Dantrolene inhibition of ryanodine channels (RyR2) in artificial lipid bilayers depends on FKBP12.6.

Dantrolene is a neutral hydantoin that is clinically used as a skeletal muscle relaxant to prevent overactivation of the skeletal muscle calcium release channel (RyR1) in response to volatile anesthetics. Dantrolene has aroused considerable recent interest as a lead compound for stabilizing calcium release due to overactive cardiac calcium release channels (RyR2) in heart failure. Previously, we found that dantrolene produces up to a 45% inhibition RyR2 with an IC50 of 160 nM, and that this inhibition requires the physiological association between RyR2 and CaM.

Calmodulin inhibition of human RyR2 channels requires phosphorylation of RyR2-S2808 or RyR2-S2814.

Calmodulin (CaM) is a Ca-binding protein that binds to, and can directly inhibit cardiac ryanodine receptor calcium release channels (RyR2). Animal studies have shown that RyR2 hyperphosphorylation reduces CaM binding to RyR2 in failing hearts, but data are lacking on how CaM regulates human RyR2 and how this regulation is affected by RyR2 phosphorylation. Physiological concentrations of CaM (100 nM) inhibited the diastolic activity of RyR2 isolated from failing human hearts by ~50% but had no effect on RyR2 from healthy human hearts.

The emerging role of calmodulin regulation of RyR2 in controlling heart rhythm, the progression of heart failure and the antiarrhythmic action of dantrolene.

Cardiac output and rhythm depend on the release and the take-up of calcium from the sarcoplasmic reticulum (SR). Excessive diastolic calcium leak from the SR due to dysfunctional calcium release channels (RyR2) contributes to the formation of delayed after-depolarizations, which underlie the fatal arrhythmias that occur in heart failure and inherited syndromes. Calmodulin (CaM) is a calcium-binding protein that regulates target proteins and acts as a calcium sensor.

Essential Role of Calmodulin in RyR Inhibition by Dantrolene.

Dantrolene is the first line therapy of malignant hyperthermia. Animal studies suggest that dantrolene also protects against heart failure and arrhythmias caused by spontaneous Ca(2+) release. Although dantrolene inhibits Ca(2+) release from the sarcoplasmic reticulum of skeletal and cardiac muscle preparations, its mechanism of action has remained controversial, because dantrolene does not inhibit single ryanodine receptor (RyR) Ca(2+) release channels in lipid bilayers.

Differences in the regulation of RyR2 from human, sheep, and rat by Ca²⁺ and Mg²⁺ in the cytoplasm and in the lumen of the sarcoplasmic reticulum.

Regulation of the cardiac ryanodine receptor (RyR2) by intracellular Ca(2+) and Mg(2+) plays a key role in determining cardiac contraction and rhythmicity, but their role in regulating the human RyR2 remains poorly defined. The Ca(2+)- and Mg(2+)-dependent regulation of human RyR2 was recorded in artificial lipid bilayers in the presence of 2 mM ATP and compared with that in two commonly used animal models for RyR2 function (rat and sheep). Human RyR2 displayed cytoplasmic Ca(2+) activation (K(a) = 4 µM) and inhibition by cytoplasmic Mg(2+) (K(i) = 10 µM at 100 nM Ca(2+)) that was similar to RyR2 from rat and sheep obtained under the same experimental conditions.

Divergent regulation of ryanodine receptor 2 calcium release channels by arrhythmogenic human calmodulin missense mutants.

Rationale: Calmodulin (CaM) mutations are associated with an autosomal dominant syndrome of ventricular arrhythmia and sudden death that can present with divergent clinical features of catecholaminergic polymorphic ventricular tachycardia (CPVT) or long QT syndrome (LQTS). CaM binds to and inhibits ryanodine receptor (RyR2) Ca release channels in the heart, but whether arrhythmogenic CaM mutants alter RyR2 function is not known.

Objective: To gain mechanistic insight into how human CaM mutations affect RyR2 Ca channels.