H2O2 and ethanol act synergistically to gate ryanodine receptor/calcium-release channel

We examined the effect of low concentrations of H2O2 on the Ca2+-release channel/ryanodine receptor (RyR) to determine if H2O2 plays a physiological role in skeletal muscle function. Sarcoplasmic reticulum vesicles from frog skeletal muscle and type 1 RyRs (RyR1) purified from rabbit skeletal muscle were incorporated into lipid bilayers. Channel activity of the frog RyR was not affected by application of 4.4 mM (0.02%) ethanol. Open probability (P-o) of such ethanol-treated RyR channels was markedly increased on subsequent addition of 10 mu M H2O2. Increase of H2O2 to 100 mu M caused a further increase in channel activity. Application of 4.4 mM ethanol to 10 mu M H2O2-treated RyRs activated channel activity. Exposure to 10 or 100 mu M H2O2 alone, however, failed to increase P-o. Synergistic action of ethanol and H2O2 was also observed on the purified RyR1 channel, which was free from FK506 binding protein (FKBP12). H2O2 at 100-500 mu M had no effect on purified channel activity. Application of FKBP12 to the purified RyR1 drastically decreased channel activity but did not alter the effects of ethanol and H2O2. These results suggest that H2O2 may play a pathophysiological, but probably not a physiological, role by directly acting on skeletal muscle RyRs in the presence of ethanol.