INTRACELLULAR CA2-TYPE CA2+ CHANNELS WITH A HILL COEFFICIENT OF SIMILAR-TO-1 AND AN INHIBITION CONSTANT OF SIMILAR-TO-4 MU-M BY REDUCING CHANNELS OPEN PROBABILITY( INACTIVATES L)

The patch-clamp technique was used to characterize the mechanism of Ca 2+-induced inactivation of cardiac L-type Ca2+ channel alpha(1C-a) + b eta(3) subunits stably expressed in CHO cells. Single Ca2+ channel act ivity was monitored with 96 mM Ba2+ as charge carrier in the presence of 2.5 mu M (-)BAYK 8644 and calpastatin plus ATP. This enabled stabil ization of channel activity in the inside-out patch and allowed for ap plication of steady-state Ca2+ concentrations to the intracellular fac e of excised membrane patches in an attempt to provoke Ca2+-induced in activation. Inactivation was found to occur specifically with Ca2+ sin ce it was not observed upon application of Ba2+. Ca2+-dependent inhibi tion of mean Ca2+ channel activity was characterized by a Hill coeffic ient close to 1. Ca2+ binding to open and closed states of the channel obtained during depolarization apparently occurred with similar affin ity yielding half-maximal inhibition of Ca2+ channel activity at simil ar to 4 mu M. 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 incre ase in first latencies and a decrease in channel opening frequency as well as channel open times. At high (12-28 mu M) 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 resu lts suggest that binding of one calcium ion to a regulatory domain ind uces a complex alteration in the kinetic properties of the Ca2+ channe l and support the idea of a single EF hand motif as the relevant Ca2binding site on the alpha(1) subunit.