Ca2+-Calmodulin Increases RyR2 Open Probability yet Reduces Ryanoid Association with RyR2

AbstractWe have shown that physiological levels of Ca2+-calmodulin (Ca2+CaM; 50–100 nM) activate cardiac ryanodine receptors (RyR2) incorporated into bilayers and increase the frequency of Ca2+ sparks and waves in cardiac cells. In contrast, it is well known that Ca2+CaM inhibits [3H]ryanodine binding to cardiac sarcoplasmic reticulum. Since the [3H]ryanodine binding technique does not reflect the effects of Ca2+CaM on RyR2 open probability (Po), we have investigated, using the reversible ryanoid, ryanodol, whether Ca2+CaM can directly influence the binding of ryanoids to single RyR2 channels independently of Po. We demonstrate that Ca2+CaM reduces the rate of ryanodol association to RyR2 without affecting the rate of dissociation. We also find that ryanodol-bound channels fluctuate between at least two distinct subconductance states, M1 and M2, in a voltage-dependent manner. Ca2+CaM significantly alters the equilibrium between these two states. The results suggest that Ca2+CaM binding to RyR2 causes a conformation change to regions of the channel that include the ryanoid binding site, thereby leading to a decrease in ryanoid association rate and modulation of gating within the ryanoid/RyR2 bound state. Our data provide a possible explanation for why the effects of Ca2+CaM at the single-channel level are not mirrored by [3H]ryanodine binding studies