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Originally published In Press as doi:10.1074/jbc.M609936200 on January 19, 2007

J. Biol. Chem., Vol. 282, Issue 12, 8667-8677, March 23, 2007
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Conformation-dependent Stability of Junctophilin 1 (JP1) and Ryanodine Receptor Type 1 (RyR1) Channel Complex Is Mediated by Their Hyper-reactive Thiols*

Andrew J. Phimister{ddagger}1, Jozsef Lango{ddagger}, Eun Hui Lee§, Michael A. Ernst-Russell{ddagger}, Hiroshi Takeshima||, Jianjie Ma**, Paul D. Allen§, and Isaac N. Pessah{ddagger}2

From the {ddagger}Department of Veterinary Molecular Biosciences and Center for Children's Environmental Health and Disease Prevention, University of California, Davis, California 95616, the §Department of Anesthesia Research, Brigham & Women's Hospital, Boston, Massachusetts 02115, the Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea, the ||Department of Biochemistry, Tohoku University, Sendai 980-8575, Japan, and the **Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

Received for publication, October 23, 2006 , and in revised form, January 3, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Junctophilin 1 (JP1), a 72-kDa protein localized at the skeletal muscle triad, is essential for stabilizing the close apposition of T-tubule and sarcoplasmic reticulum membranes to form junctions. In this study we report that rapid and selective labeling of hyper-reactive thiols found in both JP1 and ryanodine receptor type 1 (RyR1) with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin, a fluorescent thiol-reactive probe, proceeded 12-fold faster under conditions that minimize RyR1 gating (e.g. 10 mM Mg2+) compared with conditions that promote high channel activity (e.g. 100 µM Ca2+, 10 mM caffeine, 5 mM ATP). The reactivity of these thiol groups was very sensitive to oxidation by naphthoquinone, H2O2, NO, or O2, all known modulators of the RyR1 channel complex. Using preparative SDS-PAGE, in-gel tryptic digestion, high pressure liquid chromatography, and mass spectrometry-based peptide sequencing, we identified 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin-thioether adducts on three cysteine residues of JP1 (101, 402, and 627); the remaining five cysteines of JP1 were unlabeled. Co-immunoprecipitation experiments demonstrated a physical interaction between JP1 and RyR1 that, like thiol reactivity, was sensitive to RyR1 conformation and chemical status of the hyper-reactive cysteines of JP1 and RyR1. These findings support a model in which JP1 interacts with the RyR1 channel complex in a conformationally sensitive manner and may contribute integral redox-sensing properties through reactive sulfhydryl chemistry.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Junctophilins (JP)3 are a class of highly conserved proteins whose four known genetic isoforms (JP1, JP2, JP3, and JP4) are primarily responsible for stabilizing close apposition of the terminal cisternae of sarco(endo)plasmic reticulum (SR/ER) and the plasma membrane. These isoforms are differentially expressed throughout excitable cells; both JP1 and JP2 are expressed in skeletal muscle; JP2 is specifically expressed in cardiac and smooth muscle, and neurons in the brain contain both JP3 and JP4 (1–4). In skeletal muscle, the expression and proper targeting of JP1 appears to be involved in the correct formation or stabilization of "triadic junctions" between the transverse tubule (TT) membrane and terminal cisternae of SR (1, 2). The ~10 nm gap at these junctions provides an environment for the assembly of conformationally coupled calcium release units (5, 6). Calcium release units consist of clusters of four dihydropyridine receptors (i.e. CaV1.1), particles that align into tetradic arrays within the TT membrane (7–9). Dihydropyridine receptor tetrads within an array orient over every other ryanodine receptor type 1 (RyR1) channel anchored within the SR membrane. Triadic junctions of skeletal muscle therefore provide a specialized milieu where several regions within the "junctional foot" domain of RyR1 physically and functionally associate with the II–III loops of {alpha}1s-dihydropyridine receptors to engage a form of bi-directional signaling necessary for excitation-contraction coupling (orthograde signal) and maximal L-type Ca2+ current (retrograde signal) (10–14).

JPs contain MORN (membrane occupation and recognition nexus) motifs that are thought to form noncovalent interactions with membrane lipids, thereby stabilizing physical association between the membrane partners of the junctional complex (2, 15). JP1, for example, is a 72-kDa protein containing eight MORN motifs at its N-terminal cytoplasmic region and an SR-spanning segment at its C-terminal end (2). Heterologous expression of JP1 protein in amphibian embryos results in formation of artificial plasma membrane/ER junctional structures, similar in the dimensions observed in TT/SR complexes of muscle cells. However, expression of a truncated JP1 lacking the C-terminal SR spanning segment results in plasma membrane targeting without the formation of junctions. JP1 knockout mice have disrupted membrane junctional structures in their skeletal muscle fibers, limiting muscle contraction, which affirms an essential role of JP1 in skeletal muscle triad formation and function (16, 17).

