This proposal describes a continuation of our present work on the molecular characterization of the voltage-sensitive sodium channel from mammalian muscle. As in the past grant period, we will use a multidisciplinary approach incorporating biochemical, biophysical and immunological techniques to further probe the structure and function of this unique molecule. The sodium channel will be purified from rat and rabbit skeletal muscle. The relationship between the large glycoprotein and the small subunit(s) will be probed with monoclonal antibodies, peptide mapping and immunoprecipitation. The large subunit will be studied in detail, with analysis of amino acid composition, N-terminal sequence, and sugar content and composition. Peptide mapping of this subunit will be used to examine questions of molecular topography, the location of functionally active sites, and the location of determinants recognized by monoclonal antibodies. Quench-flow studies of purified, reconstituted channels will be extended to further characterize the reconstitution system, the interrelationship of neurotoxin binding sites in the reconstituted protein, and the functional integrity of channel protein. Recent work with single channel measurements on the purified protein will be pursued to measure kinetic and voltage-dependence properties of the purified reconstituted channel that are not accessible with the quench-flow system. Ongoing work with monoclonals will be continued in order to expand our panel of channel-specific antibodies, and to identify antibodies that interfere with channel function. Integration of these various approaches is emphasized with the goal of exploring structure-function relationships in the purified channel at the molecular level.
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