Oxygen-coupled Redox Regulation of the Skeletal Muscle Ryanodine Receptor/Ca²⁺ Release Channel (RyR1)

Background: Within the skeletal muscle Ca²⁺ release channel RyR1, S-oxidation and S-nitrosylation of allosterically linked Cys residues are coupled to oxygen tension (pO₂).

Results: Mass spectrometry identifies multiple Cys residues as oxidized at high versus low pO₂.

Conclusion: Endogenous H₂O₂ catalyzes pO₂-coupled disulfide formation within an allosteric Cys network that gates S-nitrosylation.

Significance: Dynamic disulfide formation subserves physiological redox regulation of RyR1.

Abstract

In mammalian skeletal muscle, Ca²⁺ release from the sarcoplasmic reticulum (SR) through the ryanodine receptor/Ca²⁺-release channel RyR1 can be enhanced by S-oxidation or S-nitrosylation of separate Cys residues, which are allosterically linked. S-Oxidation of RyR1 is coupled to muscle oxygen tension (pO₂) through O₂-dependent production of hydrogen peroxide by SR-resident NADPH oxidase 4. In isolated SR (SR vesicles), an average of six to eight Cys thiols/RyR1 monomer are reversibly oxidized at high (21% O₂) versus low pO₂ (1% O₂), but their identity among the 100 Cys residues/RyR1 monomer is unknown. Here we use isotope-coded affinity tag labeling and mass spectrometry (yielding 93% coverage of RyR1 Cys residues) to identify 13 Cys residues subject to pO₂-coupled S-oxidation in SR vesicles. Eight additional Cys residues are oxidized at high versus low pO₂ only when NADPH levels are supplemented to enhance NADPH oxidase 4 activity. pO₂-sensitive Cys residues were largely non-overlapping with those identified previously as hyperreactive by administration of exogenous reagents (three of 21) or as S-nitrosylated. Cys residues subject to pO₂-coupled oxidation are distributed widely within the cytoplasmic domain of RyR1 in multiple functional domains implicated in RyR1 activity-regulating interactions with the L-type Ca²⁺ channel (dihydropyridine receptor) and FK506-binding protein 12 as well as in “hot spot” regions containing sites of mutation implicated in malignant hyperthermia and central core disease. pO₂-coupled disulfide formation was identified, whereas neither S-glutathionylated nor sulfenamide-modified Cys residues were observed. Thus, physiological redox regulation of RyR1 by endogenously generated hydrogen peroxide is exerted through dynamic disulfide formation involving multiple Cys residues.

Introduction

Redox-based post-translational regulation of protein function is exerted principally through modification of Cys thiol side chains. S-Nitrosylation provides the best characterized example with a large number of endogenous substrates identified in the context of cellular signal transduction along myriad pathways (1, 2). Accumulating evidence also points to a potentially broad regulatory influence of S-oxidation, catalyzed by reactive oxygen species (effectively, hydrogen peroxide (H₂O₂)), which could in principle be exerted through a number of independent or coupled oxidative modifications: the reversible and dynamic formation of disulfide bonds, sulfenic (and possibly sulfinic) acid, and sulfenamide as well as S-glutathionylation and S-sulfhydration (2–4). A broad role for physiological S-oxidation would be consistent with the ubiquitous expression across multicellular organismal phylogeny and cell types of the NADPH oxidases (Noxs),³ which function to generate reactive oxygen species (5). However, although there have been significant recent advances (6, 7), there are relatively few examples in which mediation or modulation of physiological signal transduction by S-oxidation has been characterized fully, that is with respect to the (enzymatic) source of endogenous H₂O₂ and the generative stimulus, the protein targets subject to S-oxidation and the specific Cys residues modified within those targets, the chemical nature of the modification, the effect of modification on protein function, and the consequences of altered protein function in the cellular milieu.

The ryanodine receptor/Ca²⁺ release channel (RyR), which serves as the essential source of Ca²⁺ release from the sarcoplasmic reticulum (SR) that mediates excitation-contraction coupling in skeletal and cardiac striated muscle, has emerged as a paradigmatic example of redox regulation of protein function through Cys-directed post-translational modification (8–16). RyRs, which function as tetramers, are the largest channel proteins described. Monomers of RyR1, the predominant form of RyR in skeletal muscle, comprise about 5000 amino acids (∼565 kDa). One hundred of those residues are Cys, and half of those Cys residues on average are in the reduced form (sulfhydryl bearing a free thiol) under basal conditions (8, 16). Studies using application in vitro of exogenous redox agents (reduced glutathione, oxidized glutathione, S-nitrosoglutathione, nitric oxide (NO), and NO donors (NOC and NOR)) have reported modification of multiple Cys residues within RyR1 by S-glutathionylation, S-nitrosylation, and unspecified S-oxidation (8–11, 13, 14, 17, 18). It has also been reported that tetanic stimulation of skeletal muscle elicits Nox-dependent production of H₂O₂ (19) and that RyR1 can be modified by S-glutathionylation via Nox2 localized to transverse tubule membranes (20). Increasing evidence thus supports the possibility that Nox-derived reactive oxygen species may play a physiological role in skeletal muscle excitation-contraction coupling.

Physiological S-oxidation of RyR1 is coupled to endogenous S-nitrosylation of the channel, providing a paradigmatic example of allosteric cross-talk by redox-based modifications. More specifically, under physiological conditions in situ, it has been shown that RyR1 is modified by S-nitrosylation that reflects the production of NO by neuronal nitric-oxide synthase (8, 10, 12) and by S-oxidation that is dependent upon the production of H₂O₂ by Nox4 (16). Physiological S-oxidation is governed by a redox cycle intrinsic to the SR that is coupled to muscle pO₂: in isolated SR (SR vesicles) at relatively high pO₂ (21% O₂), an average of about six to eight free Cys thiols are oxidized consequent upon H₂O₂ production by Nox4, and the transition to relatively low pO₂ (1% O₂) is accompanied by reduction that reflects suppressed H₂O₂ production and the operation of an as yet unspecified reductive mechanism (8, 16). Oxygen-coupled S-oxidation activates RyR1, enhances Ca²⁺ release, and gates S-nitrosylation, which is blocked at high versus low pO₂. S-Nitrosylation at low pO₂, which also activates RyR1, targets a single Cys residue (Cys³⁶³⁵) that is not a member of the set of Cys residues oxidized at high versus low pO₂ (10, 13). Thus, S-oxidation allosterically regulates RyR1 activity, and S-oxidation and S-nitrosylation operate together to modulate Ca²⁺ release through RyR1 over a range of pO₂ that extends from the low pO₂ that characterizes working muscle to oxidative stress.

