Activation and inactivation kinetics of a Ca2+-activated Cl- current: photolytic Ca2+ concentration and voltage jump experiments |
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Authors: | Andreas Haase Klaus Hartung |
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Institution: | (1) Max-Planck-Institut für Biophysik, Max-von-Laue-Str. 3, 60438 Frankfurt am Main, Germany |
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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. |
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Keywords: | DM-nitrophen Caged Ca2+ Xenopus oocyte |
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