The long-term goals of this project are: 1) to understand the molecular basis for cell type-specific expression of voltage-sensitive calcium channel (VSCC) genes, and 2) to study the roles of VSCCs and synaptic vesicle-associated proteins in the process of secretion at peptidergic nerve terminals. Expression of diverse VSCC subtypes appears to be highly regulated both spatially and temporally during development of the central nervous system. To examine the molecular regulatory mechanisms underlying cell-specific expression of VSCC genes, we have chosen dihydropyridine- insensitive N-type and dihydropyridine-sensitive L-type alpha 1 subunit genes, since expression of N-type channels is restricted to neurons particularly in nerve endings while L-type channels are present in many excitable cell types and also predominantly in neuronal perikarya. Deletion analysis using reporter-alpha 1 subunit fusion gene constructs carried out during last year has identified several positive and negative regulatory elements located at both proximal and distal to the 5' flanking promoter regions of the N-type alpha 1B and L-type. We plan to further characterize the interactions between these regulatory elements and transcription factors that either enhance or repress expression of VSCC alpha 1 subunit genes in cell type- or tissue-specific manner. Biochemical and molecular analyses of interaction of VSCC with the proteins involved in the synaptic vesicle cycle aim to understand the roles of N-type and L-type VSCC in synaptic secretion. We and others have shown that the N-type VSCC, which is involved in fast secretion, interacts with the vesicle docking proteins syntaxin, SNAP-25, Munc-18 and the calcium binding protein synaptotagmin. To identify and characterize the putative calcium sensor in """"""""slow"""""""" neurosecretion, we have isolated a novel member of synaptotagmin gene family from rat hypothalamus. This synaptotagmin isoform (Syt B/K) is distinct from other synaptotagmins in that its mRNA is highly abundant in the rostral portion of adult rat brain and surprisingly in the kidney. The possible role of Syt B/K in stimulus-secretion coupling and vesicle trafficking will be examined. In addition, we plan to examine expression of VSCC subtypes and synaptic vesicle-associated proteins present in the peptidergic nerve terminals and characterize protein-protein interactions among them. These comparative studies will provide insights into how molecular specificity for stimulus-secretion coupling is determined.
