Low N-ethylmaleimide concentrations activate ryanodine receptors by a reversible interaction, not an alkylation of critical thiols

Previous studies proposed that N-ethylmaleimide (NEM) alkylates 3 classes o f thiols on skeletal muscle ryanodine receptors (RyRs) producing 3 phases o f channel modification, as function of time and concentration. NEM (5 mM) d ecreased, increased, and then decreased the open probability (P-o) of the c hannel by thiol alkylation, a reaction not reversed by reducing agents. We now show that low NEM concentrations (20-200 muM) elicit Ca2+ release from sarcoplasmic reticulum (SR) vesicles, but contrary to expectations, the eff ect was fully reversed by reducing agents or by washing SR vesicles, In bil ayers, NEM (0.2 mM) increased P-o of RyRs within seconds when added to the cis (not trans) side, and dithiothreitol (DTT; 1 mM) decreased P-o in secon ds. High (5 mM) NEM concentrations elicited SR Ca2+ release that was not re versed by DTT, as expected for an alkylation reaction. A non-sulfhydryl rea gent structurally related to NEM, N-ethylsuccinimide (0.1-0.5 mM), also eli cited SR Ca2+ release that was not reversed by DTT (1 mM). Other alkylating agents elicited SR Ca2+ release, which was fully (N-methylmaleimide) or pa rtially (iodoacetic acid) reversed by DTT and inhibited by ruthenium red. N itric oxide (NO) donors at concentrations that did not activate RyRs inhibi ted NEM-induced Ca2+ release, most likely by an interaction of NO with NEM rather than an inactivation of RyRs by NO. Thus, at low concentrations, NEM does not act as a selective thiol reagent and activates RyRs without alkyl ating critical thiols indicating that the multiple phases of ryanodine bind ing are unrelated to RyR activity or to NEM alkylation of RyRs,