The analysis in skeletal muscle SR provides an example of physiological S-oxidation in which the source of H₂O₂ (Nox4) and the generative stimulus (pO₂), the specific protein target (RyR1), and the effects of modification on protein function (activation of RyR1 and enhanced Ca²⁺ release from the SR) have been described. However, although it has been shown that only a relatively small subset of Cys residues on average are subject to pO₂-coupled S-oxidation, neither the identity of the targeted Cys residues nor the nature of the oxidative modification(s) are known. We developed a mass spectrometry-based scheme for analysis of regulated protein S-oxidation that we applied to identify the sites of physiological S-oxidation within RyR1 and the form of modification. Here we identify 13 Cys residues subject to oxidation at high versus low pO₂ in isolated SR and eight additional Cys residues subject to oxidation at high versus low pO₂ but only when NADPH levels are supplemented to enhance Nox4 activity. pO₂-coupled S-oxidation targets Cys residues that are distributed widely within the cytoplasmic domain of RyR1 in multiple functional domains. Furthermore, we show that disulfide formation is the likely form of S-oxidation.

EXPERIMENTAL PROCEDURES

Preparation of SR Vesicles and Purification of RyR1

All preparations here and below utilized hind limb muscle from rabbit. SR vesicles were prepared essentially as described (21). Briefly, muscle was homogenized in buffer containing 20 mm Hepes, pH 7.4, 2 mm EDTA, 0.2 mm EGTA, 0.3 m sucrose, and protease inhibitors (100 nm aprotinin, 20 μm leupeptin, 1 μm pepstatin, 0.2 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine). Homogenates were centrifuged at 9,200 × g for 20 min, and the resultant supernatant was centrifuged at 100,000 × g for 1 h. The resultant pellet (membrane fraction) was resuspended and fractionated on a continuous 20–45% (w/v) sucrose gradient by centrifugation at 100,000 × g for 14 h. Fractions containing SR vesicles were eluted, collected by centrifugation at 120,000 × g, resuspended, aliquoted, and stored in liquid nitrogen. RyR1 was purified from SR vesicles solubilized with CHAPS by sucrose density gradient centrifugation as described (22). Protein concentrations were determined with a bicinchoninic acid-based assay.

Quantification of Total RyR1 Free Thiols (Sulfhydryls)

The free thiol content of RyR1 was quantified by monobromobimane (MBB) fluorescence. As described (8, 16), SR vesicles held at 21% or 1% O₂ (glove box) were exposed to MBB in the presence of 10 μm Ca²⁺ prior to isolation of RyR1 as above and quantification of fluorescence. When used, NADPH (1 mm) was added 30 min before MBB.

Isotope-coded Affinity Tag (ICAT) Labeling

SR vesicles were incubated in 50 mm phosphate buffer containing 10 μm CaCl₂, pH 7.5 at 1 or 21% O₂ for 30 min. When used, 10 units/ml polyethylene glycol (PEG)-coupled catalase, 20 μm diphenyleneiodonium (DPI), or 1 mm NADPH was added at the beginning of the incubation. Samples were then incubated with 10× excess (over calculated Cys content) ICAT reagent for 4 h in the presence of 2% SDS. Light ICAT (¹²C) and heavy ICAT (¹³C) were used to label samples incubated at low pO₂ (1% O₂) or high pO₂ (21% O₂), respectively. Samples (1 and 21% O₂) were then mixed 1:1, and excess ICAT was removed by size exclusion filtration through a P6 gel column. Proteins were separated by reducing SDS-PAGE, and the band containing RyR1 was excised and incubated with DTT followed by iodoacetamide (IA) to alkylate previously oxidized Cys residues (23). In-gel digestion by trypsin or chymotrypsin was carried out overnight in 100 mm ammonium bicarbonate at 37 °C. ICAT-labeled peptides were purified on avidin affinity columns, and the biotin moiety was cleaved (AB Sciex). Both ICAT-labeled peptides (avidin-bound) and unlabeled peptides (flow-through) were collected, desalted with a C₁₈ UltraMicro Tip Column, and resuspended in 0.1% (v/v) formic acid for analysis by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). The ICAT-based analytic scheme is illustrated in Fig. 1A.

FIGURE 1.

The MS-based analytic scheme utilized in the identification of sites of pO₂-coupled redox regulation of RyR1. A, to identify pO₂-sensitive Cys residues within RyR1, SR vesicles maintained at 21% O₂ or 1% O₂ are solubilized in SDS, and free thiols are labeled with heavy or light ICAT, respectively. Samples are then combined, and RyR1 is isolated by SDS-PAGE under reducing conditions. The band containing RyR1 is excised, and free thiols are alkylated with iodoacetamide followed by in-gel digestion with either trypsin or chymotrypsin. Eluted peptides are passed over an ICAT affinity column, and both bound peptides and the flow-through are analyzed by LC-MS/MS. Thus, for a given peptide, an increase in the light:heavy ratio at 1% O₂ versus 21% O₂ indicates Cys residues that are oxidized at high versus low pO₂. B, ICAT fully labels RyR1 free Cys thiols. SR vesicles maintained at 1% O₂ (low pO₂) or 21% O₂ (high pO₂) were solubilized in SDS, and free thiols were labeled with the fluorescent reporter MBB. Prior incubation with ICAT reagent eliminates MBB fluorescence, demonstrating that free thiols are quantitatively labeled by ICAT reagent. Note that MBB fluorescence is decreased at high versus low pO₂, indicating a loss of free thiols to S-oxidation (8, 16). Error bars represent S.E.

Dimedone Labeling

SR vesicles were incubated with 10 mm dimedone in 50 mm phosphate buffer containing 10 μm CaCl₂, pH 7.5 at room temperature for 1 h at 21% O₂. Proteins were separated by reducing SDS-PAGE, and the band containing RyR1 was excised and incubated with DTT followed by IA. Following in-gel digestion by trypsin or chymotrypsin, eluted peptides were desalted and analyzed by LC-MS/MS. Alternatively, muscle homogenates were incubated with 10 mm dimedone at 21% O₂ for 1 h prior to isolation of SR vesicles and analysis as above.

