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Höfer, GF; Hohenthanner, K; Baumgartner, W; Groschner, K; Klugbauer, N; Hofmann, F; Romanin, C.
Intracellular Ca2+ inactivates L-type Ca2+ channels with a Hill coefficient of approximately 1 and an inhibition constant of approximately 4 microM by reducing channel's open probability.
Biophys J. 1997; 73(4):1857-1865 Doi: 10.1016/S0006-3495(97)78216-X [OPEN ACCESS]
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Co-authors Med Uni Graz: Groschner Klaus
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Abstract:: The patch-clamp technique was used to characterize the mechanism of Ca2+-induced inactivation of cardiac L-type Ca2+ channel alpha(1C-a) + beta3 subunits stably expressed in CHO cells. Single Ca2+ channel activity was monitored with 96 mM Ba2+ as charge carrier in the presence of 2.5 microM (-)BAYK 8644 and calpastatin plus ATP. This enabled stabilization of channel activity in the inside-out patch and allowed for application of steady-state Ca2+ concentrations to the intracellular face of excised membrane patches in an attempt to provoke Ca2+-induced inactivation. Inactivation was found to occur specifically with Ca2+ since it was not observed upon application of Ba2+. Ca2+-dependent inhibition of mean Ca2+ channel activity was characterized by a Hill coefficient close to 1. Ca2+ binding to open and closed states of the channel obtained during depolarization apparently occurred with similar affinity yielding half-maximal inhibition of Ca2+ channel activity at approximately 4 microM. This inhibition manifested predominantly in a reduction of the channel's open probability whereas availability remained almost unchanged. The reduction in open probability was achieved by an increase in first latencies and a decrease in channel opening frequency as well as channel open times. At high (12-28 microM) Ca2+ concentrations, 72% of inhibition occurred due to a stabilization of the closed state and the remaining 28% by a destabilization of the open state. Our results suggest that binding of one calcium ion to a regulatory domain induces a complex alteration in the kinetic properties of the Ca2+ channel and support the idea of a single EF hand motif as the relevant Ca2+ binding site on the alpha1 subunit.
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