Voltage-gated Ca2plus channels of the N-type are modulated via a number of discrete signaling pathways. The most commonly utilized pathway involves a membrane-delimited mechanism whereby receptor activation of a pertussis toxin-sensitive heterotrimeric G protein results in a distinct form of voltage-dependent inhibition. Despite intense investigation, the molecular mechanism underlying this signal transduction pathway is at present unknown. Thus, the long term goal of our research is to further our understanding of the molecular mechanisms underlying modulation of N-type Ca2plus channels. The main hypothesis to be tested is that neurotransmitter modulation of N-type Ca2plus channels is mediated by specific G-protein beta gamma-subunits which bind directly to a site on the Ca2plus channel photo1B-subunit to produce voltage- dependent channel inhibition. A combination of electrophysiological, molecular biological, and biochemical methods will be used to investigate modulation of both native and heterologously expressed N- type Ca2plus channels. Accordingly, the SPECIFIC AIMS are: 1) to identify the G protein subunit, i.e., Gphoto or G beta gamma, which mediates voltage-dependent N-type Ca2plus channel modulation; 2) to determine whether G protein subunit composition confers specificity in regard to voltage-dependent N-type Ca2plus channel modulation and receptor coupling; 3) to determine the site on the Ca2plus channel involved in G beta gamma subunit binding. These experiments will generate new information regarding the molecular mechanism of voltage-dependent N-type Ca2plus channel inhibition. The elucidation of this mechanism has broad implications as numerous G protein-coupled receptors, any of which are implicated in disease or are targets of therapeutics agents, are thought to modulate synaptic transmission in the peripheral and central nervous system, at least in part, via this mechanism.
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