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 (I-Ca,I-L) activates rapidly, then partially declines leading to a sustained inward current duri ng the plateau phase. The reason for the sustained part of I-Ca,I-L has bee n investigated here. 2. In the present study the mechanisms controlling the I-Ca,I-L during an a ction potential were investigated quantitatively in isolated guinea-pig ven tricular myocytes by whole-cell patch clamp. To measure the actual time cou rses of I-Ca,I-L and the corresponding L-type channel inactivation (f(AP)) 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 I-Ca,I-L rapidly inac tivated 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 potenti al was determined from measurements of L-type current carried by monovalent cations. This component of inactivation proceeded rather slowly and contri buted only a little to f(AP). I-Ca,I-L during an action potential is thus m ainly controlled by Ca2+-dependent inactivation. 5. In order to investigate the source of the Ca2+ controlling f(AP), intern al Ca2+ homeostasis was manipulated by the use of Ca2+ buffers (EGTA, BAPTA ), by blocking Na+-Ca2+ exchange, or by blocking Ca2+ release from the sarc oplasmic reticulum (SR). Internal BAPTA markedly reduced the L-type channel inactivation during the entire action potential, whereas EGTA affected f(A P) only during the middle and late plateau phases. Inhibition of Na+-Ca2+ e xchange markedly increased inactivation of L-type channels. Although blocki ng SR Ca2+ release decreased the fura-2-measured cytoplasmic Ca2+ concentra tion ([Ca2+](i)) transient by about 90%, it reduced L-type channel inactiva tion 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 inactivat ion process for the majority of the action potential. Nevertheless, SR Ca2 release contributes 40-50% to L-type channel inactivation during the initi al period of the action potential. However, the maximum extent of inactivat ion 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 inactivatio n has been directly determined during an action potential under various def ined [Ca2+](i) conditions. Thereby, the relative contribution to I-Ca,I-L i nactivation of voltage, Ca2+ entering through L-type channels, and Ca2+ bei ng released from the SR could be directly demonstrated.