CHARACTERISTICS OF MULTIPLE CA2+-ACTIVATED K+ CHANNELS IN ACUTELY DISSOCIATED CHICK CILIARY-GANGLION NEURONS

1. Whole-cell and single-channel recordings were used to characterize Ca2+-activated K+ channels (I(K(Ca))) in acutely dissociated chick ciliary-ganglion neurones. 2. Application of depolarizing voltage steps resulted in outward currents that could be separated according to their dependence on external Ca2+ and/or holding potential. I(K(Ca)) was the only outward current that could be evoked from holding potentials of -50 mV or less. I(K(Ca)) was eliminated by bath application of Ca2+-free salines. A voltage-dependent outward current (I(K(V))) could be evoked from more negative holding potentials in Ca2+-free salines. I(K(V)) was only partially blocked by as much as 30 mM-tetraethylammonium (TEA). 3. Tail currents associated with I(K(Ca)) reversed close to the K+ equilibrium potential (E(K)). I(K(Ca)) tail currents appeared sigmoidal, but the falling phase of the tail currents could be fitted with exponential curves that decayed faster at more negative membrane potentials. 4. I(K(Ca)) was blocked completely and reversibly by 10 mM-TEA. I(K(Ca)) was substantially reduced (80-90%) by as little as 1 mM-TEA. 5. Total I(K(Ca)) was reduced but not eliminated by saturating concentrations of apamin (200 nM). This blockade was not reversible with up to 30 min of washing. Application of 100-mu-M-d-tubocurare (dTC) also produced a partial blockade of total I(K(Ca)). 6. Whole-cell current-clamp recordings showed that I(K(Ca)) contributed to the late phases of spike repolarization and was the dominant current flowing during the spike after-hyperpolarization (AHP). Application of 200 nM-apamin caused a reduction in the duration of the AHP. This reduction was best seen when multiple spikes were evoked by prolonged (20-50 ms) injections of depolarizing current. 7. Three distinct types of I(K(Ca)) channels could be observed in inside-out patches in the presence of free Ca2+ concentrations of 2 x 10(-7) M, but not in the presence of free Ca2+ at concentrations of < 10(-9) M. These had unitary chord conductances of 190 pS (i1), 110 pS (i2), and 45 pS (i3) with [K+]o = 150 mM and [K+]i = 75 mM. Each of these three channels had distinct kinetic properties. The 45 pS channel was most sensitive to activation by Ca2+ and could be detected at free Ca2+ concentrations as low as 10(-8) M. 8. All three I(K(Ca)) channels could be observed in inside-out patches held at membrane potentials where I(K(V)) was fully inactivated. Application of 10 mM-TEA caused a complete block of I(K(Ca)) channels in outside-out patches. 9. Application of 200 nM-apamin or 100-mu-M-dTC had no effect on any of the three I(K(Ca)) channels observed directly in outside-out patches. All three I(K(Ca)) channels could also be detected in inside-out patches obtained from cells pre-treated with 200 nM-apamin. 10. All three I(K(Ca)) channels could be observed in cell-attached patches at holding potentials were I(K(V)) is fully inactivated. 11. These results suggest that a highly homogeneous population of acutely dissociated chick ciliary-ganglion neurones express as many as four distinct types of I(K(Ca)) channels. These include the three apamin-resistant I(K(Ca)) channels observed directly in single-channel recordings and a fourth class of apamin-sensitive channels inferred from the results of whole-cell voltage-clamp and current-clamp recordings.