Impaired Ligand Regulation of Native RyR2 Channels in the Catecholaminergic Polymorphic Ventricular Tachycardia Mutation, RyR2-V2475F(+/−)

Abstract

Mutations in the cardiac ryanodine receptor (RyR2) are associated with catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1), a disease that can lead to arrhythmias and sudden cardiac death. Although many CPVT1 mutations are thought to induce increased diastolic leak of Ca2+, the altered functional properties of the RyR2 channels caused by each point mutation are not fully understood. CPVT1 patients are normally heterozygous for the mutation, therefore will express a population of RyR2 channels with variable numbers of mutated subunits per tetrameric channel, yet typically, only the single-channel properties of purified recombinant homozygous mutant channels are investigated. We have therefore compared the single-channel properties of native RyR2 obtained from WT or V2475F heterozygous (+/−) mutant mouse hearts after incorporation into artificial membranes under voltage-clamp conditions. Compared to WT, greater numbers of RyR2 channels derived from V2475F(+/−) mice exhibited high open probability (Po) in response to activating cytosolic Ca2+. For example, average Po was 0.29 ± 0.04 (WT) and 0.42 ± 0.04 (V2475F(+/−)) with 1 μM cytosolic Ca2+ (SEM; n = 41-52; p< 0.05). Potentiation of Ca2+-induced activation by adenine nucleotides did not appear to be altered by the V2475F mutation as 1 mM cytosolic adenosine caused a 1.6 fold and 1.7 fold elevation of Po for channels derived from WT and V2475F(+/−) mice, respectively. The ability of Mg2+ to inhibit RyR2 channels from V2475F(+/−) mutant mice appeared to be blunted. On average, 1 mM Mg2+ was approximately 40% less effective at reducing the Po of channels from V2475F(+/−) mice than from WT mice (n = 4). We suggest that the altered response of RyR2 to both cytosolic Ca2+ and Mg2+ will be important factors driving the severity of the RyR2-V2475F phenotype.