Ca(2+)-dependent components of inactivation of unitary cardiac L-type Ca(2+) channels

A Ca(2+) ion-dependent inactivation (CDI) of L-type Ca(2+) channels (LCC) is vital in limiting and shaping local Ca(2+) ion signalling in a variety of excitable cell types. However, under physiological conditions the unitary LCC properties that underlie macroscopic inactivation are unclear. Towards this end, we have probed the gating kinetics of individual cardiac LCCs recorded with a physiological Ca(2+) ion concentration (2 mM) permeating the channel, and in the absence of channel agonists. Upon depolarization the ensemble-averaged LCC current decayed with a fast and a slow exponential component. We analysed the unitary behaviour responsible for this biphasic decay by means of a novel kinetic dissection of LCC gating parameters. We found that inactivation was caused by a rapid decrease in the frequency of LCC reopening, and a slower decline in mean open time of the LCC. In contrast, with barium ions permeating the channel ensemble-averaged currents displayed only a single, slow exponential decay and little time dependence of the LCC open time. Our results demonstrate that the fast and slow phases of macroscopic inactivation reflect the distinct time courses for the decline in the frequency of LCC reopening and the open dwell time, both of which are modulated by Ca(2+) influx. Analysis of the evolution of CDI in individual LCC episodes was employed to examine the stochastic nature of the underlying molecular switch, and revealed that influx on the order of a thousand Ca(2+) ions may be sufficient to trigger CDI. This is the first study to characterize both the unitary kinetics and the stoichiometry of CDI of LCCs with a physiological Ca(2+) concentration. These novel findings may provide a basis for understanding the mechanisms regulating unitary LCC gating, which is a pivotal element in the local control of Ca(2+)-dependent signalling processes.