Preparation of RyR1 for Native Identification of Disulfide and Other Oxidative Modifications

SR vesicles were incubated with 50 mm phosphate buffer containing 10 μm CaCl₂, pH 7.5 at 1 or 21% O₂ for 20 min. Samples were then held at 1 or 21% O₂ and incubated with a 50-fold excess of IA or N-ethylmaleimide for 20 min at room temperature in the dark followed by the addition of 2% (w/v) SDS and incubation for an additional hour. Proteins were separated by non-reducing SDS-PAGE followed by excision of the band containing RyR1, in-gel digestion with trypsin or chymotrypsin, and analysis by LC-MS/MS.

Mass Spectrometric Analysis

LC-MS/MS was performed using an LTQ Orbitrap XL linear ion trap mass spectrometer (Thermo Fisher Scientific). Reverse-phase high performance liquid chromatography (HPLC) was conducted with an Ultimate 3000 HPLC system (Dionex) equipped with a Dionex C₁₈ Acclaim PepMap 100 column (0.075 × 150 mm) in a linear gradient of acetonitrile from 2 to 60% (v/v) for a period of 60 min at a flow rate of 0.3 μl/min. Spectra were acquired in the positive ionization mode by data-dependent methods consisting of a full MS scan in high mass accuracy Fourier transform MS mode at 60,000 resolution and MS/MS on the six most abundant precursor ions in collision-induced dissociation mode with a normalized collision energy of 35%. A dynamic exclusion function was applied with repeat count of 2, repeat duration of 45 s, exclusion duration of 15 s, and exclusion size list of 350. Peptides were identified with Mascot Daemon (version 2.3.0, Matrix Science), and the data were searched against the RyR1 primary sequence. The mass tolerance was set at 10 ppm for precursor ions and 0.8 Da for product ions. Carbamidomethylation or N-ethylmaleimide labeling of Cys residues, dimedone labeling of Cys residues and sulfenic acids (when used), and oxidation of Met residues were set as variable modifications. Other possible modifications of Cys, including sulfenation, sulfination, sulfonation, glutathionylation, and sulfenamide formation, were also tested. The identification of disulfide bonds was performed using the on-line version of MassMatrix software (24). The modification(s), including disulfide bond formation, suggested by the search engine were verified by manual examination of each tandem mass spectrum and compared with corresponding unmodified peptides. For ICAT labeling analysis, both light and heavy ICAT-labeled peptides were first identified using Mascot Daemon and then confirmed by manual examination, especially in the case of peptides containing multiple Cys residues and mixed labeling with IA and ICAT.

MS Data Analysis

Quantification of ICAT labeling was conducted using Mascot Distiller (Matrix Science). All reported peptides labeled with ICAT were identified with standard error <0.1 and correlation coefficient >0.95. The two peaks from heavy and light ICAT labeling were identified as separated by 9 Da. The ratio of light ICAT (labeled at 1% oxygen) to heavy ICAT (labeled at 21% oxygen) was calculated and averaged on the basis of peptide peak heights for each individual scan. The ratios for the same peptide but with different charge and methionine oxidation state were then averaged manually. For peptides with multiple Cys residues and mixed labeling by IA and ICAT, the ratio was manually calculated from the peptide peak heights extracted from the selected ion chromatogram. Standard errors were derived from replicated biological samples, different charge and methionine oxidation states, or multiple scanning. Quantification of disulfide was conducted manually. Two native reference peptides, A (LSLPVQFHQHFR) and B (GDGGEGEGEVQFLR), that appeared unmodified in each of several repeated experiments were chosen (25). Peaks from the same disulfide-containing peptide with different methionine oxidation states were added together. After normalization with respect to the two reference peptides at the different pO₂ values, relative disulfide abundance at high versus low pO₂ was calculated as an average of three biological replicates.

RESULTS

Identification of Cys Residues within RyR1 Subject to pO₂-dependent Oxidative Modification

RyR1 is an unusually large protein (∼5,000 amino acids) that contains 100 Cys residues. We developed an approach that allowed us to assess by mass spectrometric analysis the redox state of 93 of 100 Cys residues. In this approach (Fig. 1A), an RyR1-enriched subcellular fraction (SR vesicles) is solubilized in SDS under non-reducing conditions, and free thiols are labeled with ICAT reagent. The efficacy of ICAT labeling under the conditions used is demonstrated by the finding that incubation with ICAT reagent at either low or high pO₂ eliminates subsequent labeling by the thiol-specific agent MBB (Fig. 1B). RyR1 is then enriched by SDS-PAGE under reducing conditions followed by in-gel alkylation with IA of thiols freed by reduction and in-gel digestion with either trypsin or chymotrypsin. Digests are subjected to avidin affinity purification, and both bound and unbound peptides are analyzed by LC-MS/MS. The dual enrichment provided by selective ICAT labeling of Cys-containing peptides and gel purification resulted in coverage of 93% of RyR1 Cys residues when the results of trypsin and chymotrypsin digestion were combined.

After labeling SR vesicles held at low versus high pO₂ (1% O₂ versus 21% O₂) with light (¹²C) versus heavy (¹³C) ICAT reagent, respectively, followed by reduction and labeling with IA, RyR1 Cys residues fell into one of four principal groups. Seven Cys residues were not identified in our analysis (Cys¹⁴⁶, Cys¹⁴⁸⁹, Cys¹⁴⁹¹, Cys²⁶⁵¹, Cys⁴⁶⁵⁷, Cys⁴⁸⁷⁶, and Cys⁴⁸⁸²) (including additional experiments using digestion with Asp-N), although the predicted masses of the peptides containing those Cys residues were within the detectable limit (∼800–3,000 Da). Fifteen Cys residues were labeled with IA only and were therefore identified as fully oxidized (although reducible by DTT) at both low and high pO₂ (Fig. 2), consistent with their participation in “structural” disulfides. Remarkably, 78 of 93 identified Cys residues were detected with ICAT labeling and were thus present in some proportion in reduced form. Twenty-two of those were labeled with ICAT reagent but not IA (Fig. 2) and displayed a light:heavy ratio of ∼1:1, consistent with their presence in fully reduced form at both low and high pO₂ (Table 1). An additional two Cys residues detected with ICAT only (Cys⁵⁶⁶ and Cys⁷⁶²) were pO₂-sensitive (increased light:heavy ratio) (Table 2), although no IA-labeled form was detected presumably because the IA-labeled peptide was of low abundance (consistent with the existence of those Cys residues in predominantly reduced form at low pO₂).

