ARVC-related mutations in divergent region 3 alter functional properties of the cardiac ryanodine receptor

Two single-nucleotide polymorphisms in the type 2 ryanodine receptor (RyR2) leading to the nonsynonymous amino acid replacements G1885E and G1886S are associated with arrhythmogenic right ventricular cardiomyopathy in patients who are carrying both of the corresponding RyR2 alleles. The functional properties of HEK293 cell lines isogenically expressing RyR2 mutants associated with arrhythmogenic right ventricular cardiomyopathy, RyR2-G1885E, RyR2-G1886S, RyR2-G1886D (mimicking a constitutively phosphorylated Ser¹⁸⁸⁶), and the double mutant RyR2-G1885E/G1886S were investigated by analyzing the intracellular Ca²⁺ release activity resulting from store-overload-induced calcium release. The substitution of serine for Gly¹⁸⁸⁶ caused a significant increase in the cellular Ca²⁺ oscillation activity compared with RyR2 wild-type-expressing HEK293 cells. It was even more pronounced if glycine 1885 or 1886 was replaced by the acidic amino acids glutamate (G1885E) or aspartate (G1886D). Surprisingly, when both substitutions were introduced in the same RyR2 subunit (RyR2-G1885E/G1886S), the store-overload-induced calcium release activity was nearly completely abolished, although the Ca²⁺ loading of the intracellular stores was markedly enhanced, and the channel still displayed substantial Ca²⁺ release on stimulation by 5 mM caffeine. These results suggest that the adjacent glycines 1885 and 1886, located in the divergent region 3, are critical for the function and regulation of RyR2.