Chapter 20

REDOX SENSING BY THE RYANODINE RECEPTORS

Gerhard Meissner ¹ and Jonathan S. Stamler ²

1

Depts. of Biochemistry and Biophysics, and Cell and Molecular Physiology, University of North Carolina, Chapel Hill, NC and

Depts. of Medicine and Biochemistry and Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC

INTRODUCTION

The release of ions from intracellular membrane-bound stores is a

key step in a wide variety of biological functions. In cardiac and skeletal

muscle, the release of ions through release channels into the

cytoplasm leads to muscle contraction. The release channels are also

known as ryanodine receptors (RyRs) because the plant alkaloid ryanodine

modifies their function by binding with high affinity and specificity. ¹⁰⁹ The

RyR ion channels are large protein complexes that are composed of four

RyR 560 kDa polypeptides subunits, four small 12 kDa FK506 binding

proteins (FKBP) and various associated proteins with a total molecular

weight of greater than 2,500 kDa. ^110,211 Numerous endogenous effectors

ranging from divalent cations and to small molecules (e.g. ATP)

and proteins (e.g. calmodulin, FKBP) regulate RyR function and thereby

muscle function. The RyRs are also targets for redox active molecules. This

chapter reviews the regulation of the skeletal muscle and cardiac muscle

ryanodine receptors by glutathione, oxygen tension NADH, nitric

oxide (NO) and related reactive oxygen and nitrogen species.

REGULATION OF RYRS BY OXYGEN TENSION AND GLUTATHIONE REDOX POTENTIAL

Figure 20-1. RyR1 channel activity and redox state. binding (as a measurement of RyR1 activity) and free thiol content were determined either at

or 150 mm Hg in the presence of either 5 mM reduced (GSH) or oxidized (GSSG) glutathione. binding was determined by incubating skeletal muscle sarcoplasmic reticulum vesicles with in the presence of free The free thiol content was determined by the monobromobimane method in the same condition From Xu et al.

RyR ion channels contain reactive cysteines (i.e. thiols susceptible to redox-based modifications), which modulate RyR activity. ¹¹⁰ Heavy metals, alkylating agents such as N-ethylmaleimide (NEM) and oxidants such as diamide and modulate the activity of the RyRs. 484,762,763

The

experimental results in Fig. 20-1 show that RyR1 activity and the number of

reduced cysteines (free thiols) depend on two principal determinants of

cellular redox state - oxygen tension and reduced (GSH) or oxidized

(GSSG) glutathione. ⁶¹² Studies using the lipophilic, thiol-specific probe

monobromobimane have revealed that nearly half of the 404 cysteines

within the tetrameric RyR1 channel complex are reduced in the presence of

5 mM GSH at i.e. under conditions comparable to resting

muscle. ⁷⁶⁴ In this situation, RyR1 activity was low, as measured by a ligand

binding assay using the RyR-specific probe An increase in

oxygen tension from ~10 mm Hg (simulating the tissue) to ambient air

in the presence of 5 mM GSH modifies the redox state of up to 8 thiols/RyR1 subunit without appreciably changing RyR1 activity.

Exposure of RyR1 to oxidized glutathione (GSSG) at or at ambient conditions resulted in the oxidation of up to 24 thiols/RyR1 subunit and a large increase in activity. The results of Fig. 20-1 suggest that RyR1 has a large group of functionally ‘silent’ thiols that may protect the RyR1 from low oxidative stress (as might be seen in normal working muscle), and another group of reactive thiols that may control the channel’s response to more hazardous levels of oxidants (e.g. produced during rigorous exercise).

Additional parameters determine the redox state of RyR1. Micromolar activating concentrations of lowered the redox potential of RyR1 and favored channel opening, whereas elevated inhibitory concentrations of and had opposite effects. ⁶⁰⁴ In mammalian cells, a cytosolic ratio of

creates a highly reducing redox potential. ⁷⁶⁵ However, a ratio of 3:1 to 1: 1 generates a more oxidizing environment in the ER lumen.

