Extensive Ca(2+) leak through K4750Q cardiac ryanodine receptors caused by cytosolic and luminal Ca(2+) hypersensitivity

Various ryanodine receptor 2 (RyR2) point mutations cause catecholamine-induced polymorphic ventricular tachycardia (CPVT), a life-threatening arrhythmia evoked by diastolic intracellular Ca(2+) release dysfunction. These mutations occur in essential regions of RyR2 that regulate Ca(2+) release. The molecular dysfunction caused by CPVT-associated RyR2 mutations as well as the functional consequences remain unresolved. Here, we study the most severe CPVT-associated RyR2 mutation (K4750Q) known to date. We define the molecular and cellular dysfunction generated by this mutation and detail how it alters RyR2 function, using Ca(2+) imaging, ryanodine binding, and single-channel recordings. HEK293 cells and cardiac HL-1 cells expressing RyR2-K4750Q show greatly enhanced spontaneous Ca(2+) oscillations. An endoplasmic reticulum–targeted Ca(2+) sensor, R-CEPIA1er, revealed that RyR2-K4750Q mediates excessive diastolic Ca(2+) leak, which dramatically reduces luminal [Ca(2+)]. We further show that the K4750Q mutation causes three RyR2 defects: hypersensitization to activation by cytosolic Ca(2+), loss of cytosolic Ca(2+)/Mg(2+)-mediated inactivation, and hypersensitization to luminal Ca(2+) activation. These defects combine to kinetically stabilize RyR2-K4750Q openings, thus explaining the extensive diastolic Ca(2+) leak from the sarcoplasmic reticulum, frequent Ca(2+) waves, and severe CPVT phenotype. As the multiple concurrent defects are induced by a single point mutation, the K4750 residue likely resides at a critical structural point at which cytosolic and luminal RyR2 control input converge.