Kinetics insight into the roles of the N- and C-lobes of calmodulin in RyR1 channel regulation.

Calmodulin (CaM) activates the skeletal muscle Ca release channel (ryanodine receptor, RyR1) at nanomolar Ca and inhibits it at micromolar Ca. CaM conversion from RyR1 activator to inhibitor is due to structural changes induced by Ca binding at CaM's two lobes. However, it remains unclear which lobe provides the switch for this conversion. Here, we attached the environment-sensitive fluorophore acrylodan (Acr) at either lobe of intact CaM or lobe-specific Ca-sensitive CaM mutants, and monitored the effects of Ca binding via the fluorescence change of free or RyR1-bound CaM. Using steady state measurements, we found that Ca binding to free CaM causes a dramatic structural change in the N-lobe, but only a slight effect on the C-lobe of the Ca-sensitive lobe-specific mutants, in addition to the previously known higher Ca affinity at the C-lobe versus the N-lobe. Using stopped-flow measurements, we found ∼30x faster Ca dissociation from the N- versus C-lobe, and ∼20x slower Ca association to the N-lobe versus C-lobe. These Ca binding properties hold for the CaM/RyR1 complex, and Ca affinity is enhanced at the CaM C-lobe but decreased at the N-lobe by RyR1 binding. We propose that fast Ca-binding at the C-lobe of CaM initiates its inhibition to RyR1 at high [Ca], while slow Ca binding to the N-lobe is necessary for timely enhancement of the inhibitory effect. The dysregulation of RyR1 by M124Q-CaM may be explained by the lower Ca affinity versus WT-CaM, as suggested by both steady-state and transient kinetics results.