Electrical properties of cardiovascular organs such as heart and blood vessels and the central and autonomic nervous systems which control them are known as are the whole-cell currents produced by cells in these organs. However, little is known about the voltage-gated Na+,K+ and Ca2+ channels that underlie these electrical properties. We are ignorant of how many isoforms exist, what their distributions are and what their contributions to function may be. As a result we do not have a rational basis for the design of therapeutic agents to be used in the treatment of cardiac arrhythmias or hypertension. A beginning has been made with the recent identification of one type of cardiac Na+ channel and several candidate cardiac K+ channels. From these beginnings we can now explore the correlations between structure and function for the known channels, the number of channel isoforms that exist, their distribution and their significance for function. This program project grant renewal brings together five projects and three cores which use the two techniques of molecular biology and electrophysiology to determine the properties of different voltage-gated Ca2+ and K+ channels in the cardiovascular system. Projects 1 and 2 will exploit mouse L cells, which are devoid of Ca2+ channel alpha1, alpha2, beta and gamma subunits, to determine which subunits are required for optimum function and how each subunit contributes to function. Other questions to be answered include which part of alpha1 is the voltage sensor and which the channel pore, which parts are regulated by phosphorylation, what isoforms exist for beta and gamma subunits and what structures are associated with T, N and L channels. Project 3 will generate antibodies to Ca2+ channel subunits and use immunochemical methods to determine the interactions of the subunits. This project will also map the epitopes and their relationship to the plasma membrane providing an independent measurement for the structure-function studies of Projects 1 and 2. Project 4 will clone cardiac K+ channels by homology screening with brain K+ channel probes. The tissue distribution of different K+ channel members between the conducting and contracting cells will be studied by Northern blots and by in situ hybridization. The currents in the native cells will be correlated with the currents produced by transient expression in Xenopus oocytes. Project 5 will characterize the K+ Ca2+ channel of coronary artery in bilayers and determine the regulatory effects of modulatory agents such as angiotensin and eicosanoids, and heterotrimeric G protein regulation. Studies will be initiated to clone the channel as a first step towards a detailed structure-function analysis. Three cores will support the program requirements for molecular biology, epitope mapping, stable and transient expression, electrophysiology and molecular modeling.
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