An interdisciplinary approach will continue to be used to elucidate the structrue and molecular mechanisms of the voltage-dependent sodium channel from Electrophorus electricus electroplax. The primary focus of these studies will be the role of nonprotein, post-translationally acquired domains in the function, biosynthesis, and expression of sodium channels. In reconstitution studies, the functional effects of both carbohydrate removal and changes in lipid environment will be assessed using the lipid bilayer reconstitution system. Complementary experiments will use an amphibian oocyte expression system to determine the functional consequences of pharmacologically altering post-translational synthesis of nonprotein domains. In biosynthetic studies, both amphibian oocytes and eel electrocytes will be used to study the nature and sequence of post-translational events in the processing of sodium channels. Of interest will be the glycosylation, fatty acylation, and development of higher order structural domains and the subcellular compartments in which these events occur. For these studies new methods will be developed both to fractionate the subcellular synthetic machinery along with sodium channel precursors and to assess the functional maturation of the channel during the synthetic process. In related experiments, the role of post-translational modification in the targeting and expression of sodium channels will be studied using specific inhibitors of biosynthesis. These studies will make use of single electrocytes to study the mechanisms by which nuclei in this polarized, syncitial cell are differentially regulated to direct channel synthesis. Lastly, the topography of the channel peptide will be investigated through the use of combined immunological, ultrastructural, and biochemical methods. Topographical information will be obtained with a combination of limited proteolysis, Western blot analysis with antibodies and lectins, and a novel technique using electeronmicroscopy to relate antibody binding to ultrastructural domains. Overall, these studies may be expected to address the more general question of how cells regulate the temporal mechanisms of sodium channel function and biosynthesis.
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