FIGURE 2.

RyR1 Cys residues insensitive to pO₂. Following analysis according to the scheme illustrated in Fig. 1A, 72 Cys residues were identified as pO₂-insensitive, including a set of 37 Cys residues labeled with IA or ICAT only and therefore members of a fully oxidized or a fully reduced population, respectively. Fully oxidized Cys residues are likely to participate in structural disulfides with the exception of Cys⁵³⁷, which could be labeled with dimedone (see Table 5). Note that the analysis could not discriminate between multiple Cys residues located within a single peptide. In the four cases in which multiple Cys residues were present in a single peptide (Cys^65/66, Cys^206/208/209, Cys^604/607/609, and Cys^746/747), one Cys was ICAT-labeled, and the remainder were IA-labeled in a pO₂-insensitive fashion. The locations within RyR1 of fully oxidized or reduced Cys residues are indicated. Transmembrane domains; regions implicated in the interaction of RyR1 with the L-type Ca²⁺ channel (dihydropyridine receptor (DHPR)), FK506-binding protein 12 (FKBP12), and calmodulin (CaM); leucine/isoleucine zipper (LIZ) motifs; and the SPRY1 domain as well as hot spot regions implicated in malignant hypothermia/central core disease (MH, CCD) are delineated on the basis of the human RyR1 sequence (for a recent review, see Ref. 33). Quantitative data are provided in Table 1. Black circles, fully oxidized; red circles, fully reduced; half red-half black circles, fully oxidized or reduced.

TABLE 1.

pO₂-insensitive RyR1 Cys residues

Cys residues labeled with IA alone (fully oxidized at low and high pO₂) are not shown (see Fig. 2). Cys residues labeled with ICAT alone (fully reduced at high and low pO₂) are underlined. For all remaining pO₂-independent Cys residues, some proportions were detected with IA and with ICAT labeling, and L:H ICAT ratios were ∼1:1 at low or high pO₂ and at high pO₂ with NADPH supplementation. Note anomalous values for Cys³¹⁹³ and Cys³⁶³⁵ (see Fig. 3 and text). Standard error was derived from replicated biological samples (n = 2–5), multiple peptides with different charge and/or methionine oxidation, or multiple scanning. PEG-CAT, polyethylene glycol-coupled catalase; DPI, diphenyleneiodonium; —, data not available.

Cys	Control	DPI	PEG-CAT	NADPH	Cys	Control	DPI	PEG-CAT	NADPH
24	0.94 ± 0.06	1.10 ± 0.10	0.99 ± 0.04	1.12 ± 0.06	2436	1.16 ± 0.01	1.02 ± 0.01	0.97 ± 0.02	1.13 ± 0.17
230	1.15 ± 0.01	0.98 ± 0.02	1.05 ± 0.03	1.19 ± 0.02	2565	1.20 ± 0.12	1.00 ± 0.01	0.92 ± 0.02	1.14 ± 0.16
315	1.11 ± 0.05	0.90 ± 0.02	0.98 ± 0.01	0.98 ± 0.02	3014	1.07 ± 0.07	0.94 ± 0.01	0.93 ± 0.01	1.15 ± 0.07
346	1.00 ± 0.06	1.03 ± 0.02	0.94 ± 0.05	1.06 ± 0.04	3044	1.09 ± 0.04	0.99 ± 0.05	0.94 ± 0.01	1.12 ± 0.09
393	0.96 ± 0.05	0.98 ± 0.01	1.00 ± 0.01	1.14 ± 0.09	3067	1.09 ± 0.03	1.04 ± 0.01	0.91 ± 0.02	1.20 ± 0.33
811	1.01 ± 0.07	0.98 ± 0.01	0.98 ± 0.03	1.18 ± 0.08	3165	1.01 ± 0.01	0.98 ± 0.03	0.95 ± 0.03	1.05 ± 0.01
906	0.96 ± 0.02	0.97 ± 0.03	0.94 ± 0.01	1.23 ± 0.12	3170	1.12 ± 0.04	0.96 ± 0.02	0.92 ± 0.02	1.16 ± 0.11
937	1.07 ± 0.05	1.01 ± 0.03	0.97 ± 0.02	1.16 ± 0.16	3193	0.75 ± 0.01	0.80 ± 0.01	0.83 ± 0.01	1.09 ± 0.06
1040	1.07 ± 0.11	0.84 ± 0.01	0.89 ± 0.04	1.11 ± 0.04	3216	1.13 ± 0.12	0.99 ± 0.02	0.97 ± 0.02	1.14 ± 0.10
1192	0.98 ± 0.02	1.01 ± 0.01	0.99 ± 0.01	1.17 ± 0.10	3240	1.07 ± 0.08	0.95 ± 0.03	0.94 ± 0.05	1.09 ± 0.07
1217	1.17 ± 0.03	0.96 ± 0.04	0.95 ± 0.05	1.19 ± 0.20	3278	1.09 ± 0.19	0.98 ± 0.02	0.92 ± 0.08	1.12 ± 0.17
1269	1.11 ± 0.18	0.96 ± 0.01	0.95 ± 0.01	1.12 ± 0.06	3304	1.03 ± 0.13	1.02 ± 0.01	0.99 ± 0.02	1.08 ± 0.01
1303	1.05 ± 0.13	0.97 ± 0.01	0.97 ± 0.01	1.12 ± 0.02	3402	1.11 ± 0.05	1.01 ± 0.02	0.97 ± 0.02	1.04 ± 0.05
1518	1.21 ± 0.02	1.03 ± 0.02	0.96 ± 0.06	—	3525	1.14 ± 0.04	0.97 ± 0.01	0.96 ± 0.03	0.99 ± 0.09
1591	1.04 ± 0.04	0.99 ± 0.01	0.98 ± 0.01	1.13 ± 0.10	3635	1.10 ± 0.21	0.79 ± 0.02	0.77 ± 0.01	1.11 ± 0.01
1630	1.21 ± 0.02	—	—	—	3650	1.08 ± 0.04	0.98 ± 0.05	0.96 ± 0.01	1.05 ± 0.10
1647	1.06 ± 0.05	1.05 ± 0.03	0.94 ± 0.04	1.12 ± 0.12	3733	1.03 ± 0.06	1.00 ± 0.06	0.94 ± 0.03	1.13 ± 0.05
1724	0.98 ± 0.02	—	1.02 ± 0.04	—	3786	1.16 ± 0.05	1.03 ± 0.01	1.01 ± 0.02	0.98 ± 0.10
1781	0.94 ± 0.11	0.98 ± 0.02	0.95 ± 0.04	1.08 ± 0.05	3839	—	0.98 ± 0.05	0.91 ± 0.06	1.15 ± 0.06
1947	1.13 ± 0.02	0.99 ± 0.04	0.94 ± 0.04	1.09 ± 0.03	3918	—	1.16 ± 0.05	1.07 ± 0.02	1.07 ± 0.18
2042	1.17 ± 0.02	1.02 ± 0.02	0.96 ± 0.02	1.22 ± 0.14	3973	1.09 ± 0.11	1.05 ± 0.08	1.00 ± 0.04	1.05 ± 0.04
2158	1.15 ± 0.27	1.02 ± 0.04	0.83 ± 0.01	1.21 ± 0.21	4114	0.98 ± 0.02	1.01 ± 0.03	1.02 ± 0.04	1.12 ± 0.13
2232/2233	1.19 ± 0.02	0.95 ± 0.04	0.97 ± 0.04	0.96 ± 0.07	4645	1.07 ± 0.03	1.01 ± 0.02	—	—
2237	0.94 ± 0.14	—	—	—	4958	1.06 ± 0.02	1.00 ± 0.02	1.07 ± 0.01	—
2326	1.11 ± 0.12	0.94 ± 0.01	0.95 ± 0.03	1.08 ± 0.15	4961	1.21 ± 0.03	0.91 ± 0.07	1.02 ± 0.01	—
2363	1.09 ± 0.04	0.98 ± 0.01	0.98 ± 0.01	1.06 ± 0.09	5027	0.87 ± 0.01	1.02 ± 0.01	0.97 ± 0.01	1.08 ± 0.01

