Reactive Cysteines of Ryanodine Receptor Type 1 Influence Function and Response to Oxidative Stress

Abstract

Redox modulation of the skeletal muscle ryanodine receptor1 (RyR1) plays a key role in determining the responsiveness of the Ca2+ release channel to physiological modulation. The sensitivity of RyR1 to redox stress may be conferred by seven previously identified hyper-reactive cysteines (1040, 1303, 2426, 2606, 2611, 2625 and 3635). Wild type RyR1 (wtRyR1), and seven hyper-reactive cysteine mutations of RyR1 were stably expressed in HEK-293 cells and their contribution to RyR1 function evaluated. Addition of RyR1 activator 4-chloro-m-cresol (4-CMC) elicited an increase in [Ca2+]i in the wtRyR1cells but failed to produce a Ca2+ response in the C1303S, C2606S, C2436S and C7S (expressing all seven cysteine mutations) expressing cells, while the C1040S and C2611S mutations significantly attenuated 4-CMC mediated Ca2+ response. Microsomal fractions isolated from C1040S, C2611S, C2436S and C3635S bound to 3[H]Ry while C7S and C1303S showed significantly lower, levels of RyR-binding, although significantly above preparations from RyR-null HEK 293. The sensitivity of RyR1 to 1, 4-napthoquinone (NQ) appears to depend on the expression of RyR1 and the presence of reactive cysteines. Sensitivity to NQ-induced cytotoxicity was determined by the multi-Tox fluorescence assay. NQ decreased cell viability in a dose-dependent manner, but the wtRyR1 cells were less sensitive than C2606, C1040S, C2611S and the C7S mutants. These data indicate the role of hyper-reactive cysteines in regulating RyR1 function and its response to oxidative stress. Supported by NIH AR43140.