The voltage-sensitive sodium channel purified from mammalian brain is a heterotrimeric complex of three glycoprotein subunits: alpha of 260 kDa, beta 1 of 36 kDa, and beta 2 of 33 kDa. The alpha and beta 2 subunits are linked by disulfide bonds. Alpha subunits are synthesized in excess in developing brain neurons and 70 percent accumulate in a long-lived intracellular pool of incompletely assembled alpha subunits. Formation of the disulfide linkage between alpha and beta 2 subunits is a late, and possibly rate-limiting, event in the biogenesis of mature sodium channels. Antibodies against the beta 1 and beta 2 subunits will be used to measure their biosynthesis in pulse-chase experiments and study their processing and assembly in developing brain neurons in vitro. The efficiency of assembly into the sodium channel complex will be determined and the possibility that biosynthesis of the beta 1 or beta 2 subunits of their assembly into the sodium channel complex are rate-limiting steps in the overall pathway of sodium channel biogenesis will be critically evaluated. These studies will be extended to examine sodium channel biogenesis in retinal ganglion cells. Nearly all of the immunoreactive sodium channel alpha subunits in retina are in the axon hillock, initial segments and axons of retinal ganglion cells. We will analyze the biogenesis of sodium channels in the retinal ganglion cells and their axons using pulse-chase labeling methods. Processing of each of the channel subunits to its mature form, assembly into a functional sodium channel complex, axonal transport, insertion into the plasma membrane, and degradation will be measured and the complete life-cycle of a sodium channel will be defined. Regulatory points which control the number of sodium channels incorporated into the plasma membrane will be identified and characterized. Cytosolic and cytoskeletal proteins that bind sodium channels with high affinity will be identified and isolated, and their interaction and co-localization with sodium channels will be determined. The regulation of sodium channel expression and localization in developing brain neurons and retinal ganglion cells will be examined, and the localization of sodium channels and their high affinity binding proteins will be correlated. The normal expression and localization of sodium channels will be perturbed by altering cellular activity and by microinjection of antibodies or cloned sense and anti-sense mRNA related to sodium channel subunits and binding proteins. The results will define the functional roles of these proteins in the development and maintenance of normal electrical excitability in central neurons.
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