Activation and inactivation kinetics of a Ca²⁺-activated Cl^- current: photolytic Ca²⁺ concentration and voltage jump experiments

Abstract

The activation kinetics of the endogenous Ca²⁺-activated Cl⁻ current (I _Cl,Ca) from Xenopus oocytes was investigated in excised “giant” membrane patches with voltage and Ca²⁺ concentration jumps performed by the photolytic cleavage of the chelator DM-nitrophen. Currents generated by photolytic Ca²⁺ concentration jumps begin with a lag phase followed by an exponential rising phase. Both phases show little voltage dependence but are Ca²⁺-dependent. The lag phase decreases from about 10 ms after a small Ca²⁺ concentration jump (0.1 μM) to less than 1 ms after a saturating concentration jump (55 μM). The rate constant of the rising phase is half-maximal at about 5 μM. At saturating Ca²⁺ concentrations, the rate constant is 400 to 500 s⁻¹. The Ca²⁺ dependence of the stationary current can be described by the Hill equation with n=2.3 and K _0.5=0.5 μM. The amplitude of the stationary current decreases after the excision of the membrane patch with t _1/2≈5 min (run-down). The activation kinetics of the current elicited by a Ca²⁺ concentration jump is not affected by the run-down phenomenon. At low Ca²⁺ concentration (0.3 μM), voltage jumps induce a slowly activating current with voltage-independent time-course. Activation is preceded by an initial transient of about 1-ms duration. At saturating Ca²⁺ levels (1 mM), the initial transient decays to a stationary current. The transient can be explained by a voltage-dependent inactivation process. The experimental data reported here can be described by a linear five-state reaction model with two sequential voltage-dependent Ca²⁺-binding steps, followed by a voltage-independent rate-limiting transition to the open and a voltage-dependent transition to a closed, inactivated state.