In single channel measurements, RyR1 responded to redox potentials produced by both SR lumenal and cytoplasmic glutathione, indicating that the receptor is under the control of a transmembrane redox potential. ⁶⁰⁹ Studies with skeletal muscle membranes suggested that glutathione transport across the SR membrane may be facilitated by RyR1-dependent and - independent mechanisms. ^609,766

Fig. 20-2 examines the regulation of the skeletal muscle and cardiac muscle ryanodine receptors by glutathione at ambient oxygen tension in the presence of two endogenous channel effectors, MgATP and calmodulin.

Reduced glutathione (5 mM GSH, i.e., concentrations similar to those found

in cells) resulted in low RyR1 activity, whereas the addition of oxidized

glutathione (5 mM GSSG) strongly activated binding

without appreciably affecting the of channel activity. In

contrast, a change from oxidizing to reducing conditions shift the RyR2

activation curve to the right without appreciably altering the maximal levels

of binding at optimally activating concentrations. The

results suggest that the effects of GSH/GSSG redox state on RyR1 and RyR2

activity are exerted by different mechanisms.

Figure 20-2. Redox regulation of skeletal muscle and cardiac muscle ryanodine receptors by reduced and oxidized glutathione. Specific binding to rabbit skeletal and cardiac sarcoplasmic reticulum membranes was determined at ambient oxygen tension in the presence of two endogenous channel modulators, 5 mM Mg AMPPCP (an ATP analog) and

calmodulin, in assay media containing 0.25 M KCl, 20 mM imidazole, pH 7, and the indicated concentrations of free From Balshaw et al.

MODULATION OF RYRS BY REACTIVE OXYGEN SPECIES

Working muscle produces reactive oxygen species (ROS) at a low basal rate. ⁷⁶⁷ Enzymes that scavenge ROS such as superoxide anion and hydrogen peroxide attenuate force development in muscle exposed to 95% (standard bioassay conditions), supporting a functional role. During strenuous muscle exercise or short episodes of ischemia followed by the resupply of oxygen, increased levels of ROS impose an oxidative stress by altering various cellular functions. Cells protect themselves against the excessive production of reactive oxygen species via the action of superoxide dismutase, which converts to and and catalase and glutathione peroxidase, which detoxify Excessive levels of decreased cardiac SR content by lowering SR pump activity ⁷⁶⁸ and displacing calmodulin from RyR2, resulting in increased release of from SR. ⁷⁶⁹

Reactive oxygen species can be formed by several mechanisms,

including the mitochondrial electron transport chain, xanthine oxidase and

NADH oxidase. In skeletal and cardiac muscle, SR-associated NADH oxidases are potential sources of production. NADH can stimulate RyR1 activity. ⁷⁷⁰ Activation of RyR1 was observed in the presence of mitochondrial electron transport inhibitors but was inhibited by superoxide dismutase, suggesting that a NADH oxidase activates RyR1 by producing The skeletal muscle RyR1 has an N-terminal oxidoreductase-like domain and binds to sites other than the ATP binding site. ¹⁰³ However, it remains to be established that the oxidoreductase-like domain is enzymatically active. Contrary to the results of Xia et al. ⁷⁷⁰ , Baker et al. ¹⁰³ observed that NADH had only minor effects on RyR1 activity.

