Oxidation and reduction of pig skeletal muscle ryanodine receptors

Time-dependent effects of cysteine modification were compared in skeletal r yanodine receptors (RyRs) from normal pigs and RyR(MH) (Arg(615) to Cys(615 )) from pigs susceptible to malignant hyperthermia, using the oxidizing rea gents 4,4'-dithiodipyridine (4,4'-DTDP) and 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) dr the reducing agent dithiothreitol (DTT). Normal and RyR(MH) channels responded similarly to all reagents. DTNB (1 mM), either cytoplas mic (cis) or luminal (trans), or 1 mM 4,4'-DTDP (cis) activated RyRs, intro ducing an additional long open time constant. 4,4'-DTDP (cis), but not DTNB , inhibited channels after >5 min. Activation and inhibition were relieved by DTT (1-10 mM). DTT (10 mM, cytoplasmic or luminal), without oxidants, ac tivated RyRs, and activation reversed with 1 mM DTNB. Control RyR activity was maintained with 1 mM DTNB and 10 mM DTT present on the same or opposite sides of the bilayer. We suggest that 1) 4,4'-DTOP and DTNB covalently mod ify RyRs by oxidizing activating or inhibiting thiol groups; 2) a modified thiol depresses mammalian? skeletal RyR activity under control conditions; 3) both the activating thiols and the modified thiols, accessible from eith er cytoplasm or lumen, reside in the transmembrane region; 4) some cardiac sulfhydryls are unavailable in skeletal RyRs; and 5) Cys(615) in RyR(MH) is functionally unimportant in redox cycling.