in human red cells. Biophys. J. 45: 693-698. Hamill, 0. P. (198 1) Potassium channel currents in human red blood cells. J. Physiol. (Lond.). 319: 97P-98P. Hamill, 0. P., A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth (198 1) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluegers Arch. 391: 85-100. Hartzell, H. C. (1981) Mechanisms of slow postsynaptic potentials. Nature 291: 539-544. Hartzell, H. C., S. W. Kuffler, R. Stickgold, and D. Yoshikami (1977) 1 1 25 2.5 3.75 5.0 Synaptic excitation and inhibition resulting from direct action of ace- OPEN TIME (ms) tylcholine on two types of chemoreceptors on individual amphibian parasympathetic neurones. J. Physiol. (Lond.). 271: 817-846. Figure 8C. Frequency vs open-time histogram from 300 single G&(*+, Kameyama, M., T. Kiyosue, and M. Soejima (1983) Single-channel channel events at an isolated patch potential of -60 mV. The histogram analysis of the inward rectifier K current in the rabbit ventricular of channel open times was fitted with a single exponential with a time cells. Jpn. J. Physiol. 33: 1039-1056. constant of0.76 msec. The coincidentally determined P,,, equaled 0.33, Katayama, Y., and S. Nishi (1984) Sites and mechanisms of actions and 0 freqequaled 790/set of closed time. of enkephalin in the feline parasympathetic ganglion. J. Physiol. (Lond.). 351: 11 l-121. Kostyuk, P. G., N. S. Veselovsky, S. A. Fedulova, and A. Y. Tsyndrenko References (198 1) Ionic currents in the somatic membrane of rat dorsal root Adams, P. R., D. A. Brown, and A. Constanti (1982) M-currents and ganglion neurons. III. Potassium currents. Neuroscience 6: 2439- other potassium currents in bullfrog sympathetic neurones. J. Physiol. 2444. (Lond.). 330: 537-572. Krylov, B. V., and V. S. Makovsky (1978) Spike frequency adaptation Bader, C. R., D. Bertrand, and A. C. Kato (1982) Chick ciliary ganglion in amphibians’ sensory tibres is probably due to slow K-channels. in dissociated cell culture. II. Electrophysiological properties. Dev. Nature 275: 549-55 1. Biol. 94: 131-141. Latorre, R., and C. Miller (1983) Conduction and selectivity in po- Barrett, E. F., and J. N. Barrett (1976) Separation of two voltage- tassium channels. J. Membr. Biol. 71: 1 l-30. sensitive potassium currents, and demonstration of a tetrodotoxin- Latorre, R., C. Vergara, and C. Hidalgo (1982) Reconstitution in pla- resistant calcium current in frog motoneurones. J. Physiol. (Lond.). nar lipid bilayers of a Ca*+-dependent K+ channel from transverse 255: 737-774. tubule membrane isolated from rabbit skeletal muscle. Proc. Natl. Barrett, E. F., J. N. Barrett, and W. E. Crill (1980) Voltage sensitive Acad. Sci. USA 79: 805-809. outward currents in cat motoneurones. J. Physiol. (Lond.). 304: 25 l- Llano, I., and F. Bezanilla (1983) Bursting activity of potassium chan- 276. nels in the cut-open axon. Biophys. J. 41 (2, pt.2): 38a. Barrett, J. N., K. L. Magleby, and B. S. Pallotta (1982) Properties of Lux, H. D., E. Neher, and A. Marty (198 1) Single channel activity single calcium-activated potassium channels in cultured rat muscle. associated with calcium-dependent outward current in Helixpomatia. J. Physiol. (Lond.). 331: 21 l-230. Pfluegers Arch. 389: 293-295. Belluzzi, O., 0. Sacchi, and E. Wanke (1985) A fast transient outward Magleby, K. L., and B. S. Pallotta (1983a) Calcium dependence of current in the rat sympathetic neurone studied under voltage-clamp open and shut interval distributions from calcium-activated potas- conditions. J. Physiol. (Lond.). 358: 91-108. sium channels in cultured rat muscle. J. Physiol. (Lond.). 344: 585- Benham, C. D., and T. B. Bolton (1983) Patch-clamp studies of slow 604. potential-sensitive potassium channels in longitudinal smooth muscle Magleby, K. L., and B. S. Pallotta (1983b) Burst kinetics of single cells of rabbit jejunum. J. Physiol. (Lond.). 340: 469-486. calcium-activated potassium channels in cultured rat muscle. J. Phys- Clapham, D. E., and L. J. DeFelice (1984) Voltage-activated K+ chan- iol. (Lond.). 