Voltage-dependent calcium channels play a key role in contraction of the heart and are targets for therapeutic agents used in treating several cardiovascular disorders. This application will use a molecular genetic approach to address two questions of fundamental significance for understanding the function of these channels and for developing new therapeutic agents for the treatment of cardiovascular disease. The first question is: """"""""What is the functional significance of the molecular diversity of calcium channel subunits?"""""""" These studies will build on preliminary results which show that in Drosophila, as in other species, channel diversity arises from multiple genes encoding calcium channel subunits and from alternative splicing of each of these subunit genes. PCR amplification of cDNA from different body parts and various stages in development will be used to identify regions of a subunit gene that display alternative splice products and automated DNA sequencing will be used to deduce the amino acid sequence changes produced by each splice variant. Baculovirus expression, Xenopus oocyte expression and transposon- mediated gene transformation studies will be used to determine the physiological significance of a subset of the most interesting splice variants. A genetic approach will be used to define the null phenotype of each cloned calcium channel subunit in Drosophila in order to determine whether individual subunit genes play a unique role or are functionally redundant. Studies will initially focus on the recently sequenced DroCa alpha1 subunit for which a mutant gene has been identified. The second question to be addressed is: """"""""What is the molecular nature of binding domains for the therapeutically important organic calcium channel blockers?"""""""" This question will be addressed by defining the pharmacological profile for the newly cloned Drosophila alpha1 subunit by expressing it with baculovirus and then comparing it wi+h similarly expressed mammalian alpha1 subunits. For ligands which show similar affinities across species, sequence comparisons will identify amino acids which are free to vary and thus play little role in ligand/channel interactions. For ligands with different affinities across species, sequence comparisons will help to identify amino acids which play a key role in binding sites. Models developed from these studies will be tested in chimeric constructs. Such detailed analysis of drug binding sites will aid in the development of new therapeutic agents. A PCR strategy will be used to identify genes encoding other alpha1 subunits as well as other subunits (alpha2-delta, beta, and gamma) known to comprise mammalian calcium channels. Subunit combinations expressed in Xenopus oocytes will be used to define possible interactions among subunits while the power of Drosophila genetics will be used to define which interactions actually occur within the organism. These studies will set the stage for future genetic studies aimed at identifying endogenous calcium channel ligands which will have the potential to be developed into new therapeutic agents for the treatment of cardiovascular diseases.