The previous studies did not examine the possibility that JP1 actively interacts with RyR1 or that its presence directly participates in regulating Ca2+ release from SR. In this study, we tested the hypothesis that JP1 and RyR1 interact in a conformationally sensitive manner that coincides with changes in the reactivity of specific thiol residues residing on both proteins. The rationale for this investigation is derived from the observation that a small number of highly reactive cysteines are coordinately labeled within RyR1 and a protein of Mr 90,000 previously identified with a fluorescent cysteine-labeling technique that selectively forms thioether adducts between the most reactive cysteines present in junctional SR (JSR) and a discriminating concentration of the coumarin-maleimide probe 7-diethylamine-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) (18, 19). Reactive cysteines appear to represent components of an integral redox sensor that modulates RyR1 Ca2+ channel function and SR Ca2+ release (20) and confer sensitivity to xenobiotic quinones (21–23). The most reactive cysteines within the Ca2+ channel complex also appear to confer responsiveness to local changes in GSH:GSSG redox potential (24, 25) and represent specific sites of S-nitrosylation (26, 27) and S-glutathionylation (28). Therefore, hyper-reactive cysteines within the RyR1 macromolecular complex are likely to be important sites of physiological and pathophysiological regulation of SR Ca2+ release.

Recently, seven of the most reactive RyR1 cysteines toward CPM were isolated as fluorescent CPM-thioether adducted tryptic peptides and identified by mass spectrometry (29). The identity of the Mr 90,000 protein that possesses hyper-reactive cysteines and whose reactivity appears coordinately regulated with RyR1 conformation was not identified. Results from co-immunoprecipitation and Western blot analyses provided evidence that triadin may possess these highly reactive cysteines and interacts with RyR1 (18); however, these studies were not conclusive because two proteins having similar size on SDS-PAGE were precipitated with RyR1 monoclonal antibody 34C (see Fig. 7 in Ref. 19). Here we use the CPM labeling method to unambiguously identify three highly reactive cysteines at Mr 90,000 that map to thiol residues in JP1. The reactivity of JP1 cysteines is regulated by physiological cations (Ca2+ and Mg2+) and relevant sulfhydryl-active compounds (1,4-NQ, H2O2, NO, or O2) in a manner coincident with the highly reactive cysteines identified in RyR1. Conditions that stabilize the closed state of the RyR1 channel complex enhance thiol reactivity of both RyR1 and JP1 and weaken their interaction when measured by co-immunoprecipitation. By contrast, conditions that stabilize the open state of the RyR1 complex dramatically diminish thiol reactivity of both proteins and stabilize their interaction when measured by co-immunoprecipitation. These findings are the first to identify a conformationally sensitive interaction between JP1 and the RyR1 channel complex. Our data suggest that this interaction may contribute to integral redox-sensing properties of both proteins through reactive sulfhydryl reactions.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—CPM was obtained from Molecular Probes (Eugene, OR). MS sequencing grade trypsin was from Promega (Madison, WI). Low protein binding ultra-smooth polypropylene tubes were from Island Scientific (Bainbridge Island, WA). All other chemicals and solvents were reagent grade or higher purity.

Preparation of JSR Vesicles—JSR membrane vesicles were prepared from rabbit fast-twitch skeletal muscle by the method of Saito et al. (30). JSR pellets were resuspended at 10–15 mg/ml, snap-frozen in liquid nitrogen, and stored at –80 °C. All buffers were de-oxygenated with argon to limit protein oxidation during handling. Protein was quantified by the BCA method (Pierce) using bovine serum albumin as a standard.

