Ca2+-calmodulin increases RyR2 open probability yet reduces ryanoid association with RyR2.
We have shown that physiological levels of Ca(2+)-calmodulin (Ca(2+)CaM; 50-100 nM) activate cardiac ryanodine receptors (RyR2) incorporated into bilayers and increase the frequency of Ca(2+) sparks and waves in cardiac cells. In contrast, it is well known that Ca(2+)CaM inhibits [(3)H]ryanodine binding to cardiac sarcoplasmic reticulum. Since the [(3)H]ryanodine binding technique does not reflect the effects of Ca(2+)CaM on RyR2 open probability (Po), we have investigated, using the reversible ryanoid, ryanodol, whether Ca(2+)CaM can directly influence the binding of ryanoids to single RyR2 channels independently of Po. We demonstrate that Ca(2+)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, M(1) and M(2), in a voltage-dependent manner. Ca(2+)CaM significantly alters the equilibrium between these two states. The results suggest that Ca(2+)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 Ca(2+)CaM at the single-channel level are not mirrored by [(3)H]ryanodine binding studies.
Department of Physiology and Pharmacology and Bristol Heart Institute, University of Bristol, Bristol, United Kingdom.
Charalambos
C
Sigalas
Maria Belen
MB
Mayo-Martin
David E
DE
Jane
Rebecca
R
Sitsapesan
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D015640:Ion Channel Gating; D007700:Kinetics; D009206:Myocardium / Q000166:cytology; D011336:Probability; D011485:Protein Binding; D012433:Ryanodine / Q000737:chemistry / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D014316:Tritium / Q000737:chemistry
D002147:Calmodulin; D019837:Ryanodine Receptor Calcium Release Channel; D014316:Tritium; D012433:Ryanodine; D002118:Calcium