The long-term objective of this proposal is to elucidate new knowledge about the slow inward current that is thought to be associated with the plateau of the ventricular action potential. This current is believed to reflect calcium entry into the myocardial cell and may serve as the trigger for myocardial contraction. The specific aims are: (1) to discover the in vitro manifestation (in sarcolemma enriched preparations from canine ventricle) of the process responsible for the slow inward current; (2) to develop improved sarcolemma enriched preparations to study the process; and (3) to characterize the process in isolated vesicles in terms of time- and voltage-dependent activation and inactivation parameters, possible links to other sarcolemma systems, molecular constituents and the effect of cations and selected drugs. Recent studies in this laboratory with a highly enriched sarcolemma preparation from canine ventricle showed that a transition in potential (relative depolarization) of electrically polarized vesicles (inside negative) markedly enhanced calcium uptake by the vesicles. This reaction was inhibited by verapamil, manganous ion and lanthanum. It was concluded that these findings are consistent with the presence of a calcium channel in the sarcolemma enriched preparation and, as such, may be the in vitro manifestation of the process responsible for the slow inward current. Techniques to be used include monitoring membrane potentials with voltage-sensitive fluorescent dyes and shifts of calcium, sodium, potassium and chloride between the extravesicular and intravesicular spaces with radioisotopes. Stopped-flow instrumentation will be employed to measure the activation and inactivation kinetics of ion flow through the putative calcium channel and/or in association with any other processes which may be related to the slow inward current. The specificity of the process with regard to sarcolemma will be tested by a search for similar processes in other membrane preparations from cardiac tissue. Effects of catecholamines and antagonists, muscarinic agents, verapamil, cardiac glycosides and local anesthetics will be determined.