A role for cysteine 3635 for RYR1 in redox modulation and calmodulin binding

Oxidation of the skeletal muscle Ca2+ release channel (RYR1) increases its activity, produces intersubunit disulfide bonds, and blocks its interaction with calmodulin. Conversely, bound calmodulin protects RYR1 from the effec ts of oxidants (Zhang, J.-Z., Wu, Y., Williams, B. Y., Rodney, G., Mandel, F., Strasburg, G. M., and Hamilton, S. L. (1999) Am. J. Physiol, 276, Cell Physiol. C46-C53), In addition, calmodulin protects RYR1 from trypsin cleav age at amino acids 3630 and 3637 (Moore, C. P., Rodney, G., Zhang, J.-Z., S antacruz-Toloza, L., Strasburg, G. M., and Hamilton, S. L. (1999) Biochemis try 38, 8532-8537), The sequence between these two tryptic sites is AVVA (C ) under bar FR. Alkylation of RYR1 with N-ethylmaleimide (NEM) blocks both S-35-apocalmodulin binding and oxidation-induced intersubunit cross-linking , In the current work, we demonstrate that both cysteines needed for the ox idation-induced intersubunit crosslink are protected from alkylation with N -ethylmaleimide by bound calmodulin, We also show, using N-terminal amino a cid sequencing together with analysis of the distribution of [H-3]NEM label ing with each sequencing cycle, that cysteine 3635 of RYR1 is rapidly label ed by NEM and that this labeling is blocked by bound calmodulin, We propose that cysteine 3635 is located at an intersubunit contact site that is clos e to or within a calmodulin binding site, These findings suggest that calmo dulin and oxidation modulate RYR1 activity by regulating intersubunit inter actions in a mutually exclusive manner and that these interactions involve cysteine 3635.