CPM Labeling of JSR—CPM was dissolved in anhydrous Me2SO and stored as a concentrated stock at –20 °C until use. Kinetic measurement of the formation of CPM-thioether adducts was made fluorometrically using an LS55 luminescence spectrometer (PerkinElmer Life Sciences) with excitation set at 397 nm and emission measured at 465 nm as described previously (31). Vesicles were diluted to 50 µg/ml in 100 mM KCl, 20 mM MOPS, pH 7.4, supplemented with RyR1 activators or inhibitors as indicated in the figure legends. The presence of 10 mM MgCl2 constituted the RyR1 "closed" buffer, whereas the presence of 100 µM CaCl2, 10 mM caffeine, 5 mM ATP constituted the RyR1 "open" buffer. JSR were diluted into either closed or open buffers at 37 °C with stirring for 3 min before adding 10 nM CPM (1 pmol/µg JSR protein) to initiate thiol labeling. After 1 min the solutions were cooled on ice, and JSR microsomes were collected at 150,000 x g for 1 h. Pellets were resuspended in PBS, and protein content was determined by BCA assay. Recovery rates for JSR protein were typically ~75%.

Co-immunoprecipitation—JSR membranes diluted in either open or closed buffer were treated with CPM, 1,4-naphthoquinone (1,4-NQ), or an equivalent volume of Me2SO (solvent control) as described above. After recovering microsomes by centrifugation, pellets were lysed in an IP buffer (150 mM NaCl, 5 mM Na2EDTA, 1 mM Na3VO4, 10% glycerol (v/v), 10 mM Tris, pH 7.4) containing 1% Triton X-100 (v/v) for 4 h at 4 °C with gentle mixing. After centrifugation of the lysate to remove insoluble material, protein concentration of the supernatant was measured. Lysate protein (500 or 1000 µg) was incubated with monoclonal anti-RyR1 antibody 34C (Developmental Studies Hybridoma Bank University of Iowa, Iowa City) overnight at 4 °C and subsequent incubation with protein G-Sepharose beads for 4 h at 4 °C. Beads were washed three times with Triton X-100-free lysis buffer to remove nonspecifically bound proteins. The immune complexes were subjected to SDS-PAGE and immunoblot analysis with anti-RYR1, anti-triadin (GE 4.90; Affinity BioReagents, Golden, CO), anti-calsequestrin (MA3-913; Affinity BioReagents), anti-JP1, or anti-JP2 (32). Signal detection was visualized by enhanced chemiluminescence.

CPM-labeled JP1 Isolation—CPM-labeled JSR protein was separated on preparative (16 x 20 cm) SDS-polyacrylamide gels, 3% stacking gel, 7.5% resolving gel, under constant current (50 mA). CPM-labeled proteins were easily identified by their fluorescence under UV light (320 nm). In some experiments, gels were stained with SYPRO Ruby (Sigma). Bands of interest were excised with razor blades and processed for in-gel trypsin digestion according to published methods (33, 34). Processing included extensive washing, reduction with dithiothreitol (10 mM), and alkylation with iodoacetamide (55 mM), before drying under vacuum. Trypsin diluted in 25 mM NH4HCO3 was added to the dried gel plugs and digestion proceeded overnight at 37 °C. Tryptic peptides were extracted with 5% formic acid, 50% acetonitrile and concentrated under vacuum for further purification. Alternatively, peptide extracts were desalted using micro-C18 ZipTips (Millipore, Billerica, MA) and analyzed by mass spectrometry (see below).

CPM Peptide Purification—Individual CPM-labeled peptides were purified from pooled JP1 tryptic digests by microbore HPLC with a C18 reverse-phase column (1 x 250 mm; Vydac, Hesperia, CA). Chromatographs were monitored for both peptide absorbance (UV 214 nm) and CPM fluorescence (ex 384/em 469 nm). Buffers and gradients were as reported previously (29). Purified peptides were concentrated under vacuum for further mass spectral analysis.

Mass Spectrometry—Both crude tryptic digests and purified CPM-labeled peptides were analyzed by MALDI/TOF/TOF mass spectrometry (ABI 4700, Applied Biosystems, Foster City, CA) in positive ion mode. The instrument was equipped with a Nd:YAG laser (PowerChip, JDS Uniphase, San Jose, CA) operating at 200 Hz and controlled by Applied Biosystems Explorer version 1.1 software. The mass resolution of the instrument was better than 15,000 in MS mode. The mass accuracy in the MS mode, using internal calibration was ±50 mDa, whereas in the MS/MS mode, the mass accuracy was better than ± 100 mDa. Both the MS and MS/MS spectra were acquired in the full profile mode with ~130,000 and 80,000 data points per spectrum, respectively. The data were acquired in reflector mode from a mass range of 700–6000 Da, and 2500 laser shots were averaged for each mass spectrum. Each sample was internally calibrated to trypsin autolysis fragments if present.

