The Journal of Neurosctence July 1986, 6(7): 2106-2116 Single-Channel Recordings of Three K+-Selective Currents in Cultured Chick Ciliary Ganglion Neurons 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 pharmacological manipulations. 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 components can 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 conductance of 64 pS gate outward K+ currents into unitary channel currents. The under physiological recording conditions and gating that was individual K+ channelpopulation can then be characterizedwith affected by membranepotential but was not dependent on 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-time constants of 0.09 (61%) This paper reports experiments using the patch-clamp tech- and 0.35 (39%) msec, at +40 mV membrane potential. The nique to study K+ channels in 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 channel opening. 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 msec at - 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 transmitters that have beenlocalized populationsin the membraneof cultured chick CG neurons. by immunohistochemical study to preganglionic nerve 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 me- dium (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 1 Om’ 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-at- tached 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 re- cording 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.