Control of L-type calcium current during the action potential of guinea-pig ventricular myocytes.

1. During an action potential the L-type Ca2+ current (ICa,L) activates rapidly, then partially declines leading to a sustained inward current during the plateau phase. The reason for the sustained part of ICa,L has been investigated here. 2. In the present study the mechanisms controlling the ICa,L during an action potential were investigated quantitatively in isolated guinea-pig ventricular myocytes by whole-cell patch clamp. To measure the actual time courses of ICa,L and the corresponding L-type channel inactivation (fAP) during an action potential, action potential-clamp protocols combined with square pulses were applied. 3. Within the first 10 ms of the action potential the ICa,L rapidly inactivated by about 50 %; during the plateau phase inactivation proceeded to 95 %. Later, during repolarization, the L-type channels recovered up to 25 %. 4. The voltage-dependent component of inactivation during an action potential was determined from measurements of L-type current carried by monovalent cations. This component of inactivation proceeded rather slowly and contributed only a little to fAP. ICa,L during an action potential is thus mainly controlled by Ca2+-dependent inactivation. 5. In order to investigate the source of the Ca2+ controlling fAP, internal Ca2+ homeostasis was manipulated by the use of Ca2+ buffers (EGTA, BAPTA), by blocking Na+-Ca2+ exchange, or by blocking Ca2+ release from the sarcoplasmic reticulum (SR). Internal BAPTA markedly reduced the L-type channel inactivation during the entire action potential, whereas EGTA affected fAP only during the middle and late plateau phases. Inhibition of Na+-Ca2+ exchange markedly increased inactivation of L-type channels. Although blocking SR Ca2+ release decreased the fura-2-measured cytoplasmic Ca2+ concentration ([Ca2+]i) transient by about 90 %, it reduced L-type channel inactivation only during the initial 50 ms of the action potential. Thus, it is Ca2+ entering the cell through the L-type channels that controls the inactivation process for the majority of the action potential. Nevertheless, SR Ca2+-release contributes 40-50 % to L-type channel inactivation during the initial period of the action potential. However, the maximum extent of inactivation reached during the plateau is independent of Ca2+ released from the SR. 6. For the first time, the actual time course of L-type channel inactivation has been directly determined during an action potential under various defined [Ca2+]i conditions. Thereby, the relative contribution to ICa,L inactivation of voltage, Ca2+ entering through L-type channels, and Ca2+ being released from the SR could be directly demonstrated.