Abstract
The ryanodine receptor (RyR) calcium release channel functions as a redox sensor that is sensitive to channel modulators. The FK506-binding protein (FKBP) is an important regulator of channel activity, and disruption of the RyR2-FKBP12.6 association has been implicated in cardiac disease. In the present study, we investigated whether the RyR-FKBP association is redox-regulated. Using co-immunoprecipitation assays of solubilized native RyR2 from cardiac muscle sarcoplasmic reticulum (SR) with recombinant [(35)S]FKBP12.6, we found that the sulfydryl-oxidizing agents, H(2)O(2) and diamide, result in diminished RyR2-FKBP12.6 binding. Co-sedimentation experiments of cardiac SR vesicles with [(35)S]FKBP12.6 also demonstrated that oxidizing reagents decreased FKBP binding. Matching results were obtained with skeletal muscle SR. Notably, H(2)O(2) and diamide differentially affected the RyR2-FKBP12.6 interaction, decreasing binding to approximately 75 and approximately 50% of control, respectively. In addition, the effect of H(2)O(2) was negligible when the channel was in its closed state or when applied after FKBP binding had occurred, whereas diamide was always effective. A cysteine-null mutant FKBP12.6 retained redox-sensitive interaction with RyR2, suggesting that the effect of the redox reagents is exclusively via sites on the ryanodine receptor. K201 (or JTV519), a drug that has been proposed to prevent FKBP12.6 dissociation from the RyR2 channel complex, did not restore normal FKBP binding under oxidizing conditions. Our results indicate that the redox state of the RyR is intimately connected with FKBP binding affinity.
Highlights
Activity is regulated by Ca2ϩ, Mg2ϩ, ATP, phosphorylation and redox status, and a number of accessory proteins
Glutathione-induced Aggregation of ryanodine receptor (RyR) and FK506-binding protein (FKBP)—Initially, we tested the effect of redox reagents (i.e. 2 mM DTT, 5 mM GSH, 2 mM GSSG, 1 mM H2O2, 200 M diamide) on the solubilization of the RyR
RyR could not be solubilized from sarcoplasmic reticulum (SR) samples that had been treated with glutathione
Summary
Materials—Radioactive sulfur was obtained from Amersham Biosciences in the form of Pro-Mix containing ϳ70% L-[35S]methionine and 30% L-[35S]cysteine. Co-immunoprecipitation Assays—Cardiac heavy SR vesicles (1 mg), prepared as described previously [12], were solubilized in 200 l of IP buffer (20 mM Tris, 150 mM NaCl, 0.4% CHAPS, pH 7.4, and Complete protease inhibitors (Roche Applied Science)) by overnight incubation at 4 °C with continuous mixing. For the experiments to test whether oxidizing conditions induce FKBP12.6 dissociation, redox reagents were applied for 30 min at room temperature or for 6 h at 4 °C at the end of the IP protocol, once RyR2-FKBP12.6 immunocomplexes had formed following overnight incubation. Co-sedimentation Assays—Cardiac (200 g) or skeletal muscle (100 g) heavy SR vesicles were resuspended in 200 l of buffer (10 mM Na2-Pipes, 120 mM KCl, pH 7.4, and Complete protease inhibitors (Roche Applied Science)) and treated with an appropriate redox reagent (2 mM DTT, 5 mM GSH, 2 mM GSSG, 1 mM H2O2, 200 M diamide) for 30 min at room temperature.
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