This proposal uses a combination of genetic and molecular biological approaches to analyze the structure and regulation of the voltage-sensitive calcium channels which play a key role in cardiac function. The goals of these studies are to determine how many genetically distinct classes of calcium channels exist and to determine whether any of these are expressed specifically in mammalian cardiac tissue. If there are unique aspects of structural and regulatory sequences for cardiac calcium channels, it may be possible to use this knowledge to design more specific drugs for treating cardiovascular diseases. The general approach for identifying genes affecting calcium channels will be isolate behavioral mutants and/or verapamil resistant mutants in Drosophila melanogaster and identify those which affect verapamil binding parameters. Such mutants will be mapped by recombination and deletion mapping and the genes cloned by chromosome walking or by microdissection. Cloned Drosophila genes will be used to isolate evolutionarily conserved sequences from bovine and rat cardiac cDNA libraries. As an alternative approach, a method will be developed to use ligand binding to directly screen for calcium channel structural gene components in Drosophila and rat cardiac cDNA expression libraries. Identification of functionally important regions of the calcium channel will be facilitated by identifying highly conserved sequences between Drosophila and mammalian channels. In addition, analysis of Drosophila mutants will pinpoints single amino acid residues which play a crucial role in calcium channel function. Chimeric genes consisting of a combination of normal and mutant Drosophila calcium channel genes and between Drosophila and mammalian cardiac calcium channel sequences will be used for stable germ line transformation studies to dissect structure/function relationships. The cloned cardiac cDNA sequences will be used as probes for genomic Southern blotting to determining whether calcium channels represent a family of related genes. The cardiac cDNA clones will also be used to determine the tissue specific expression of each calcium channel gene. The clones developed in this study will be useful as probes to analyze the effect of various hereditary cardiac disorders on expression and structures of calcium channels.