TABLE 2.

pO₂-sensitive RyR1 Cys residues

For most pO₂-dependent Cys residues, some proportions were detected with IA and with ICAT labeling, and ICAT L:H ratios ≥1:1.24 (L represents light ICAT, low pO₂; H represents heavy ICAT, high pO₂) indicate enhanced oxidation at high versus low pO₂. In two cases (indicated by underlining), only ICAT labeling was detected. In all cases, the increase in L:H ratio at high versus low pO₂ was eliminated by inhibiting NADPH oxidase activity with DPI or scavenging H₂O₂ with polyethylene glycol-coupled catalase (PEG-CAT) and was enhanced by supplementation with NADPH. Standard error was derived from replicated biological samples (n = 2–5), multiple peptides with different charge and/or methionine oxidation, or multiple scanning. —, data not available.

Cys	Control	DPI	PEG-CAT	NADPH
36	1.33 ± 0.02	1.04 ± 0.02	0.98 ± 0.03	1.42 ± 0.22
566	1.25 ± 0.04	1.04 ± 0.02	1.00 ± 0.03	1.47 ± 0.08
762	1.27 ± 0.05	—	1.03 ± 0.06	1.28 ± 0.23
845/854	1.28 ± 0.06	1.14 ± 0.02	1.06 ± 0.01	1.53 ± 0.28
1674	1.27 ± 0.08	1.03 ± 0.01	0.94 ± 0.08	1.56 ± 0.03
2305/2310	1.47 ± 0.05	0.84 ± 0.01	0.93 ± 0.08	1.30 ± 0.11
2555	1.27 ± 0.08	1.08 ± 0.03	0.94 ± 0.03	1.32 ± 0.17
2606	1.30 ± 0.08	1.22 ± 0.01	1.05 ± 0.02	1.33 ± 0.01
2611	1.39 ± 0.35	1.10 ± 0.01	0.86 ± 0.01	1.35 ± 0.01
2704	1.25 ± 0.03	—	0.98 ± 0.03	—
4238	1.24 ± 0.03	1.08 ± 0.01	0.98 ± 0.02	—

Each of the remaining 54 Cys residues were labeled with IA or ICAT reagent and identified as members of a population that contained both reduced and oxidized Cys residues at both low and high pO₂. The observed light ICAT labeling versus heavy ICAT labeling (L:H) ratios ranged from 0.75 to 1.47 with the large majority distributed from 0.94 to 1.21 (Fig. 3A). Greater relative abundance of light ICAT labeling versus heavy ICAT labeling identified Cys residues subject to oxidation at high versus low pO₂. Based upon an arbitrary threshold L:H of 1.24 (which in terms of tetrameric RyR1 would represent at the extremes a population of ∼25% of RyR where all subunits are modified or 100% of RyR where a single monomer is modified), the redox states of 13 Cys residues (L:H of 1.24–1.47) were coupled to pO₂ (Fig. 4 and Table 2). Importantly, as illustrated in Table 2, in every case examined, the increase in L:H ratio at high versus low pO₂ was eliminated when SR vesicles were exposed to 20% O₂ in the presence of either PEG-coupled catalase to remove H₂O₂ or the Nox inhibitor DPI, confirming the essential role of endogenous H₂O₂ production by SR-resident Nox4 (16).

FIGURE 3.

