Time-dependent effects of
cysteine modification were compared in skeletal
ryanodine receptors (RyRs) from normal pigs and RyR(MH) (Arg(615) to Cys(615)) from pigs susceptible to
malignant hyperthermia, using the oxidizing
reagents 4,4'-dithiodipyridine (4, 4'-DTDP) and 5,5'-dithio-bis(2-nitrobenzoic acid) (
DTNB) or the
reducing agent dithiothreitol (DTT). Normal and RyR(MH) channels responded similarly to all
reagents.
DTNB (1 mM), either cytoplasmic (cis) or
luminal (trans), or 1 mM 4,4'-DTDP (cis) activated RyRs, introducing 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, activated 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'-DTDP and
DTNB covalently modify 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 either 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.