Ca channels transduce the cell surface electrical signals in neurons and other excitable cells into intracelular regulatory processes which control contraction, secretion, neurotransmission and gene expression. Studies of neuronal Ca channels in our laboratory and others show that the N-type and L-type channels have distinct subcellular localizations and serve distinct functions in neuronal signal transduction. These channel types are generally similar in their voltage-dependent activation, deactivation, and ion selectivity, but they have physiologically significant differences in their rates and mechanisms of inactivation, their modulation by protein phosphorylation and G proteins, and their interactions with effector proteins including synaptic membrane proteins and cellular signaling proteins. Inactivation, modulation, and interaction with cellular effectors and signaling proteins are likely to be interactive processes, and all three are likely to be determined substantially by protein-protein interactions at the intracellular surface of the Ca channels. While the membrane-associated regions of the alpha1 subunits of neuronal Ca channels have highly homologous primary structures, the intracellular loops connecting the transmembrane domains are highly divergent. Thus, the distinct localization and function of these Ca channels likely depends primarily on the unique structures of their intracellular surface. In the experiments proposed in this application, we will determine the molecular basis for the unique function and modulation of L-type and N-type neuronal Ca channels focussing on the intracellular surface of the Ca channel protein as a primary site at which these events are initiated.
Our Specific Aims are to define the sites and mechanisms of modulation of the class B N-type and class C L-type Ca channels by protein phosphorylation; to define the sites and mechanisms of modulation of class B N-type Ca channels by direct interaction with G proteins; to determine the mechanism and physiological significance of receptor-dependent proteolytic processing of the carboxyl terminal domain of the alpha1 subunit of the class C L-type Ca channel; and to identify the sites of interaction of N- type Ca channels with synaptic membrane and synaptic vesicle proteins, examine the physiological role of these interactions in the processes of docking and exocytosis of neurotransmitters from synaptic vesicles, explore their regulation by protein phosphorylation and G proteins; and search for other intracellular signaling proteins which interact with Ca channels. The results of these experiments will provide essential new information required to understand function of Ca channels in cellular signal transduction in neurons and other cell types.
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