Extensive Ca2+ leak through K4750Q cardiac ryanodine receptors caused by cytosolic and luminal Ca2+ hypersensitivity

Abstract

Various ryanodine receptor 2 (RyR2) point mutations cause catecholamine-induced polymorphic ventricular tachycardia (CPVT), a life-threatening arrhythmia evoked by diastolic intracellular Ca²⁺ release dysfunction. These mutations occur in essential regions of RyR2 that regulate Ca²⁺ 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 Ca²⁺ imaging, ryanodine binding, and single-channel recordings. HEK293 cells and cardiac HL-1 cells expressing RyR2-K4750Q show greatly enhanced spontaneous Ca²⁺ oscillations. An endoplasmic reticulum-targeted Ca²⁺ sensor, R-CEPIA1er, revealed that RyR2-K4750Q mediates excessive diastolic Ca²⁺ leak, which dramatically reduces luminal [Ca²⁺]. We further show that the K4750Q mutation causes three RyR2 defects: hypersensitization to activation by cytosolic Ca²⁺, loss of cytosolic Ca²⁺/Mg²⁺-mediated inactivation, and hypersensitization to luminal Ca²⁺ activation. These defects combine to kinetically stabilize RyR2-K4750Q openings, thus explaining the extensive diastolic Ca²⁺ leak from the sarcoplasmic reticulum, frequent Ca²⁺ 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.