The long~term goals of this project are to: (1) understand the molecular basis for cell type-specific expression of voltage-sensitive calcium channel (VSCC) genes; and (2) study the roles of VSCCs and synaptic vesicle-associated proteins in the process of secretion at peptidergic nerve terminals. Electrophysiologic and pharmacologic studies have classified voltage-sensitive Ca2+ channels (VSCC) into five major types: an emerging family of a low voltage-activated channels, and 4 classes of high voltage-activated channels (L-, N-, P-type, and the recently identified dihydropyridine~insensitive, omega-conotoxin-insensitive Q- type channels). Expression of these and other Ca2+ channel 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 human N-type (alpha1B) and L-type (alpha1D) alpha1 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. Although they are comprised of similar polypeptide subunits, these two high voltage-activated calcium channels are encoded by distinct genes located on separate chromosomes. During the past year we have carried out molecular analysis of the 5' flanking promoter regions of human L- and N-type VSCC genes to identify and characterize important regulatory elements that either enhance or repress expression of these genes in cell type- or tissue-specific manner. In addition, using subunit-specific antisera that we have prepared past year, we have purified rat brain N- type Ca2+ channels and identified polypeptide subunit components. The 160 KDa alpha2delta and 55 kDa beta3 subunit proteins were copurified with the 125I-omega-CgTx~labeled alpha1 subunit of N~type Ca2+ channels. The purified rat brain N~type Ca2+ channel fractions contained the vesicle docking proteins syntaxin and Munc-18 proteins, but not the synaptic vesicle proteins synapsin and synaptophysin. Thus, our results suggest that certain presynaptic membrane proteins can physically associate with the N-type Ca2+ channels, and this interaction may be an important regulatory mechanism for neurotransmitter release at the presynaptic terminals. In the coming year we plan to identify VSCC subtypes present in the peptidergic nerve terminals and characterize their interactions with synaptic vesicle~associated proteins.
