The skeletal muscle calcium release channel, ryanodine receptor, is activated by calcium-free calmodulin and inhibited by calcium-bound calmodulin. Previous biochemical studies from our laboratory have shown that calcium-free calmodulin and calcium bound calmodulin protect sites at amino acids 3630 and 3637 from trypsin cleavage (Moore, C. P., Rodney, G., Zhang, J. Z., Santacruz-Toloza, L., Strasburg, G., and Hamilton, S. L. (1999) Biochemistry 38, 8532-8537). We now demonstrate that both calcium-free calmodulin and calcium-bound calmodulin bind with nanomolar affinity to a synthetic peptide matching amino acids 3614 -3643 of the ryanodine receptor. Deletion of the last nine amino acids (3635-3643) destroys the ability of the peptide to bind calcium-free calmodulin, but not calcium-bound calmodulin. We propose a novel mechanism for calmodulin's interaction with a target protein. Our data suggest that the binding sites for calcium-free calmodulin and calcium-bound calmodulin are overlapping and, when calcium binds to calmodulin, the calmodulin molecule shifts to a more N-terminal location on the ryanodine receptor converting it from an activator to an inhibitor of the channel. This region of the ryanodine receptor has previously been identified as a site of intersubunit contact, suggesting the possibility that calmodulin regulates ryanodine receptor activity by regulating subunit-subunit interactions.

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In skeletal muscle the L-type Ca 2؉ channel directly controls the opening of the sarcoplasmic reticulum Ca 2؉ release channel (RYR1), and RYR1, in turn, prevents L-type Ca 2؉ channel inactivation. We demonstrate that the two proteins interact using calmodulin binding regions of both proteins. A recombinant protein representing amino acids 1393-1527 (D1393-1527) of the carboxyl-terminal tail of the skeletal muscle L-type voltage-dependent calcium channel binds Ca 2؉ , Ca 2؉ calmodulin, and apocalmodulin. In the absence of calmodulin, D1393-1527 binds to both RYR1 and a peptide representing the calmodulin binding site of RYR1 (amino acids 3609-3643). In addition, biotinylated R3609-3643 peptide can be used with streptavidin beads to pull down [ 3 H]PN200-110-labeled L-type channels from detergent-solubilized transverse tubule membranes. The binding of the L-type channel carboxyl-terminal tail to the calmodulin binding site on RYR1 may stabilize the contact between the two proteins, provide a mechanism for Ca 2؉ and/or calmodulin regulation of their interaction, or participate directly in functional signaling between these two proteins. A unique aspect of this study is the finding that calmodulin binding sequences can serve as specific binding motifs for proteins other than calmodulin.

Calcium and calmodulin both regulate the skeletal muscle calcium release channel, also known as the ryanodine receptor, RYR1. Ca(2+)-free calmodulin (apocalmodulin) activates and Ca(2+)-calmodulin inhibits the ryanodine receptor. The conversion of calmodulin from an activator to an inhibitor is due to Ca(2+) binding to calmodulin. We have previously shown that the binding sites for apocalmodulin and Ca(2+)-calmodulin on RYR1 are overlapping with the Ca(2+)-calmodulin site located slightly N-terminal to the apocalmodulin binding site. We now show that mutations of the calcium binding sites in either the N-terminal or the C-terminal lobes of calmodulin decrease the affinity of calmodulin for the ryanodine receptor, suggesting that both lobes interact with RYR1. Mutation of the two C-terminal Ca(2+) binding sites of calmodulin destroys calmodulin's ability to inhibit ryanodine receptor activity at high calcium concentrations. The mutated calmodulin, however, can still bind to RYR1 at both nanomolar and micromolar Ca(2+) concentrations. Mutating the two N-terminal calcium binding sites of calmodulin does not significantly alter calmodulin's ability to inhibit ryanodine receptor activity. These data suggest that calcium binding to the two C-terminal calcium binding sites within calmodulin is responsible for the switching of calmodulin from an activator to an inhibitor of the ryanodine receptor.