In contrast to activation of RyR1, NADH decreased single RyR2 ion channel activity ⁷⁷¹ and SR release. ⁷⁷² Furthermore, NADH inhibition of RyR2 was not affected by superoxide dismutase and thus was independent on production. ⁷⁶⁸ Another remarkable difference was that mitochondrial electron transport inhibitors relieved the inhibition of RyR2 activity by NADH. ⁷⁷² How then does NADH inhibit RyR2? RyR2 may sense changes in redox potential that may be influenced by either or cellular respiration or both. Alternatively, NADH may transfer reducing equivalents to RyR2 to reduce regulatory thiol groups. Such a mechanism would require a high local NADH concentration because the inhibition of RyR2 (as measured by sparks in permeabilized cells and in single channel measurements) was only seen at ^768,771 By comparison, the cytosolic in aerobically perfused working hearts is low (~0.1%). ⁷⁷³ Even during the extreme anaerobic condition of sustained ischemia, the cytosolic ratio ⁷⁷³ is below that required to inhibit SR release. ^768,771 It may be of biological or experimental interest that thiols will add directly to nicotinamide at the 1:4 position of the nicotinamide ring of

MODULATION OF RYRS BY NO AND RELATED NITROGEN SPECIES

NO is formed by one of three major classes of nitric oxide synthases

(NOSs): endothelial (eNOS), neuronal (nNOS) and inducible (iNOS). In

skeletal muscle, the predominant isoform, nNOS, is targeted to neuronal

postsynaptic densities by interacting with postsynaptic proteins, and to

specialized invaginations of the sarcolemma, called caveolae, by binding to

caveolin 3 and dystrophin-associated proteins. ⁷⁷⁴ In cardiac muscle, eNOS is

the predominent NOS isoform. The enzyme is primarily expressed in the

coronary and endocardial endothelia but has also been localized to caveolae

and mitochondria of cardiomyocytes. Immunoelectron microscopy showed

association of nNOS with cardiac but not skeletal muscle SR membranes. ⁷⁷⁵ A selective association of nNOS with RyR2 was described and the use of and mice demonstrated that nNOS has a specific role in regulating SR release. ⁷⁷⁶ RyR1 ⁶¹² and RyR2 ⁶¹¹ are endogenously S- nitrosylated, supporting a role of NO in skeletal and cardiac muscle EC coupling. The third isoform, iNOS, is transcribed in inflammatory cells and muscles in response to cytokines and bacterial endotoxins.

NO exerts its cellular effects via cGMP-dependent or -independent pathways. ⁷⁷⁷ In the cGMP-dependent pathway, the binding of NO to the heme group of guanylate cyclase results in enhanced production of intracellular cGMP and activation of cGMP-dependent kinase. The principal mechanism by which NO operates independently of cGMP is through S- nitrosylation, which occurs most often at a single cysteine residue within an acid-base or hydrophobic structural motif ⁷⁷⁸ (compounds such as glutathione which shuttles NO bioactivity in the form of GSNO may serve as intermediates). Pathological oxidation of RyR may involve other species, such as peroxynitrite which extensively oxidizes the RyRs ^611,779 and has been implicated in postischemic injury. ⁷⁸⁰

NO or NO related species have been reported to activate ⁷⁸¹ or inhibit ⁶⁶⁰ the skeletal RyR1. NO generated in situ from arginine by endogenous NOS and the NO donor S-nitroso-N-acetylpenicillamine reduced the rate of release from SR vesicles and the activity of single skeletal RyR1 channels incorporated in lipid bilayers. ⁶⁶⁰ In contrast, Stoyanovsky et al. ⁷⁸¹ found that NO, delivered in the form of NO gas, and NO donors (NONOates, S- nitrosothiols) activated single RyR1 ion channels and release from SR vesicles. Aghdasi and colleagues ⁷⁶³ reported that NO donors had no detectable effects at low concentrations, but at higher concentrations were able to block intersubunit cross-links and prevented activation of the skeletal RyR channel by the disulfide inducing agent diamide. In two other studies, NO-generating agents both activated and inhibited RyR1 in lipid bilayers, depending on donor concentration, membrane potential, and the presence of channel agonists and other sufhydryl modifying reagents. ^782,783

In vitro, S-nitrosylation of the skeletal muscle RyR1 by NO depends on NO concentration and tension. ⁶¹² Physiological concentrations

of NO S-nitrosylated and activated RyR1 at but not in ambient air Changes in oxygen tension oxidized/reduced as many as 6-8 thiols in each RyR1 subunit, which may explain the responsiveness of RyR1 to NO at tissue but not ambient air. ⁶¹² In contrast, other NO related molecules such as 3-morpholinosydnonimine (SIN-1), S-nitrosoglutathione or NOC12, which generate a variety of reactive nitrogen oxides can activate RyR1 independently of oxygen tension.

