A Noncontiguous, Intersubunit Binding Site for Calmodulin on the Skeletal Muscle Ca²⁺ Release Channel*

Abstract

Both apocalmodulin (Ca²⁺-free calmodulin) and Ca²⁺-calmodulin bind to and regulate the activity of skeletal muscle Ca²⁺ release channel (ryanodine receptor, RYR1). Both forms of calmodulin protect sites after amino acids 3630 and 3637 on RYR1 from trypsin cleavage. Only apocalmodulin protects sites after amino acids 1982 and 1999 from trypsin cleavage. Ca²⁺-calmodulin and apocalmodulin both bind to two different synthetic peptides representing amino acids 3614–3643 and 1975–1999 of RYR1, but Ca²⁺-calmodulin has a higher affinity than apocalmodulin for both peptides. Cysteine 3635, within the 3614–3643 sequence of RYR1, can form a disulfide bond with a cysteine on an adjacent subunit within the RYR1 tetramer. The second cysteine is now shown to be between amino acids 2000 and 2401. The close proximity of the cysteines forming the intersubunit disulfide to the two sites that bind calmodulin suggests that calmodulin is binding at a site of intersubunit contact, perhaps with one lobe bound between amino acids 3614 and 3643 on one subunit and the second lobe bound between amino acids 1975 and 1999 on an adjacent subunit. This model is consistent with the finding that Ca²⁺-calmodulin and apocalmodulin each bind to a single site per RYR1 subunit (Rodney, G. G., Williams, B. Y., Strasburg, G. M., Beckingham, K., and Hamilton, S. L. (2000) Biochemistry 39, 7807–7812).