Effect of pO₂ on redox state of Cys residues within RyR1. A, SR vesicles held at low pO₂ (1% O₂) or high pO₂ (21% O₂) were labeled with light or heavy ICAT reagent, respectively, before isolation of RyR1, trypsin digest, and analysis by LC-MS/MS (see Fig. 1A). The graph includes the 71 Cys residues that were identified as ICAT-labeled under control conditions (low and high pO₂ with no exogenous additives) in more than one biological replicate and that were contained in peptides that included only a single Cys (allowing unambiguous identification). Light:heavy ratios >1 indicate Cys oxidation (loss of free thiol) at high versus low pO₂. A light:heavy cutoff value of 1.24:1 (indicated by the upper dotted line) yielded a set of 13 Cys residues. An asterisk indicates Cys³¹⁹³ and Cys³⁶³⁵, which exhibited anomalous behavior (see Table 1). B, Cys residues identified as subject to oxidation at high versus low pO₂ are listed with reference to their location within RyR1. Note that the L:H ratio was enhanced for all listed Cys residues by supplemental NADPH (see Table 2), but that the L:H ratio for Cys residues listed in the right-hand column was ∼1:1 in the absence of supplemental NADPH; that is, pO₂-dependent oxidation required supplemental NADPH (see Table 3). Identified disulfides (2305/2310 and 2606/2611) are indicated by brackets (see Figs. 5 and 6).

FIGURE 4.

An illustrative example of ICAT-based identification and quantification of pO₂-coupled oxidation of RyR1 Cys (Cys³⁶). Mass spectra of RyR1 tryptic peptide ³⁵LCLAAEGFGNR⁴⁵ (M²⁺) derived from SR vesicles incubated at 1% O₂ or 20% O₂ (A–D) with the addition of PEG-coupled catalase (CAT; 10 units/ml) (B), DPI (100 μm) (C), or NADPH (1 mm) (D). In A–D, the left-hand and right-hand peaks represent labeling by light (at 1% O₂) or heavy (at 20% O₂) ICAT (difference of 4.5 m/z), respectively. The L:H ratio of 1.26 at 1% O₂ versus 20% O₂ (A) indicates a 26% increase in the yield of peptides containing oxidized Cys³⁶ at high versus low pO₂, and this difference is eliminated by diminishing H₂O₂ with catalase (B) or NADPH oxidase activity with DPI (C). The NADPH-coupled increase in L:H ratio to 1.55 (D) indicates increased oxidation when pO₂-dependent NADPH oxidase activity is enhanced. E, peptide MS/MS spectra identify the site of ICAT labeling as Cys³⁶.

It is of note that the two Cys residues exhibiting anomalous behavior, Cys³¹⁹³ and Cys³⁶³⁵, include the identified site of endogenous S-nitrosylation (Cys³⁶³⁵) (Fig. 3A and Table 1). Cys³⁶³⁵ is pO₂-insensitive (L:H ∼ 1 under control conditions), consistent with the previous finding that Cys³⁶³⁵ is not subject to pO₂-coupled S-oxidation (13). However, the L:H ratio drops to ∼0.78 when H₂O₂ is removed at high pO₂ (Table 1). One possible explanation for this finding is that, inasmuch as some proportion of Cys³⁶³⁵ is present in isolated SR vesicles in the S-nitrosylated form (not shown), enhanced denitrosylation (26) at high pO₂ in the absence of H₂O₂ (as in the hypothetical case of a denitrosylase that is inhibited by reactive oxygen species) would increase ICAT labeling at high versus low pO₂ and thereby decrease the L:H ratio.

We reported previously that both generation of H₂O₂ and RyR1 activity in SR vesicles were enhanced at 21 versus 1% oxygen and that these effects were associated with pO₂-coupled oxidation of a set of thiols within RyR1 (16). In addition, we reported that supplementation with NADPH (to enhance Nox4 activity) enhanced H₂O₂ production (16). Here we confirmed directly that the loss of free thiols within RyR1 in SR vesicles exposed to high versus low pO₂ was enhanced by addition of NADPH (Table 3). When SR vesicles were incubated with NADPH (1 mm), the L:H ratios were also enhanced for 6 of 11 tested Cys residues of the 13 Cys residues that were oxidized at high versus low pO₂ (Fig. 4 and Table 2). Furthermore, addition of NADPH resulted in an increase in L:H ratio from ∼1:1 to >1.24:1 in a population of eight additional Cys residues (Fig. 3B; Table 4). Thus, 21 Cys residues within RyR1 are subject to pO₂-coupled redox regulation.

TABLE 3.

Oxidation of RyR1 Cys thiols at high pO₂ is enhanced by NADPH

In SR vesicles incubated at 1% O₂ or 20% O₂ and in the presence or absence of supplementary NADPH (1 mm), RyR1 free thiols were labeled with MBB prior to purification by density gradient centrifugation and fluorescence quantification. Values are mol of free thiol/mol of RyR1. Note that about five free thiols on average are oxidized at high versus low pO₂ and that oxidation is enhanced by NADPH at high but not at low pO₂. p values are from paired t tests (n = 5).

	High pO2 (21% O2)	Low pO2 (1% O2)
Control	35.30 ± 1.56	40.31 ± 1.59
+NADPH	32.98 ± 1.42	39.42 ± 2.07
p value vs. control	<0.05	0.41

TABLE 4.

NADPH-dependent oxidation of Cys thiols in RyR1 at high pO₂

In SR vesicles incubated at 1% O₂ or 20% O₂ and in the presence or absence of supplementary NADPH (1 mm), a set of Cys residues was identified whose members were oxidized at high versus low pO₂ only in the presence of supplemental NADPH (L:H ratios ≥1:1.24) (L represents light ICAT, low pO₂; H represents heavy ICAT, high pO₂). All of these Cys residues were detected with both IA and ICAT labeling. Standard errors are derived from replicated biological samples (n = 2–5), peptides with different charge and methionine oxidation, or multiple scanning. PEG-CAT, polyethylene glycol-coupled catalase. —, data not available.

