Kinetic analysis of acetylcholine-induced chloride current in isolatedAplysia neurones

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Abstract
(1) Kinetics of activation and desensitization phases of the ACh-induced chloride current (I Cl) were studied in isolated single neurones ofAplysia kurodai, using the ‘concentration clamp’ technique which combines internal perfusion and rapid exchange of the external solution within a few milliseconds (2) The dose-response curve for the peakI Cl gave a dissociation constant of 6.7×10−6 M a Hill coefficient of 1.7. (3) The current-voltage relationship was linear in the voltage range examined (−70to +30mV). The reversal potential (E ACh) was −7.1±1.8mV (n=14). The value was close to the calculated equilibrium potential for chloride ions (E Cl) (4) The activation phase of theI Cl was single exponential and the desensitization proceeded double exponentially to a steady state level. The time constants of both phases decreased with increasing concentrations of ACh but showed no potential dependency. The desensitizing component of theI Cl was generated by activation of a single population of the receptor-channel complex. (5) The recovery from desensitization of theI Cl induced, by 6×10−6 M ACh proceeded double exponentially, with time constants of 6.5 and 43 s at a holding potential of −30 mV. (6) Noise analysis performed on the steady state ofI Cl induced by low concentrations of ACh (3×10−7 M to 3×10−6 M) showed that the steadyI Cl was due to activation of a single population of the receptor-channel complex with a single channel conductance of 23.3±4.3 pS (n=9). (7) The closing (α) and opening [β'(A)] rate constants in a simple sequential model were obtained in the low concentration range of ACh. α was not altered by changing the ACh concentration [ACh]. The slope of the β'(A)-[ACh]relationship gave the Hill coefficient of 1.8 for the steady stateI Cl. The α-[ACh] and the β'(A)-[ACh] plots crossed at 2.4×10−6 M ACh, yielding theK D value. (8) Our results suggest that the ACh-inducedI Cl inAplysia neurones consists at least of two components: a minor-desensitizing component to form the steady stateI Cl and a desensitizing component which contributes to the peakI Cl. The receptor-channel complexes apparently have two binding sites for ACh.