LUMENAL SITES AND C-TERMINUS ACCESSIBILITY OF THE SKELETAL-MUSCLE CALCIUM-RELEASE CHANNEL (RYANODINE RECEPTOR)

The membrane topology of the skeletal muscle ryanodine receptor (RyR1) was investigated using site-directed antibodies directed against amin o acid sequences 2804-2930, 4581-4640, 4860-4886, and 4941-5037, Ab(28 04-2930) bound with identical affinity to either closed or permeabiliz ed sarcoplasmic reticulum vesicles, confirming the cytoplasmic locatio n of this segment. Ab(4581-4640) did not bind to closed vesicles but b ound well to permeabilized vesicles, supporting a lumenal location for this segment. Ab(4860-4886) did not bind to closed vesicles but exhib ited weak binding to the permeabilized vesicles, suggesting that a por tion of the epitope may be exposed on the lumenal surface. The C-termi nal antibody (Ab(4941-5037)) bound weakly to closed vesicles, and bind ing was not significantly enhanced by permeabilizing vesicles with low concentrations of non-denaturing detergent. However, the C-terminal a ntibodies bound efficiently to vesicles which were transiently incubat ed 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 Takeshi ma et al. (Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H., Ka ngawa, K., Minamino, N., Matsuo, H., Ueda, M., Hanaoka, M., Hirose, T. , and Numa, S. (1989) Nature 339, 439-445). The results also suggest t hat the native conformation of the C terminus is inaccessible to antib odies.