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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 withN-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 [14C]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 ∼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 intrasubunit 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.

Two sulfhydryl reagents, N-ethylmaleimide (NEM), an alkylating agent, and diamide, an oxidizing agent, were examined for effects on the skeletal muscle Ca2+release channel. NEM incubated with the channel for increasing periods of time displays three distinct phases in its functional effects on the channel reconstituted into planar lipid bilayers; first it inhibits, then it activates, and finally it again inhibits channel activity. NEM also shows a three-phase effect on the binding of [3H]ryanodine by first decreasing binding (phase 1), followed by a recovery of the binding (phase 2), and then a final phase of inhibition (phase 3). In contrast, diamide 1) activates the channel, 2) enhances [3H]ryanodine binding, 3) cross-links subunits within the Ca2+release channel tetramer, and 4) protects against phase 1 inhibition by NEM. All diamide effects can be reversed by the reducing agent, dithiothreitol. Diamide induces intersubunit dimer formation of both the full-length 565-kDa subunit of the channel and the 400-kDa generated by endogenous calpain digestion, suggesting that the cross-link does not involve sulfhydryls within the N-terminal 170-kDa fragment of the protein. NEM under phase 1 conditions blocks the formation of the intersubunit cross-links by diamide. In addition, single channels activated by diamide are further activated by the addition of NEM. Diamide either cross-links phase 1 sulfhydryls or causes a conformational change in the Ca2+release channel which leads to inaccessibility of phase 1 sulfhydryls to NEM alkylation. The data presented here lay the groundwork for mapping the location of one of the sites of subunit-subunit contact in the Ca2+release channel tetramer and for identifying the functionally important sulfhydryls of this protein.