Purified peptides were also analyzed by a quadrupole orthogonal time-of-flight (TOF) mass spectrometer with QqT geometry (QSTAR, Applied Biosystems) for ESI product ion experiments, operating in positive ion mode. The peptides were loaded onto a reverse-phase Poros R2 microcolumn (Applied Biosystems, Foster City, CA) in 5% formic acid and eluted with 5% formic acid, 50% methanol directly into a nanospray ionization source (Protana, Odense, Denmark). Voltage on the Protana capillary tips was 900 V. The instrument was controlled by Analyst-QS version 1.0 software with Service Pack 3 installed. External two-point calibration was performed using CsI ions and a peptide with mass of 828.5 Th. Mass accuracy was typically better 10 mDa, and resolution was typically better than 7000 (full width half-maximum). The flow rate of the nanospray source was ~25–50 nl/min. The TOF analyzer was set to acquire spectra at a rate of 7 kHz over the mass range 100–3000 Da. The resultant spectra were the averages of 100–300 consecutive spectra.


Figure 1
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  		FIGURE 1.
Schematic showing structures and masses of CPM and hydrolyzed CPM molecules adducted to a generic protein thiol. Note water is added in a hydrolysis reaction to the lactam ring of coumarin, producing the hydrolyzed CPM species (mass increase of 18.01 Da).

 
MS Data Analysis—All mass spectral data were transformed into ASCII format for analysis using MassLynx 4.0 sp3 software (Waters, Billerica, MA). Theoretical masses for peptides (1–3 positive charges) from full and incomplete (up to 3 missed cleavages) tryptic digestion of JP1 were generated using the MassLynx peptide editor module. Peptide masses were also calculated for all cysteine-containing peptides modified by adduction with CPM (402.16 Da), hydrolyzed-CPM (420.17 Da), or iodoacetamide (carboxyamidated; 57.02 Da) (see Fig. 1). All of the theoretical masses were merged into one Microsoft Excel data base used to screen raw MS data. In MS mode, matches between observed and theoretical masses were only considered significant with less than 30 ppm error. Peptide sequence information obtained from MS/MS experiments was used whenever possible.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
CPM Labeling
Prior to CPM labeling experiments, the quality of individual preparations of JSR membranes was assessed via standardized assays for ATP-dependent Ca2+ uptake, [3H]ryanodine binding, and single RyR channel gating behavior (19, 24). Furthermore, CPM labeling reactions were only carried out using discriminating amounts of CPM (1 pmol/µg protein) at 37 °C. Under these conditions, CPM labeling proceeded 12-fold more rapidly in the presence of 10 mM Mg2+, which stabilized the closed RyR1 channel conformation (i.e. channel closed buffer) compared with the presence of 100 µM Ca2+, 20 mM caffeine, 5 mM ATP that stabilized the open state of the channel (i.e. channel open buffer) (Fig. 2A). As reported previously (18) this difference in reactivity is only apparent at low CPM concentrations when JSR thiols are in great excess to maleimide, and it represents formation of thioether adducts with the most highly reactive thiols present in JSR preparations. Fifteen seconds after addition of CPM, labeling was 73% complete under channel closed conditions but only 7% complete for channel open conditions. The addition of reducing equivalents in the form of tributylphosphine, chosen for its nonreactivity toward the maleimide moiety of CPM, dramatically accelerated the CPM labeling rate for channel open conditions (Fig. 2B). This correlates with previous findings on the effect that reducing equivalents have on single channel gating behavior, showing that changing from an oxidizing to a reducing environment by shifting GSH:GSSG ratios promotes channel closure and hence thiol reactivity (24).


Figure 2
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  		FIGURE 2.
JSR microsomal vesicles were incubated under RyR1 (open) (i.e. 100 µM Ca2+, 10 mM caffeine, 5 mM ATP) or (closed) (i.e. 10 mM Mg2+) conditions (A and B). CPM (5 nM final concentration) was added at a final ratio of 1 pmol of CPM/µg of protein to initiate JSR thiol labeling. Under closed conditions labeling proceeded 12 times faster than under open conditions (A). Preincubation with the reducing agent tributylphosphine (TBP; 1 µM final concentration) dramatically accelerated the rate of CPM labeling under open conditions (B). Following CPM labeling, JSR microsomes were recovered by centrifugation and analyzed by 7.5% SDS-polyacrylamide gels (C). Only a fraction of proteins are CPM-labeled, with differences in labeling patterns apparent between JSR proteins labeled under open (OP) or closed (CL) conditions. The intense band labeled with an arrowhead was later identified as junctophilin 1 (JP1) with >60% sequence coverage by MS.

