ARVC-Related Mutations in Divergent Region 3 Alter Functional Properties of the Cardiac Ryanodine Receptor

¹ Inst. Physiol. Chem., Ruhr-University Bochum
² Dept. Physiol. and Biophysics, University of Calgary

^* To whom correspondence should be addressed. E-mail: magdolna.varsanyi{at}ruhr-uni-bochum.de.

Submitted on September 20, 2007
Revised on October 17, 2007
Accepted on 30 January 2008

	Abstract

Two single nucleotide polymorphisms in the type 2 ryanodine receptor (RyR2) leading to the non-synonymous amino acid replacements G1885E and G1886S are associated with arrhythmogenic right ventricular cardiomyopathy (ARVC) in patients who are carrying both of the corresponding RyR2 alleles. The functional properties of HEK293 cell lines isogenically expressing RyR2 mutants associated with ARVC, RyR2-G1885E, RyR2-G1886S, RyR2-G1886D (mimicking a constitutively phosphorylated Ser1886), and the double mutant RyR2-G1885E/G1886S were investigated by analysing the intracellular Ca2+ release activity resulting from store-overload-induced calcium release (SOICR). The substitution of serine for glycine 1886 caused a significant increase in the cellular Ca²⁺ oscillation activity compared to 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 SOICR activity was nearly completely abolished although the Ca²⁺ loading of the intracellular stores was markedly enhanced and the channel still displayed substantial Ca2+ release upon 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.

Key Words: ARVC, Ca2+ transients, SOICR, cardiac ryanodine receptor, single-cell Ca imaging, stable inducible HEK293 cell line