Site-selective modification of hyperreactive cysteines of ryanodine receptor complex by quinones

Quinones undergo redox cycling and/or arylation reactions with key biomolec ules involved with cellular Ca2+ regulation. The present study utilizes nan omolar quantities of the fluorogenic maleimide 7-diethylamino-3-(4'-maleimi dylphenyl)-4 coumarin (CPM) to measure the reactivity of hyperreactive sulf hydryl moieties on sarcoplasmic reticulum (SR) membranes in the presence an d absence of quinones by analyzing the kinetics of forming CPM-thioether ad ducts and localization of fluorescence by SDS-polyacrylamide gel electropho resis. Doxorubicin, 1,4-naphthoquinone (NQ), and 1,4-benzoquinone (BQ) are found to selectively and dose-dependently interact with a class of hyperrea ctive sulfhydryl groups localized on ryanodinesensitive Ca2+ channels [ryan odine receptor (RyR)], and its associated protein, triadin, of skeletal typ e channels. NQ and BQ are the most potent compounds tested for reducing the rate of CPM labeling of hyperreactive SR thiols (IC50 = 0.3 and 1.8 mu M, respectively) localized on RyR and associated protein. The reduced forms of quinone, tert-butylhydroquinone, and 5-imino-daunorubicin do not alter sig nificantly the pattern or kinetics of CPM labeling up to 100 mu M, demonstr ating that the quinone group is essential for modulating the state of hyper reactive SR thiols, Nanomolar NQ is shown to enhance the association of [H- 3]ryanodine for its high-affinity binding site and directly enhance channel -open probability in bilayer lipid membrane in a reversible manner. By cont rast, micromolar NQ produces a time-dependent biphasic action on channel fu nction, leading to irreversible channel inactivation. These results provide evidence that nanomolar quinone selectively and reversibly alters the redo x state of hyperreactive sulfhydryls localized in the RyR/Ca2+ channel comp lex, resulting in enhanced channel activation. The Ca2+-dependent cytotoxic ities observed with reactive quinones formed at the microsomal surface by o xidative metabolism may be related to their ability to selectively modify h yperreactive thiols regulating normal functioning of microsomal Ca2+ releas e channels.