344: 605-623. nels in embryonic chick heart. Biophys. J. 45: 40-42. Colauhoun, D.. and B. Sakmann (1981) Fluctuations in the micro- Marty, A. (1981) Ca-dependent K channels with large unitary con- second time range of currents through the acetylcholine receptor ion ductance in chromaffin cell membranes. Nature 291: 497-500. channels. Nature 294: 464-466. McAfee, D. A., and P. J. Yarowsky (1979) Calcium-dependent po- Colquhoun, D., and F. Sigworth (1983) Fitting and statistical analysis tentials in the mammalian sympathetic neurone. J. Physiol. (Lond.). of single channel records. In Single Channel Recording, E. Neher and 290: 507-523. B. Sakmann, eds., pp. 191-263, Plenum, New York. Meech, R. W., and N. B. Standen (1975) Potassium activation in Helix Connor, J. A., and C. F. Stevens (1971) Voltage clamp studies of a aspersa neurones under voltage clamp: A component mediated by transient outward membrane current in gastropod neural somata. J. calcium influx. J. Physiol. (Lond.) 249: 2 1 l-239. Physiol. (Lond.). 213: 21-30. Moczydlowski, E., and R. Latorre (1983) Gating kinetics of Ca2+- Conti, F., and E. Neher (1980) Single channel recordings of K+ currents activated K+ channels from rat muscle incorporated into planar lipid in squid axons. Nature 285: 140-143. bilayers. Evidence for two voltage-dependent Ca binding reactions. Coronado, R., R. Latorre, and H. G. Mautner (1984) Single potassium J. Gen. Physiol. 82: 5 1 l-542. channels with delayed rectifier behavior from lobster axon mem- Momose, Y., G. Szabo, and W. Giles (1983) An inwardly rectifying branes. Biophys. J. 45: 289-299. K+ current in bullfrog atria1 cells. Biophys. J. 41: 3 1 la. Dubois, J. M. (1981) Evidence for the existence of three types of Neher, E. (197 1) Two fast transient current components during voltage potassium channels in the frog Ranvier node membrane. J. Physiol. clamp on snail neurones. J. Gen. Physiol. 58: 36-53. (Lond.). 318: 297-316. Nishi, R., and D. K. Berg (1981) Two components from eye tissue E&hsen; J. T., A. Reiner, and H. J. Karten (1982) Co-occurrence of that differentially stimulate the growth and development of ciliary substance P-like and leu-enkephalin-like immunoreactivities in neu- ganglion neurons in cell culture. J. Neurosci. I: 505-5 13. rones and fibres of avian nervous system. Nature 295: 407-410. Ohmori, H., S. Yoshida, and S. Hagiwara (1981) Single K+ channel Fukushima, Y. (198 1) Single-channel potassium currents ofthe anom- currents__ . of anomalous- ~~. ~.rectification in cultured rat myotubes. Proc. alous rectifier. Nature 294: 368-371. Natl. Acad. Sci. USA. 78: 4960-4964. 2116 Gardner Vol. 6, No. 7, Jul. 1986

Sakmann, B., and G. Trube (1984a) Conductance properties of single and cyclic AMP close single K+ channels in Aplysiu sensory neurones. inwardly rectifying potassium channels in ventricular cells from guinea- Nature 299: 4 13-4 17. pig heart. J. Physiol. (Land.). 347: 641-657. Thompson, S. H. (1977) Three pharmacologically distinct potassium Sakmann, B., and G. Trube (1984b) Voltage-dependent inactivation channels in molluscan neurones. J. Physiol. (Lond.). 265: 465-488. of inward-rectifying single-channel currents in the guinea-pig heart Trube, G., B. Sakmann, and W. Trautwein (198 1) Inward rectifying cell membrane. J. Physiol. (Land.). 347: 659-683. potassium currents recorded from isolated heart cells by the patch Sakmann, B., A. Noma, and W. Trautwein (1983) Acetylcholine ac- clamp method. Pfluegers Arch. 391: R28. tivation of single muscarinic K+ channels in isolated pacemaker cells Vergara, C., and R. Latorre (1983) Kinetics of Caz+-activated K+ chan- of the mammalian heart. Nature 303: 250-252. nels from rabbit muscle incorporated into planar bilayers. Evidence Siegelbaum, S. A., J. S. Camardo, and E. R. Kandel (1982) Serotonin for a Ca*+ and Ba2+ blockade. J. Gen. Physiol. 82: 543-568.