Cys	Control	DPI	PEG-CAT	NADPH
120	1.14 ± 0.08	0.86 ± 0.02	0.98 ± 0.01	1.24 ± 0.21
253	1.07 ± 0.11	0.97 ± 0.01	0.99 ± 0.01	1.26 ± 0.35
305	1.13 ± 0.23	0.89 ± 0.04	0.90 ± 0.03	1.27 ± 0.09
490	1.10 ± 0.05	0.96 ± 0.05	0.90 ± 0.05	1.55 ± 0.07
1686	1.10 ± 0.02	1.04 ± 0.03	0.97 ± 0.01	1.35 ± 0.19
2021	1.02 ± 0.04	0.97 ± 0.01	0.94 ± 0.01	1.27 ± 0.08
3892	1.18 ± 0.01	1.03 ± 0.04	0.99 ± 0.01	1.25 ± 0.16
4663	—	1.03 ± 0.04	—	1.31 ± 0.06

Identification of the Form of pO₂-coupled S-Oxidation

To investigate the form of pO₂-coupled S-oxidation, we incubated SR at low or high pO₂, blocked free thiols with IA or N-ethylmaleimide, and then purified RyR1 by non-reducing SDS-PAGE prior to trypsin or chymotrypsin digest and analysis by LC-MS/MS. We did not detect S-glutathionylation at either low or high pO₂. Western blot analysis of intact RyR1 using anti-glutathione antibodies also failed to detect S-glutathionylation, although S-glutathionylation of RyR1 was detected by Western blot when SR vesicles were incubated with oxidized glutathione as a positive control (data not shown). We also did not detect sulfenamide (27), and in addition, neither sulfenic (SOH) nor sulfinic acid (SO₂H) was detected. We identified the sulfonic acid form (SO₃H) of Cys¹¹⁹² and Cys³⁰⁶⁷, but quantification revealed that sulfonation was not pO₂-coupled (not shown).

Intrapeptide disulfide linkages were identified between Cys²³⁰⁵ and Cys²³¹⁰ and between Cys²⁶⁰⁶ and Cys²⁶¹¹ (Figs. 3B, 5, and 6). Both disulfide pairs were identified in each of three separate biological samples. In both cases, Cys^2305/2310 and Cys^2606/2611 could also be alkylated by N-ethylmaleimide or IA at both low and high pO₂ (Fig. 5). Quantification of disulfide formation with a native peptide reference method (see “Experimental Procedures”) (25) yielded H:L ratios of 1.23 ± 0.16 and 1.45 ± 0.04 (n = 3 experiments) for Cys^2305/2310 and Cys^2602/2611, respectively. We did not detect disulfide formation between Cys residues located in separate peptides, but theoretical analysis of the results of trypsin/chymotrypsin digest indicates that very few composite peptides resulting from disulfide formation would possess a mass low enough to allow their detection by MS/MS.

FIGURE 5.

pO₂-coupled disulfide formation within RyR1 (Cys^2305/2310). A–D, MS/MS spectra illustrate the four forms of peptide ²²⁹⁸VVSYLAGCGLQSCPMLLAK²³¹⁶ observed. An intrapeptide disulfide between Cys²³⁰⁵ and Cys²³¹⁰ (A) was also seen in conjunction with oxidation (Ox) of Met²³¹² (B). IA alkylation of Cys²³⁰⁵ and Cys²³¹⁰ (C) also seen in conjunction with oxidation (Ox) of Met²³¹² (D) indicates that both Cys residues were in the reduced state prior to initial blocking by alkylation. Note that all four forms are detected in samples prepared from SR vesicles held at high or low pO₂ but that the disulfide form is more abundant (1.23-fold) in samples prepared at high pO₂ (see “Results”).

FIGURE 6.

Schematic illustration of the distribution within RyR1 of Cys residues subject to pO₂-coupled redox modification. Among the 100 Cys residues of rabbit RyR1, color-coding identifies Cys residues unaffected by pO₂ (n = 72; black circles), oxidized at high versus low pO₂ (enhanced by exogenous NADPH) (n = 13; red circles), and oxidized at high pO₂ only with addition of exogenous NADPH (n = 8; green circles). Cys residues not detected in our analysis are also indicated (n = 7; blue circles). Identified disulfides formed more abundantly at high versus low pO₂ are indicated by brackets (Cys²³⁰⁵-Cys²³¹⁰ and Cys²⁶⁰⁶-Cys²⁶¹¹), and for reference, the previously identified site of S-nitrosylation is indicated (Cys³⁶³⁵). The following functional domains within RyR1 are delineated: transmembrane domains; regions implicated in the interaction of RyR1 with the L-type Ca²⁺ channel (dihydropyridine receptor (DHPR)), FK506-binding protein 12 (FKBP12), and calmodulin (CaM); leucine/isoleucine zipper (LIZ) motifs; and the SPRY1 domain as well as hot spot regions implicated in malignant hypothermia/central core disease (MH, CCD).

Although we did not observe sulfenic acid at either low or high pO₂, we considered the possibility that sulfenic acid might represent a transient intermediate en route to disulfide formation. We did not identify sulfenic acid by dimedone labeling (28) following incubation of SR vesicles at low or high pO₂. However, if muscle homogenates were incubated with dimedone (in room air; 21% O₂) prior to the isolation of SR vesicles and purification of RyR1, then 13 dimedone-modified Cys residues were detected (Table 5). Eleven of those 13 Cys residues were not among the set of pO₂-sensitive Cys residues (listed in Tables 2 and 4). However, Cys³⁶ and Cys¹²⁰ were identified as both dimedone-labeled and pO₂-sensitive, consistent with a role for sulfenic acid as a transient intermediate to higher oxidation, presumably disulfide formation.

TABLE 5.

Dimedone labeling of Cys thiols in RyR1

Muscle homogenates were incubated with dimedone prior to preparation of SR vesicles and analysis. Dimedone labeling (red) was identified by MS/MS. Mass is given as Daltons.

DISCUSSION

It has been established that the redox state of pO₂-sensitive RyR1 Cys residues is regulated by a cycle of dynamic S-oxidation and reduction that operates within the SR, but the identity of pO₂-sensitive Cys residues has remained unknown (16, 17). We show here that pO₂-coupled redox regulation of RyR1 is exerted through S-oxidation of 21 Cys residues that are distributed widely within RyR1. Previous reports based upon thiol labeling have reported pO₂-coupled oxidation of a set of approximately six to eight thiols on average (8). The analytic scheme used here based upon ICAT labeling and mass spectrometric analysis revealed that this set of thiols represents substoichiometric oxidation of a larger set. (Indeed, only two of the Cys residues we identified as pO₂-sensitive appeared to be fully reduced at low pO₂, and more generally, 54 of the 93 Cys residues we identified comprise mixed populations of reduced and oxidized Cys residues at both low and high pO₂). In the best characterized cases of redox regulation of protein function by S-oxidation, including most prominently protein-tyrosine phosphatases, modification preferentially targets a single, active site Cys within a minor population of protein (27). Thus, our results provide an apparently novel example of multisite redox regulation by physiological S-oxidation.

