Voltage-dependent sodium channels mediate the propagating action potential of nerve and muscle. Molecularly, sodium channels from a number of tissues are comprised of single large polypeptide that is heavily modified by carbohydrate and hydrophobic domains inferred to be lipid. This application proposes to continue investigations into the role of these nonprotein domains in the molecular mechanisms that underlie voltage-sensitive gating and ion conductance. There is strong preliminary evidence from reconstitution studies that negatively charged sialic acid residues attached to the channel significantly affect the local electrical field near an activation gating sensor. If so, then biosynthetic attachment of sialic acid residues may represent an adaptive mechanism that allows certain cells to determine channel gating characteristics appropriate to the membrane properties and functional requirements of that cell type. this hypothesis will be addressed using a complementary interdisciplinary approach in which channel carbohydrate compositions will be manipulated in the following ways: 1) through removal of sugars from channels expressed at the cell surface using neuraminidases; 2) inhibition of glycosylation during biosynthesis using selective metabolic inhibitors; 3) expression of channels in mutant cell lines lacking various elements of the glycosylation machinery, and 4) mutagenesis of cloned channel cDNAs to modify, move or eliminate glycosylation sites. In these studies channel cDNAs will be transfected into cell lines and the affect of the above manipulations on channel synthesis, expression, and function will be studied using biochemical, immunological, and biophysical recording techniques. Further insight into the roles of glycosylation will be obtained by comparing the results from similar studies performed on channels expressed in Xenopus oocytes and mammalian muscle fibers. Overall, the studies in this application will address the question of how channel function may be modified at the posttranslational level. Such information has applications to the understanding of sodium channel roles in degenerative and developmental disorders of the neuromuscular system and in the design of more affective drugs for use in anesthesia and cardiac arrhythmias.
Showing the most recent 10 out of 34 publications
