The carboxy-terminal calcium binding sites of calmodulin control calmodulin's switch from an activator to an inhibitor of RYR1

Calcium and calmodulin both regulate the skeletal muscle calcium release ch annel, also known as the ryanodine receptor, RYR1. Ca2+-free calmodulin (ap ocalmodulin) activates and Ca2+-calmodulin inhibits the ryanodine receptor. The conversion of calmodulin from an activator to an inhibitor is due to C a2+ binding to calmodulin. We have previously shown that the binding sites for apocalmodulin and Ca2+-calmodulin on RYR1 are overlapping with the Ca2-calmodulin site located slightly N-terminal to the apocalmodulin binding s ite. 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 c almodulin for the ryanodine receptor, suggesting that both lobes interact w ith RYR1 Mutation of the two C-terminal Ca2+ binding sites of calmodulin de stroys 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 Ca2+ concentrations. Mutating the two N-terminal calcium binding sites of calmodulin does not significantly alte r calmodulin's ability to inhibit ryanodine receptor activity. These data s uggest that calcium binding to the two C-terminal calcium binding sites wit hin calmodulin is responsible for the switching of calmodulin from an activ ator to an inhibitor of the ryanodine receptor.