Nitric Oxide, NOC-12, and S-Nitrosoglutathione Modulate the Skeletal Muscle Calcium Release Channel/Ryanodine Receptor by Different Mechanisms: AN ALLOSTERIC FUNCTION FOR O2 INS-NITROSYLATION OF THE CHANNEL

Junhui Sun,Le Xu,Jerry P Eu,Jonathan S Stamler,Gerhard Meissner

doi:10.1074/jbc.m211940200

Abstract

The skeletal muscle Ca(2+) release channel/ryanodine receptor (RyR1) contains approximately 50 thiols per subunit. These thiols have been grouped according to their reactivity/responsiveness toward NO, O(2), and glutathione, but the molecular mechanism enabling redox active molecules to modulate channel activity is poorly understood. In the case of NO, very low concentrations (submicromolar) activate RyR1 by S-nitrosylation of a single cysteine residue (Cys-3635), which resides within a calmodulin binding domain. S-Nitrosylation of Cys-3635 only takes place at physiological tissue O(2) tension (pO(2); i.e. approximately 10 mm Hg) but not at pO(2) approximately 150 mm Hg. Two explanations have been offered for the loss of RyR1 responsiveness to NO at ambient pO(2), i.e. Cys-3635 is oxidized by O(2) versus O(2) subserves an allosteric function (Eu, J. P., Sun, J. H., Xu, L., Stamler, J. S., and Meissner, G. (2000) Cell 102, 499-509). Here we report that the NO donors NOC-12 and S-nitrosoglutathione both activate RyR1 by release of NO but do so independently of pO(2). Moreover, NOC-12 activates the channel by S-nitrosylation of Cys-3635 and thereby reverses channel inhibition by calmodulin. In contrast, S-nitrosoglutathione activates RyR1 by oxidation and S-nitrosylation of thiols other than Cys-3635 (and calmodulin is not involved). Our results suggest that the effect of pO(2) on RyR1 S-nitrosylation is exerted through an allosteric mechanism.

Highlights

The skeletal muscle Ca2؉ release channel/ryanodine receptor (RyR1) contains ϳ50 thiols per subunit
NOC-12 and GSNO The stock solutions of nitric oxide (NO) donors (10 mM) were prepared fresh, and NO release by 0.1 mM each NO donor was recorded with a NO electrode (WPI Instruments) in the buffer used for [3H]ryanodine binding at 24 °C in room air
Data are the mean Ϯ S.E. of the number of recordings indicated in parentheses. *, p Ͻ 0.05 compared with control

Summary

The abbreviations used are

RyR, ryanodine receptor; RyR1, skeletal muscle isoform of RyR; pO2, O2 tension; CaM, calmodulin; NO, nitric oxide; SNO, S-nitrosothiol; GSNO, S-nitrosoglutathione; SR, sarcoplasmic reticulum; NOC-12, N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)ethanamine; WT, wild type; HEK293, human embryonic kidney 293; ing domain of RyR1 (6 – 8), confers responsiveness to NO. S-nitrosylation of Cys3635 only occurs at low O2 tension (pO2 ϳ10 mm Hg, comparable with that found in skeletal muscle in vivo) [1, 6] At this pO2, 6 – 8 (of ϳ50) thiols per RyR1 subunit are actively maintained in the reduced state [1]. We examined the activation of the skeletal muscle Ca2ϩ release channel by NOC-12 and GSNO, an endogenous S-nitrosothiol, and compared their effects to solutions of NO. We found that both NOC-12 and GSNO activated RyR1 independently of O2 tension and that the NO scavenger, C-PTIO, blocked the effects of both. NO, NOC-12, and GSNO activate the prototypic redox-sensitive RyR1 channel by different mechanisms, and the effect of O2 tension on S-nitrosylation by NO is best rationalized by an allosteric mechanism

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION