Ca2+-calmodulin increases RyR2 open probability yet reduces ryanoid association with RyR2

Journal article

We 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 Ca2²⁺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.

Authors: Sigalas, C; Mayo-Martin, MB; Jane, DE; Sitsapesan, R

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Elsevier
• Journal: Biophysical Journal
• Volume: 97
• Issue: 7
• Pages: 1907-1916
• Publication date: 2009-10-01
• Acceptance date: 2009-07-20
• DOI: 10.1016/j.bpj.2009.07.027
• EISSN: 1542-0086
• ISSN: 0006-3495
• Pmid: 19804721
• Language: English
• Keywords: probability; calmodulin; calcium; animals; tritium; ion channel gating; protein binding; ryanodine receptor calcium release channel; ryanodine; myocardium; kinetics