Key result
Mutations in calmodulin's C-terminal domain altered its Ca2+ affinity, demonstrating that Ca2+ binding to the C-terminal acts as the switch converting calmodulin from a RyR1 activator to an inhibitor.
Ca2+ binding to CaM's C-terminal acts as the switch converting CaM from a RyR1 activator into a channel inhibitor, suggesting targeting CaM's Ca2+ affinity may tune ion channel activation.
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May guide calmodulin-targeted RyR1 modulation; leaves open translation from animal models to human disease.
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.
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Jiang et al. (2010) studied this question. Mutant calmodulins (T26D, N60D, N97D, Q135D) vs. Wild-type calmodulin was evaluated on Ca2+ binding affinity and RyR1 activation profile. Mutations in calmodulin's C-terminal domain altered its Ca2+ affinity, demonstrating that Ca2+ binding to the C-terminal acts as the switch converting calmodulin from a RyR1 activator to an inhibitor.
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