NOC-12 activated by S-nitrosylation, ⁶¹⁷ SIN-1 by oxidation of thiols, ⁶¹⁷ and

S-nitrosoglutathione by S-nitrosylation/oxidation ⁶¹⁷ and S-thionylation. ⁶¹³ Site-directed mutagenesis studies demonstrated that at physiological concentrations, NO specifically S-nitrosylates Cys3635 among ~50 free cysteines per RyR1 subunit. ⁶¹⁶ C3635 is located in the CaM binding domain of RyR1, which provides an explanation for the observation that NO transduces its functional effect only in the presence of calmodulin (Fig. 20- 3).

Figure 20-3. NO modulates single RyR1 channel activity under physiological muscle tension in the presence of calmodulin. A. Channel currents of two RyR1s, shown as downward deflections from closed (c--) levels, were recorded in a lipid bilayer chamber pre- equilibrated to with voltage held at -35 mV. Top trace: control with 100 nM CaM and 4 mM free channel open probability (Po) = 0.06; second trace: 2 min after the addition of 0.75 mM NO to cytosolic side, Po=0.13. B. As compared to controls (open bars), 0.75 mM NO (filled bars) significantly increased Po of RyR1 (*, p < 0.05) at

but not NO did not significantly alter Po of RyR1 at of ~10 mm Hg in the presence of 1 mM myosin light chain-derived calmodulin binding peptide that dissociates endogenously bound calmodulin (-CaM). From Eu et al.

A recent study addressed the role of NO and tension in controlling SR release and contractile force generated by skeletal muscle. Eu et al. ⁷⁶⁴ compared the role of on contractility of extensor digitorum longus muscle and on transients and cell shortening of flexor digitorium brevis myocytes, both prepared from normal and nNOS-deficient mice. Included were measurements at 95% because studies with isolated skeletal muscle fibers are usually done at high concentrations.

The measurements revealed an enhancement in muscle performance and the

amplitude of the transients at low physiological tension, which depended on endogenous nNOS activity. At 95% which produced a high non-physiological core muscle force production was enhanced but response to NO was diminished. The findings show that contractility depends on tension and NO modulates dependence.

NO also fulfills many of the criteria of a physiological modulator of cardiac muscle EC coupling. 776,777,784,785

In in vitro studies, NO and NO- related molecules activated ^611,781 or inactivated ⁶⁶¹ RyR2, leading to reversible or irreversible alteration of RyR2 channel activity. The two NO-related species GSNO and CysNO S-nitrosylated and/or oxidized up to 12 sites per RyR2 subunit in ambient tension. ⁶¹¹ The level of S-nitrosylation appeared to be dependent on channel conformation because a RyR inhibitor, reduced SNO content. 3-Morpholinosydnonimine (SIN-1), which produces effector peroxynitrite, activated and oxidized but did not S-nitrosylate RyR2.

CONCLUDING REMARKS

Current evidence suggests that the activity of the RyRs depends critically on redox state both at the whole cell level and at the level of the RyRs themselves: oxygen tension, GSH/GSSG, and NO can each modulate channel activity. Future work will need to address the isoform and tissue specificity of interaction of the RyRs with redox active molecules, the molecular basis of this specificity, and how “redox” regulation of the RyRs by relates to the roles of NO and in overall muscle function.

ACKNOWLEDGMENTS

Support by National Institutes of Health grants AR18687 and HL27540 is

gratefully acknowledged.