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The skeletal muscle isoform of the ryanodine receptor Ca2+-release channel (RyR1) is regulated by Ca2+ and CaM (calmodulin). CaM shifts the biphasic Ca2+-dependence of RyR1 activation leftward, effectively increasing channel opening at low Ca2+ and decreasing channel opening at high Ca2+. The conversion of CaM from a RyR1 activator into an inhibitor is due to the binding of Ca2+ to CaM; however, which of CaM's four Ca2+-binding sites serves as the switch for this conversion is unclear. We engineered a series of mutant CaMs designed to individually increase the Ca2+ affinity of each of CaM's EF-hands by increasing the number of acidic residues in Ca2+-chelating positions. Domain-specific Ca2+ affinities of each CaM variant were determined by equilibrium fluorescence titration. Mutations in sites I (T26D) or II (N60D) in CaM's N-terminal domain had little effect on CaM Ca2+ affinity and regulation of RyR1. However, the site III mutation N97D increased the Ca2+-binding affinity of CaM's C-terminal domain and caused CaM to inhibit RyR1 at a lower Ca2+ concentration than wild-type CaM. Conversely, the site IV mutation Q135D decreased the Ca2+-binding affinity of CaM's C-terminal domain and caused CaM to inhibit RyR1 at higher Ca2+ concentrations. These results support the hypothesis that Ca2+ binding to CaM's C-terminal acts as the switch converting CaM from a RyR1 activator into a channel inhibitor. These results indicate further that targeting CaM's Ca2+ affinity may be a valid strategy to tune the activation profile of CaM-regulated ion channels. [ABSTRACT FROM AUTHOR]