H2O2and ethanol act synergistically to gate ryanodine receptor/calcium-release channel

We examined the effect of low concentrations of H(2)O(2) on the Ca(2+)-release channel/ryanodine receptor (RyR) to determine if H(2)O(2) 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 microM H(2)O(2). Increase of H(2)O(2) to 100 microM caused a further increase in channel activity. Application of 4.4 mM ethanol to 10 microM H(2)O(2)-treated RyRs activated channel activity. Exposure to 10 or 100 microM H(2)O(2) alone, however, failed to increase P(o). Synergistic action of ethanol and H(2)O(2) was also observed on the purified RyR1 channel, which was free from FK506 binding protein (FKBP12). H(2)O(2) at 100-500 microM 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 H(2)O(2). These results suggest that H(2)O(2) may play a pathophysiological, but probably not a physiological, role by directly acting on skeletal muscle RyRs in the presence of ethanol.