A missense mutation makes a mess of Ca2+ sensing

175 JGP | Research News The human heart beats more than 2.5 billion times over the course of an average lifetime. The ryanodine receptor type 2 (RyR2) helps regulate these beats, and mutations in this ion channel cause catecholamine-induced polymorphic ventricular tachycardia (CPVT). Uehara et al. describe how one particular disease-causing mutation dramatically affects RyR2 behavior (1). The arrival of a cardiac action potential allows extracellular Ca to enter the cell and bind to RyR2 channels embedded in the membrane of the endoplasmic reticulum (called the sarcoplasmic reticulum, or SR, in muscle cells), causing the channels to open and release Ca from the SR into the cytoplasm. The resultant rise in cytoplasmic Ca activates the cardiomyocyte’s actomyosin contractile apparatus and also jumpstarts mechanisms that pump Ca back out of the cytoplasm to prepare the cell for the arrival of the next action potential. Mutations in human RyR2 can allow excessive SR Ca leak, provoking contractions out of sync with cardiac action potentials and causing CPVT patients to develop an abnormally rapid heartbeat in response to exercise or stress (2). A 2009 study (3) identified a young girl with a point mutation in one of her RyR2 alleles that replaced the lysine residue at amino acid 4750 with a glutamine residue. “The patient harboring this mutant exhibits the most severe clinical phenotype of CPVT reported thus far, which likely shows us a clear molecular dysfunction of the RyR2,” says Dr. Akira Uehara, from Fukuoka University in Japan. To investigate how the K4750Q mutation affects RyR2 behavior, Uehara et al. expressed recombinant RyR2 in HEK 293 or cardiac-derived HL-1 cells and monitored intracellular Ca dynamics using a cytoplasmic Ca sensor. Whereas cells expressing wild-type RyR2 occasionally experienced brief bursts of SR Ca release and reuptake as rising external Ca levels drove Ca into the cytoplasm, cells expressing RyR2-K4750Q experienced these oscillations at lower external Ca concentrations and with much greater frequency. For a clearer view of how cells’ internal calcium stores are handled by RyR2K4750Q, Uehara and colleagues used a novel technique, imaging two Ca-sensing dyes to simultaneously visualize calcium levels in both the ER lumen and the cytoplasm. The authors observed that mutant RyR2 is extremely leaky. “Our imaging experiment demonstrated that the lumenal Ca concentration is significantly decreased by the K4750Q mutation,” notes Uehara.