Sulfhydryls associated with H₂O₂-induced channel activation are on luminal side of ryanodine receptors

Abstract

The mechanism underlying H2O2-induced activation of frog skeletal muscle ryanodine receptors was studied using skinned fibers and by measuring single Ca2+-release channel current. Exposure of skinned fibers to 3–10 mM H2O2 elicited spontaneous contractures. H2O2 at 1 mM potentiated caffeine contracture. When the Ca2+-release channels were incorporated into lipid bilayers, open probability ( P o) and open time constants were increased on intraluminal addition of H2O2 in the presence of cis catalase, but unitary conductance and reversal potential were not affected. Exposure to cis H2O2 at 1.5 mM failed to activate the channel in the presence of trans catalase. Application of 1.5 mM H2O2 to the transside of a channel that had been oxidized by cis p-chloromercuriphenylsulfonic acid (pCMPS; 50 μM) still led to an increase in P o, comparable to that elicited by trans 1.5 mM H2O2 without pCMPS. Addition of cis pCMPS to channels that had been treated with or without trans H2O2 rapidly resulted in high P o followed by closure of the channel. These results suggest that oxidation of luminal sulfhydryls in the Ca2+-release channel may contribute to H2O2-induced channel activation and muscle contracture.

Further Characterization of the Type 3 Ryanodine Receptor (RyR3) Purified from Rabbit Diaphragm

“…In 3 of 14 experiments, cis-pCMPS elicited irreversible closure after transient increase in P o , which could not be recovered by any ligands including a large amount of DTT (data not shown), suggesting the existence of at least two different sulfhydryls associated with activation and inactivation of the channel in the RyR1 molecule. Similar results on sulfhydryl reagents were observed with RyR channels in frog skeletal muscle SR (43). The conversion between low P o and high P o channels occurred immediately after addition of these reagents.…”

“…It has been demonstrated that a number of sulfhydryl-oxidizing reagents activate RyR channels (40,41,43). Sulfhydrylreducing reagents such as glutathione and DTT have been shown to reduce the RyR channel activity (39,42).…”

Classes of Thiols That Influence the Activity of the Skeletal Muscle Calcium Release Channel

“…The skeletal muscle isoform of RyR (RyR1) contains a large number of free thiols: as many as 50 out of a total of 101 cysteine residues/subunit (100 cysteines/ RyR1 subunit (3) and 1 cysteine/FK506-binding protein subunit (4)) (5). RyR1 channel activity is dramatically altered by redox modifications of critical thiols (oxidation, S-nitrosylation, or alkylation) (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Conversely, RyR1 has thiols whose redox potential is dependent on effectors that regulate RyR1 activity such as Ca 2ϩ and Mg 2ϩ (15).…”

Involvement of ryanodine-operated channels intert-butylhydroperoxide-evoked Ca2+ mobilisation in pancreatic acinar cells

“…This conclusion is supported by our results showing that ryanodine (at a concentration of 50·mol·l -1 , which blocks ryanodine receptors), abolishes tBHP-induced Ca 2+ -release from nonmitochondrial Ca 2+ pools, thus suggesting that tBHP sensitises ryanodine receptors, at least in pancreatic acinar cells. In fact, it has been reported that other oxidising agents, like H 2 O 2 , release Ca 2+ from intracellular stores by activation of the ryanodine receptor (Favero et al, 1995;Oba et al, 1998) and that sulphydryl groups (susceptible to oxidation) have a critical role in the ryanodine-sensitive Ca 2+ channel (Oba et al, 1998). In addition, ryanodine shows 'in vitro' sensitisation in the presence of the sulphydryl group oxidising agent thimerosal (Abramson et al, 1995;Wu et al, 1996).…”