Redox Sensitivity of the Ryanodine Receptor Interaction with FK506-binding Protein

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 redoxregulated. Using co-immunoprecipitation assays of solubilized native RyR2 from cardiac muscle sarcoplasmic reticulum ( and diamide differentially affected the RyR2-FKBP12.6 interaction, decreasing binding to ϳ75 and ϳ50% 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. Ryanodine receptors (RyRs)2 are tetrameric intracellular Ca 2ϩ channels that mediate the release of Ca 2ϩ from the sarco/ endoplasmic reticulum in muscle and nonmuscle cells (1). Three genes coding for mammalian RyRs have been identified: RyR1 in skeletal muscle, RyR2 in heart and brain, and RyR3 in a number of tissues. The deduced primary structure of all RyRs suggests a hydrophobic C terminus forming the channel pore, with the remaining ϳ80% being cytoplasmic. RyR channel activity is regulated by Ca 2ϩ , Mg 2ϩ , ATP, phosphorylation and redox status, and a number of accessory proteins.The immunophilin, FK506-binding protein (FKBP), a receptor protein for the immunosuppressants, FK506 and rapamycin, is an essential component of the RyR-Ca 2ϩ release channel complex in both skeletal and cardiac muscle (2, 3). RyR1 binds to both FKBP12 and FKBP12.6 with similar affinities (4), whereas RyR2 associates specifically with the FKBP12.6 isoform (3, 5). The stoichiometry of the association is four molecules of FKBP per RyR tetrameric channel (i.e. one FKBP molecule for each RyR protomer) (3, 6). Mapping studies have failed to identify a unique FKBP-binding site, with evidence presented for three distinct RyR regions (N-terminal, central, and C-terminal domains) (7-12), suggesting that FKBP interaction may be stabilized by multiple physical contacts and/or may be conformation-sensitive. The functional effects of FKBP association with RyR have been suggested to include stabilization of the full conductance state (7,13,14), channel closure (5, 6, 15), and coupled gating between neighboring channels (16,17). These effects are highlighted in abnormal or disease states, where defective regulation of the RyR-FKBP association has been implicated in cardiomyopathy (18), cardiac hypertrophy (19), h...

Selenoprotein N is required for ryanodine receptor calcium release channel activity in human and zebrafish muscle

“…Oxidation of selected ''hyperreactive'' cysteines can occur through formation of disulfides, by Snitrosylation, or by S-glutathionylation, and the redox state of these cysteines correlates closely with the open state of the calcium release channel (35,36). Redox modification of specific cysteine residues also affects the ability of calcium release channel components to maintain their association with the RyR core protein (37). Moreover, well characterized modulators of calcium release channel activity affect the redox potential of the channel, whether through direct or indirect means (26,38).…”

Role of chronic ryanodine receptor phosphorylation in heart failure and β-adrenergic receptor blockade in mice

“…H 2 O 2 (1 mM) resulted in oxidation of RyR2 (determined by assessment of DNP-derivatized RyR2) and a reduction in the amount of calstabin2 in the RyR2 complex in cardiac microsomes from WT mice by approximately 20% (similar to an earlier report; ref. 38). In contrast, oxidation of RyR2-S2808D (which mimics constitutively PKA-phosphorylated channels) by 1 mM H 2 O 2 caused nearly complete depletion of calstabin2 from the RyR2 complex in cardiac microsomes from 1.5-month-old RyR2-S2808D mice ( Figure 3, A and B).…”

Redox Regulation of Sodium and Calcium Handling

“…Thiol-oxiziding agents such as H 2 O 2 activate RyR Ca release after oxidation of more than 7 thiols per subunit (1, 6, 21, 45, 84,147,155). Since the RyR2 has multiple sites for regulation by phosophorylation and/or interaction with Ca, Mg, ATP, CaM, or regulatory proteins (FKBP12.6 also called calstabin), oxidation may interfere with these regulators.…”

“…Since the RyR2 has multiple sites for regulation by phosophorylation and/or interaction with Ca, Mg, ATP, CaM, or regulatory proteins (FKBP12.6 also called calstabin), oxidation may interfere with these regulators. Indeed, the mechanism of ROS-induced increase in RyR Ca release involves a change in RyR sensitivity to cytosolic Ca and ATP (42, 93), the alteration of the RyR interaction with triadin, which regulates RyR sensitivity to luminal Ca (84), and the disturbance of FKBP12.6 binding (155). For activation to occur, it requires the oxidation of more than 7 thiols per subunit (147).…”