Analysis of protease-sensitive regions in the skeletal muscle sodium channel in vitro and implications for channel tertiary structure

S. J. Zwerling, S. A. Cohen, R. L. Barchi

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The tertiary structure of the rat skeletal muscle sodium channel was probed in vitro by determining regions of sensitivity to V-8 protease, trypsin, and chymotrypsin. Resultant channel fragments were identified with antibodies to defined sequences distributed along the primary structure. The temporal pattern of proteolysis was followed with channel protein in either detergent-phospholipid micelles or membrane fragments as well as with channel exposed to sodium dodecyl sulfate. Proteolysis in micelles and membranes occurred in discrete, reproducible steps that were similar in both systems. Although the size of intermediates varied slightly, their sequence of appearance was similar for all enzymes, suggesting that the observed pattern was determined by the relative accessibility of selected sites in the tertiary structure. No major change in channel organization appeared to occur after solubilization of membranes in nonionic detergents. Highly accessible sites in the native structure included the carboxyl terminus and the region linking the second and third internal repeat domains, while the amino terminus and the repeat domains themselves were relatively resistant to proteolysis unless the protein was denatured. Kinetically, interdomain II-III was the most readily cleaved; interdomains I-II and especially III-IV were less easily accessible. While domains I and IV appeared to remain intact throughout our experiments, limit fragments for epitopes associated with domains II and III suggest that cleavage eventually occurs at sites between the putative S5 and S6 helices in these domains.

Original languageEnglish (US)
Pages (from-to)4574-4580
Number of pages7
JournalJournal of Biological Chemistry
Issue number7
StatePublished - 1991
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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