In order to understand the physiological basis of impulse propagation in nerve and muscle, it is necessary to define the molecular and cellular characteristics of the sodium channel. The overall objectives of this research program are to elucidate the molecular organization of the Na+ channel, its mechanism of action and regulation, and its distribution and dynamics on the nerve cell surface. I. Molecular Structure of the Na+ Channel. We propose to chemically modify and electrophysiologically assess important functional groups of the purified and reconstituted Na+ channel protein to gain insight into those groups critical for ion selection and gating. Conformational states accessible to the channel for ion selection and gating will be done by placing spectroscopic probes at functional sites and measuring the relaxation properties of the channel during action potential propagation by simultaneous optical and electrical recordings. Delayed fluorescence and photobleaching will be applied to examine the rotational and lateral diffusion of the reconsituted and native Na+ channel with the goals of reconstructing in three-dimensions the molecular structure given our other data, and to define the interactions between channels and with other cellular components. II. Cellular Distribution and Mobility of Na+ Channels. Because the sodium channel system lends such unique excitability characteristics to nerve and muscle cell membranes, and since the regulation of the distribution and density is associated with pathological changes that accompany certain neuromuscular disorders, we wil continue to localize and quantitate sodium channels at specific sites on myelinated and demyelinated nerve fibers by immuno-fluorescence and -electron microscopy. To examine the mobility of Na+ channels during neuronal differentiation, fluorescence recovery after photobleaching will be used in an in vitro myelination system of purified spinal cord neurons and Schwann cells to elucidate mechanism in the plasma membrane organization of Na+ channels, and their interactions with cytoskeletal and/or extracellular elements important in maintaining the segregation of Na+ channels on the axon surface.
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