The primary focus of this project is to elucidate the structure and function of the receptor for three classes of calcium channel inhibitor drugs, viz., DIHYDROPYRIDINES, (e.g., nifedipine), PHENYLALKYLAMINES (e.g., verapamil), and BENZOTHIAZEPINES (e.g., diltiazem) in heart, vascular smooth muscle and brain. These drugs are used to treat coronary artery disease and hypertension and may be valuable in related conditions such as cerebral vasospasm. The drug binding sites are located in specific domains of a large protein, the alpha 1 subunit of the L type voltage-dependent calcium channel (VDCC). The central hypothesis is that tissue-specific alpha 1 isoforms contain the drug binding domains and form the calcium channel. Other subunits, however, may be required for function and regulation. The three specific aims are: (1) to isolate and sequence full-length alpha 1 cDNA clones from heart, vascular smooth muscle and brain; (2) to express VDCC activity with appropriate pharmacology using alpha 1 cDNA clones alone or in combination with cDNA clones encoding the other VDCC subunits; (3) to identify the drug binding domains and the sites for regulation of VDCC within the alpha 1 subunit. Skeletal muscle alpha 1 cDNAs have been isolated and sequenced (skeletal muscle T-tubules are the richest source of VDCC and calcium antagonist binding sites) and will be used to identify alpha 1 isoforms in other tissues. The alpha 1 subunit alone or in combination with other subunits will be expressed in Xenopus oocytes and/or mammalian cells. Function of the expressed protein(s) will be examined using radioligand binding and electrophysiological techniques. Based upon deduced primary amino acid sequence of the alpha 1 isoforms and the known tissue-specific pharmacological characteristics, chimeric constructs and selected mutants will be made, expressed and functionally evaluated for VDCC activity and pharmacology. These proposed studies are relevant to understanding the nature of the receptors for, and hence, bear on the mechanism of the calcium antagonists. Further, if VDCC are altered in certain cardiovascular disorders as has been suggested by published studies, the nature of the putative defects can be explored by genetic means. The data derived from the proposed experiments should be useful in understanding the function and regulation of the receptor and of channel activity.
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