Site-specific modification of calmodulin Ca²⁺ affinity tunes the skeletal muscle ryanodine receptor activation profile

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.