 


Figure 3
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  		FIGURE 3.
JSR microsomal vesicles were incubated under RyR1 (closed) (i.e. 10 mM Mg2+) conditions at 37 °C in the presence of various oxidizing agents for 3 min. CPM (5 nM final concentration) was added at a final ratio of 1 pmol of CPM/µg of protein to initiate JSR thiol labeling. Exposure to 500 nM NQ or high oxygen tension (bubbling with 95% O2(g) for 2 min) decreased the extent of CPM labeling by nearly 80% (A). Likewise, brief treatment with hydrogen peroxide (H2O2; B) or the nitric oxide donor spermineNONOate (C) retarded CPM labeling kinetics.

 
In addition to the vastly different reaction rates observed under channel open and closed conditions, we observed striking differences in protein labeling patterns when CPM-thioether adducts were size-separated by reducing SDS-PAGE and visualized under a UV lamp (Fig. 2C). A representative gel image in Fig. 2C illustrates that in addition to RyR1 at the top of the gel, only a small number of the JSR proteins possess thiols that are reactive enough to form CPM-thioether adducts under the experimental conditions used. Most notable is a particularly intense band at Mr ~90,000 (Fig. 2C, arrowhead) that consistently reacted with CPM under channel closed but not open buffer conditions coincident with the CPM labeling characteristics of RyR1 (Fig. 2C). Importantly channel closed and open buffer conditions did not detectably alter the staining pattern of JSR proteins when the same gels were visualized with SYPRO Ruby protein stain (Fig. 2C, right panel).

The reactivity of thiols on RyR1 and the Mr 90,000 protein toward CPM was also extremely sensitive to the thiol-oxidizing/reactive agents 1,4-NQ, 95% oxygen (Fig. 3A), or hydrogen peroxide (Fig. 3B), or the NO generator spermineNONOate (Fig. 3C). The decreased reactivity of hyper-reactive JSR thiols in the presence of H2O2 or spermineNONOate was concentration-dependent and quantitatively mirrored their previously reported effects on RyR1 channel activity (26–28, 36).

Identity of Protein Near Mr 90,000 in JSR
The major protein bands immediately below SERCA I at Mr 100,000 encompassing the region of strong CPM labeling (Fig. 2C, arrowhead in CL lane) were detected with SYPRO Ruby protein staining and then excised. In-gel tryptic digests were prepared from these bands, and the proteins to which they belonged were identified by mass spectrometry. Peptide mass fingerprinting was performed using the SEQUEST algorithm searching against the Swiss Protein Database. Fig. 4A shows a representative gel with the region of interest magnified (inset). In addition to identifying JP1, several proteins such as SERCA I, 6-phosphofructokinase, NADH dehydrogenase, and glycerol-3-phosphate dehydrogenase were identified in the region of the gel adjacent to JP1. Calsequestrin, the major Ca2+-binding protein of the JSR lumen, was also identified at Mr 51,000 (Fig. 4A). MASCOT sequence searches identified each of these six protein bands with high confidence (99–100%). These results are consistent with proteins known to be present in JSR microsomes, and the predicted masses compared well to the migration patterns observed on the SDS-polyacrylamide gels. However an unexpected finding was that JP1 was consistently detected at Mr 90,000 instead of its 72-kDa mass predicted by its amino acid sequence. Migration of JP1 to Mr 90,000 was consistent whether the proteins were size-separated from JSR labeled with CPM or unlabeled JSR. The majority of the CPM fluorescent signal from JSR labeled under open conditions is associated with the SERCA protein, which possesses 22 cysteines and accounts for 68% of the protein within the JSR preparations (Fig. 2C). When CPM labeling reactions were quenched 1 min after the reaction was initiated by addition of either 10 mM GSH or 1 mM N-ethylmaleimide, the pattern of fluorescently labeled bands was indistinguishable from unquenched reactions in the channel closed buffer condition, whereas fluorescence was barely detectable in the channel open buffer (data not shown). This indicates that for unquenched reactions (shown in Fig. 2C), the labeling observed under open conditions is directed toward much less reactive thiols than those labeled with CPM under closed conditions. Additionally we investigated whether JSR proteins in the region of Mr 90,000 were glycosylated by oxidizing samples with periodic acid-Schiff followed by staining for carbohydrate (Pro-Q glycoprotein detection kit, Molecular Probes, Eugene, OR). Several proteins were positive for glycosyl-adducts in these JSR preparations, including calsequestrin that is known to be glycosylated (37). By contrast evidence for glycosylation of JP1 was not detected using this method (Fig. 4B).


