This project explores the molecular structure and diversity of L-type Ca2+ channels. In specific. we will determine its subunit structure, its genetic complexity, and explore its tissue specific patterns of expression. To date the only Ca2+ channel biochemically purified is that of skeletal muscle, which is responsible for a dihydropyridine (DHP)-sensitive, slow. long-lasting, voltage-gated Ca2+ currents. This channel is referred to as either the DHP receptor or the L-type channel, the latter to differentiate it from T-type channels responsible for rapidly inactivating transient currents, and from N-type channels that have intermediary properties and a distinct pharmacological profile. The purified skeletal muscle DHP receptor appears to be a pentamer of composition alpha 1, alpha 2, beta, gamma and delta, of which alpha 2 and delta are made as a single pro[alpha 2 delta] molecule, and alpha 1 is both the channel proper and the DHP binding unit. The approaches used are almost exclusively those offered by molecular biology and cell biology techniques, in which new molecules are characterized by molecular cloning and expression in appropriate cells. Electrophysiological studies are essential for proper interpretations of structural studies. The work proposed is a continuation of ongoing experimentations. During the last three years we were able to accomplish the following: 1) to demonstrate that the alpha 1 subunit of the DHP receptor is indeed a Ca2+ channel and the DHP receptor, in the absence of any of the other subunits with which it co-purifies; and 2) to apply the polymerase chain reaction (PCR) to the study of molecular diversity of L-type Ca2+ channels in tissues other than skeletal muscle, such as heart, brain, ovaries, and selected cell lines. The result indicated that molecular diversity arises from both the existence of at least five non-allelic genes and alternative splicing of single gene products. Taken together the results give us confidence to continue and expand these avenues of research.
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