Trans Complementation with the Common G1885E Polymorphism Attenuates Mutant RyR2 L433P Channel Dysfunction

Abstract

A channelopathic Leu433Pro (L433P) mutation in the cardiac ryanodine receptor (RyR2) alters intracellular Ca2+ handling and is associated with malignant arrhythmia. This mutation is reported to exclusively co-inherit with a common single nucleotide polymorphism (SNP) Gly1885Glu (G1885E) present on the same allele (i.e. in cis). We investigated the functional impact of the G1885E SNP on L433P channels. RyR2-null HEK cells were used as the background for the systematic expression of homo- and heteromeric RyR2 channels composed of recombinant wild-type (WT) subunits, and those containing L433P and G1885E substitutions. Homomeric G1885E channels were functionally normal, but Ca2+ handling abnormalities associated with the L433P mutation were exacerbated by complementation with the G1885E in cis. At the cellular level, channel dysfunction manifested as reduced caffeine-evoked Ca2+ release and decreased ER Ca2+ store content. These findings suggest that although L433P is not a typical gain-of-function mutation it augments basal channel activity. Co-expression of L433P subunits with those containing G1885E (i.e. in trans complementation) attenuated L433P mutant channel dysfunction. Cells expressing heteromeric assemblies of L433P and G1885E subunits exhibited caffeine-activation profiles, homeostatic and post-activation Ca2+ fluxes and ER Ca2+ loads that were comparable to those typical of WT channels. Our data show that the genotype, allelic distribution and the resultant mode of L433P and G1885E channel assembly (i.e. cis or trans subunit complementation) has divergent effects on RyR2 channel (dys)function. The G1885E SNP may be a complex determinant of channel behaviour in situ and the potential therapeutic benefits of normalising mutant channel dysfunction via trans-complementation strategies should be further explored.