Functional interactions between cytoplasmic domains of the skeletal muscle Ca2+ release channel

The skeletal muscle Ca2+ release channel (RYR1), which plays a critical role in excitation-contraction coupling, is a homotetramer with a subunit molecular mass of 565 kDa, Oxidation of the channel increases its activity and produces intersubunit cross-links within the RYR1 tetramer (Aghdasi, B., Zhang, J., Wu, Y., Reid, M. B., and Hamilton, S. L. (1997) J. Biol. Chem. 272, 3739-3748), Alkylation of hyperreactive sulfhydryls on RYR1 with N-ethylmaleimide (NEM) inhibits channel function and blocks the intersubunit cross-linking, We used calpain and tryptic cleavage, two dimensional SDS polyacrylamide gel electrophoresis, N-terminal sequencing, sequence-specific antibody Western blotting, and [C-14]NEM labeling to identify the domains involved in these effects, Our data are consistent with a model in which 1) diamide, an oxidizing agent, simultaneously produces an intermolecular cross-link between adjacent subunits within the RYR1 tetramer and an intramolecular cross-link within a single subunit; 2) all of the cysteines involved in both cross-links are in either the region between amino acids similar to 2100 and 2843 or the region between amino acids 2844 and 4685; 3) oxidation exposes a new calpain cleavage site in the central domain of the RYR1 (in the region around amino acid 2100); 4) sulfhydryls that react most rapidly with NEM are located in the N-terminal domain (between amino acids 426 and 1396); 5) alkylation of the N-terminal cysteines completely inhibits the formation of both inter- and intra-subunit cross-links, In summary, we present evidence for interactions between the N-terminal region and the putatively cytoplasmic central domains of RYR1 that appear to influence subunit subunit interactions and channel activity.