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Abstract:
The activation kinetics of the endogenous Ca(2+)-activated Cl(-) current (I (Cl,Ca)) from Xenopus oocytes was investigated in excised "giant" membrane patches with voltage and Ca(2+) concentration jumps performed by the photolytic cleavage of the chelator DM-nitrophen. Currents generated by photolytic Ca(2+) concentration jumps begin with a lag phase followed by an exponential rising phase. Both phases show little voltage dependence but are Ca(2+)-dependent. The lag phase decreases from about 10 ms after a small Ca(2+) concentration jump (0.1 microM) to less than 1 ms after a saturating concentration jump (55 microM). The rate constant of the rising phase is half-maximal at about 5 microM. At saturating Ca(2+) concentrations, the rate constant is 400 to 500 s(-1). The Ca(2+) dependence of the stationary current can be described by the Hill equation with n=2.3 and K (0.5)=0.5 microM. The amplitude of the stationary current decreases after the excision of the membrane patch with t (1/2) approximately 5 min (run-down). The activation kinetics of the current elicited by a Ca(2+) concentration jump is not affected by the run-down phenomenon. At low Ca(2+) concentration (0.3 microM), 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 Ca(2+) 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 Ca(2+)-binding steps, followed by a voltage-independent rate-limiting transition to the open and a voltage-dependent transition to a closed, inactivated state.
