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 H₂O₂elicited spontaneous contractures. H₂O₂at 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 H₂O₂in the presence of cis catalase, but unitary conductance and reversal potential were not affected. Exposure to cis H₂O₂at 1.5 mM failed to activate the channel in the presence of trans catalase. Application of 1.5 mM H₂O₂to 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 H₂O₂without pCMPS. Addition of cis pCMPS to channels that had been treated with or without trans H₂O₂rapidly resulted in high P_ofollowed 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.