Physiological S-Oxidation of Cys Residues in RyR1

There are few analyses of oxidative modification of protein function in situ that have identified the source and nature of oxidizing equivalents, the protein targets and Cys residues modified, the nature of the modification, and the consequences of modification for protein function. Skeletal muscle RyR1, which contains 100 Cys residues, has long provided a model of Cys-based redox regulation. In particular, it has been reported that Nox2 is localized to transverse tubule membranes in skeletal muscle and that addition of NADPH to isolated triads (containing transverse tubule as well as SR membranes) results in the glutathionylation of RyR1 (20). However, we did not identify S-glutathionylated Cys residues within RyR1 following pO₂-coupled activation of RyR1, consistent with previous work showing that Nox2 may play a role in tetanic contraction but is not a source of pO₂-coupled reactive oxygen species production (16). In contrast, Nox4 was identified previously as the source of pO₂-coupled S-oxidation of RyR1 that activates RyR1 to enhance Ca²⁺ release from the SR (16). Here, in addition to identifying pO₂-sensitive Cys residues within RyR1, we identified disulfide as a stable product of S-oxidation but not other potential, reversible oxidative modifications, including S-glutathionylation, sulfenic acid, sulfinic acid, or sulfenamide (a form of modification within protein-tyrosine phosphatases (29, 30)). Thus, glutathionylation and disulfide modification may represent signatures of contraction- and oxygen-regulated RyR1 activity, respectively.

Previous studies using mass spectrometric analysis but relying primarily upon the administration of exogenous oxidants reported modification of multiple Cys residues within RyR1 by glutathionylation or unidentified S-oxidation as well as (by inference) disulfide formation (14). Although it is not possible to deduce the coverage of RyR1 Cys residues in previous analyses, remarkably, none of the Cys residues reported previously to be oxidatively modified are among the set of Cys residues shown here to be subject to physiological, pO₂-coupled S-oxidation with the exception of Cys³⁶, which was identified previously as a possible participant in disulfide formation catalyzed by the addition of high concentrations of H₂O₂ (5 mm) (14). Conversely, with the exception of Cys³⁶, Cys residues identified here as pO₂-sensitive do not appear among the Cys residues reported to be oxidatively modified by exogenous agents (14), although Cys^2606/2611, which we identified as participating in a pO₂-sensitive disulfide, were identified as hyperreactive by selective labeling with a thiol-reactive coumarin (31). Interestingly, a recent crystal structure of the N-terminal domain of RyR2 in which Cys³⁶ is conserved indicated a conformation in which Cys³⁶ could form a vicinal pair with Cys⁶⁵ (32). However, we did not identify Cys⁶⁵ as pO₂-sensitive.

Generally, studies of oxidative modification of Cys residues within proteins that are involved in cellular signal transduction have relied on the in vitro application of oxidants, principally H₂O₂, to address the form of modification. In the much studied case of protein-tyrosine phosphatases, in vitro analysis has identified multiple possible oxidative modifications, including intramolecular disulfides and sulfenamide (27). However, there is little evidence bearing on the nature of oxidative modification of Cys residues induced by endogenously generated H₂O₂, and more generally, the nature of oxidative modification that may subserve physiological signal transduction remains largely unexplored (2). Our results indicate that disulfide formation serves as a principal dynamic modification of Cys residues within RyR1 that is governed by endogenous oxidative and reductive mechanisms to regulate RyR1 function allosterically.

Our finding that ∼21 Cys residues contribute to a total of 6–8 mol of thiol/mol of RyR monomer that are subject to pO₂-coupled oxidation is in keeping with the evolving understanding that regulatory post-translational modifications of proteins are typically present in substoichiometric amounts. Subpopulations of proteins may be subject to differential regulation, and sub-stoichiometric oxidative modification of RyR1 may be viewed as providing a selective gain of function. However, we cannot determine whether a subpopulation of RyR1 is subject to privileged modification (e.g. by Nox4 that may be associated with a fraction of RyR1) or whether redox modifications are in fact evenly distributed across the entire population of RyR1 in which case oxidation of single subunits of the RyR1 tetramer may cooperatively influence the activity of other subunits to subserve pO₂-coupled regulation of calcium release.

The Distribution of Physiologically S-Oxidized Cys residues in RyR1

The localization of identified Cys residues within RyR1 is illustrated in Fig. 6. All pO₂-sensitive Cys residues are located in cytoplasmic portions of RyR1 and with a single exception (Cys⁴⁶⁶³) within the N-terminal domain. Cys residues subject to pO₂-dependent S-oxidation are located within multiple functional domains (for a recent review, see Ref. 33), including domains implicated in activity-regulating interactions of RyR1 with FK506-binding protein 12 (33, 34) and the dihydropyridine receptor (33, 35). In addition, pO₂-sensitive Cys residues are located in domains implicated in malignant hyperthermia and central core disease (33). It is not possible to deduce which identified Cys residues play a role in pO₂-dependent enhancement of RyR1 activity or may be subject to dysregulated S-oxidation in disease, and concerted effects involving multiple Cys residues seem likely. However, it should be noted that mutation of Cys³⁶ (Cys³⁵ in the human sequence) has been implicated directly in malignant hyperthermia and central core disease (36) and that oxidative stress associated with aging and in a model of muscular dystrophy results in the displacement of FK506-binding protein 12 from RyR1 and consequently Ca²⁺ leakage through RyR1 that contributes to loss of muscle function (37, 38). It is also of interest that Cys³⁶³⁵, which is not a target of pO₂-coupled S-oxidation but which serves as the principal target of endogenous S-nitrosylation that is gated allosterically by RyR1 S-oxidation of alternative Cys residues (10, 13), is located distantly in the primary sequence from identified pO₂-sensitive Cys residues within RyR1 (Fig. 6).

Ninety-eight of 100 RyR1 Cys residues are conserved in mammals, including all pO₂-sensitive Cys residues identified here with the exception of Cys³⁰⁵ (oxidized at high pO₂ only in the presence of NADPH). Furthermore, the majority of RyR1 Cys residues that we identified as subject to physiological redox regulation are also conserved in RyR2 and RyR3, consistent with the finding that the activity of RyR2 is enhanced at high versus low pO₂ (15). Thus, our findings suggest that S-oxidation of Cys residues within the RyRs is likely to serve as a mechanism for physiological redox regulation of RyR function across many mammalian cell types and tissues.