The long~term goals of this project are to: 1) understand the molecular basis for the structure, function and tissue~specific expression of voltage~sensitive calcium channels (VSCCs), and 2) elucidate the functional roles and expression patterns of VSCCs during development. Multiple types of VSCCs (i.e., T, N, L, and P~type) are present in both excitable and nonexcitable cells. They are important for excitation~contraction coupling, secretion of neurotransmitters and hormones, and regulation of gene expression. During the past year, we have developed several molecular reagents useful for analysis of calcium channels. They include: 1) brain cDNAs corresponding to the alpha1~subunits of the dihydropyridine (DHP)~sensitive L~type as well as DHP~insensitive N~ and P~type calcium channels; 2) cDNA clones encoding the alpha2~ and beta~subunits of calcium channels; and 3) polyclonal antisera specific for each type of VSCC subunit proteins. In situ hybridization histochemistry and immunocytochemistry techniques were utilized to examine spatiotemporal expression patterns of multiple VSCC subunit mRNAs and proteins in adult and developing rat brains. Our results have indicated that heterogeneous types of VSCC are expressed at an early embryonic stage (E12), suggesting that they may play important roles in developing central nervous system (CNS). Ongoing analysis of genetic heterogeneity of various VSCCs expressed during CNS development are in progress. In addition, Northern blot hybridization analysis revealed that L~type VSCC mRNA was present in a number of human tissues examined including brain, heart, lung, kidney, pancreas, and skeletal muscle, whereas the N~type VSCC transcript was exclusively present in nervous tissues. As a first step toward understanding the genetic bases for the tissue~specific expression of VSCCs, we have isolated the human genomic clones for the L~ and N~type calcium channel alpha1~subunits. The 5' flanking promoter regions of the L~type and N~type VSCCs will be analyzed to identify and characterize important regulatory elements that either enhance or repress expression of these channel genes in cell type~ or tissue~specific manner.
