Skeletal muscle ryanodine receptor channels are activated by the fungal metabolite, gliotoxin

Interactions between the reactive disulfide fungal metabolite, gliotoxin (G TX), and rabbit skeletal ryanodine receptor (RyR) calcium release channels have been examined. RyRs in terminal cisternae vesicles formed a covalent c omplex with 100 mu M S-35-GTX, which was reversed by 1 mM dithiothreitol (D TT) or 1 mM glutathione. GTX (80-240 mu M), added to either cytoplasmic (ci s) or luminal (trans) solutions, increased the rate of Ca2+ release from SR vesicles and the frequency of opening of single RyR channels in lipid bila yers. Channel activation was reversed upon addition of 2 mM DTT to th cis s olution, showing that the activation was due to an oxidation reaction (2 mM DTT added to the cis solution in the absence of GTX did not affect RyR act ivity). Furthermore, RyRs were not activated by trans GTX if the cis chambe r contained DTT, suggesting that GTX oxidized a site in or near the membran e. In contrast to cis DTT, 2 mM DTT in the trans solution increased RyR act ivity when added either alone or with 200 mu M trans GTX. The results sugge st that (i) GTX increases RyR channel activity by oxidizing cysteine residu es that are close to the membrane and located on RyR, or associated protein s, and (ii) a disulfide bridge or nitrosothiol, accessible only from the lu minal solution, normally suppresses RyR channel activity. Some of the actio ns of GTX in altering Ca2+ homeostatsis might depend on its modification of RyR calcium channels.