Lumenal sites and C terminus accessibility of the skeletal muscle calcium release channel (ryanodine receptor).

Published

Journal Article

The membrane topology of the skeletal muscle ryanodine receptor (RyR1) was investigated using site-directed antibodies directed against amino acid sequences 2804-2930, 4581-4640, 4860-4886, and 4941-5037. Ab(2804-2930) bound with identical affinity to either closed or permeabilized sarcoplasmic reticulum vesicles, confirming the cytoplasmic location of this segment. Ab(4581-4640) did not bind to closed vesicles but bound well to permeabilized vesicles, supporting a lumenal location for this segment. Ab(4860-4886) did not bind to closed vesicles but exhibited weak binding to the permeabilized vesicles, suggesting that a portion of the epitope may be exposed on the lumenal surface. The C-terminal antibody (Ab(4941-5037)) bound weakly to closed vesicles, and binding was not significantly enhanced by permeabilizing vesicles with low concentrations of non-denaturing detergent. However, the C-terminal antibodies bound efficiently to vesicles which were transiently incubated at alkaline pH or subjected to trypsinolysis, conditions where few of the vesicles were permeabilized. These results support a model for the membrane topology of the ryanodine receptor as proposed by Takeshima et al. (Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H., Kangawa, K., Minamino, N., Matsuo, H., Ueda, M., Hanaoka, M., Hirose, T., and Numa, S. (1989) Nature 339, 439-445). The results also suggest that the native conformation of the C terminus is inaccessible to antibodies.

Full Text

Duke Authors

Cited Authors

  • Grunwald, R; Meissner, G

Published Date

  • May 1995

Published In

Volume / Issue

  • 270 / 19

Start / End Page

  • 11338 - 11347

PubMed ID

  • 7744771

Pubmed Central ID

  • 7744771

Electronic International Standard Serial Number (EISSN)

  • 1083-351X

International Standard Serial Number (ISSN)

  • 1083-351X

Digital Object Identifier (DOI)

  • 10.1074/jbc.270.19.11338

Language

  • eng