investigator's application): The integration of incoming synaptic signals, the threshold for action potential generation, and the frequency of firing of central neurons depend critically on the location, cell surface density, and functional properties of sodium channels. Previous results suggest that assembly, cell surface insertion, and differential targeting of sodium channel subtypes all play a role in determining cell surface density and localization of sodium channels. Cloning and functional analysis of b1 and b2 subunits of brain sodium channels have implicated these two proteins in modulation of sodium channel gating, assembly of functional channels, and expression on the cell surface. The b2 subunit may also be involved in targeting sodium channels to specific cellular compartments and in interaction with extracellular matrix proteins. The overall objective is to explore the hypothesis that b1 and b2 subunits are important determinants of sodium channel function, expression, and localization. The investigator will determine the kinetics and mechanism of assembly and cell surface expression of sodium channel a subunit with b1 and b2 subunits in mammalian cells in culture; identify the sites of interaction and define the mechanisms of channel modulation by b1 and b2 subunits; analyze the coexpression of these subunits on gating of Na channels; measure the binding of the b2 subunit to tenascin and related extracellular matrix proteins, search for novel interactions of b1 and b2 with extracellular proteins, and identify their site of interactions; and examine the ability of interactions with b1 and b2 subunits to modulate the expression and localization of sodium channels in heterologous cells in rat brain neurons in cell culture. These results will be essential for understanding the molecular basis of expression and localization of sodium channels.
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