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

Calmodulin (CaM) activates the skeletal muscle Ca2+ release channel (ryanodine receptor, RyR1) at nanomolar Ca2+ and inhibits it at micromolar Ca2+. CaM conversion from RyR1 activator to inhibitor is due to structural changes induced by Ca2+ 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 Ca2+-sensitive CaM mutants, and monitored the effects of Ca2+ binding via the fluorescence change of free or RyR1-bound AcrCaM. Using steady state measurements, we found that Ca2+ binding to free CaM causes a dramatic structural change in the N-lobe, but only a slight effect on the C-lobe of the Ca2+-sensitive lobe-specific mutants, in addition to the previously known higher Ca2+ affinity at the C-lobe versus the N-lobe. Using stopped-flow measurements, we found ∼30x faster Ca2+ dissociation from the N- versus C-lobe, and ∼20x slower Ca2+ association to the N-lobe versus C-lobe. These Ca2+ binding properties hold for the CaM/RyR1 complex, and Ca2+ affinity is enhanced at the CaM C-lobe but decreased at the N-lobe by RyR1 binding. We propose that fast Ca2+-binding at the C-lobe of CaM initiates its inhibition to RyR1 at high [Ca2+], while slow Ca2+ 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 Ca2+ affinity versus WT-CaM, as suggested by both steady-state and transient kinetics results.