Figure 4
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  		FIGURE 4.
Unlabeled JSR microsomes were separated by denaturing SDS-PAGE. Bands were excised, digested with trypsin, and analyzed by MALDI-TOF-TOF MS analysis to generate peptide mass fingerprints (>35% coverage and two fully sequenced peptides to identify each band indicated), which were matched to the Swiss Protein Database (A). Unlabeled JSR proteins separated in a denaturing SDS-PAGE were also stained first for glycosidic groups (Pro-Q Emerald glycoprotein kit, Molecular Probes), imaged, counterstained for all proteins with Sypro Ruby, and re-imaged (B). Arrowhead indicates JP1 band, which does not appear to be glycosylated.

 


Figure 5
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  		FIGURE 5.
Tryptic peptides from in-gel digestion of CPM-labeled JP1 were purified by microbore reverse-phase HPLC (1 x 250 mm) using trifluoroacetic acid in water and acetonitrile. The upper trace shows the UV signal identifying all peptides, and the lower trace shows the fluorescence signal (384ex/469em) identifying only CPM-peptides. The CPM-peptides peaks were collected and concentrated under vacuum for MS analysis.

 
JP1 Proteomics and Location of Reactive CPM-Thioether Adducts
We proceeded to unambiguously identify the location of the hyper-reactive cysteines that react with CPM under channel closed conditions at Mr 90,000. Tryptic digests of JP1 (662 amino acids; NCBI Entrez Protein data base, entry code Q9GKY8) can produce 75 peptide fragments, but only a handful of these contain cysteine(s). We separated CPM-labeled peptides by microbore HPLC with detection for both UV absorbance at 214 nm (all peptides) and CPM fluorescence emission at 469 nm (coumarin thioether adducts) (Fig. 5). Seventeen reproducible fractions were collected during 18 consecutive HPLC runs, and individual fractions were pooled after collection. During the consolidation of fractions, each microcentrifuge tube was rinsed with 5% formic acid, 50% acetonitrile, water (v/v/v) to minimize sample losses. Several individual fractions were re-injected to verify that the elution pattern and peak profile remained unchanged (data not shown). The sum of the fluorescence peak areas from the collected fractions represented >85% of the total fluorescence signal applied to the HPLC column. The remainder of the CPM fluorescence was accounted by a peak eluting at the void volume that was examined by ESI-MS and SDS-PAGE without success.

Tryptic in-gel digests of the intensely fluorescent band at Mr 90,000 (Fig. 2C, arrowhead) were analyzed by MALDI/TOF (Fig. 6A), and yielded more than 45 peptides that map to JP1 (Tables 1 and 2). Peptide sequencing in TOF/TOF mode of several intense peaks further confirmed the identity of the protein to be JP1. We performed mass spectral experiments on samples alkylated with iodoacetamide before or after SDS-PAGE analysis, with identical results. This was to ensure cysteines were not being oxidized or alkylated with gel components prior to isolation. These results gave us positive identification of JP1 cysteine residues that appear carboxyamidated, and therefore not modified by CPM or Hy-CPM thioether adducts (see Fig. 1). Because the peptides containing cysteines modified by CPM could not be accounted for by using existing automated data bases, we constructed a custom data base. Using our data base, more than 45 tryptic peptides were detected with a mean error of less than 20 ppm in MS mode, yielding 60% coverage for the JP1 protein. Five peptides were completely sequenced in MS/MS mode producing exact matches to the JP1 sequence. When unlabeled Mr 90,000 protein was excised from gels using SYPRO Ruby to visualize its location (data not shown), automated data base matching consistently identified JP1. Tissue preparations from three separate rabbits were prepared and analyzed in this same manner and consistently identified the CPM-labeled protein from Fig. 2C as JP1.


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  		TABLE 1
Summary of JP1 tryptic peptides identified by mass

Values are given in Thomson units for [M + H]+1 ions. The average error of mass measurement is better than 10 ppm. The average coverage of the protein (without redundant fragments) is better than 60%.

 


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  		TABLE 2
JP1 cysteine residue reactivity

Blank columns infer not detected or not found.

 


Figure 6
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  		FIGURE 6.
MS and MS/MS sequencing of CPM-labeled JP1 peptides. A full mass spectral scan of a tryptic digest of CPM-labeled JP1 (MALDI-TOF) identifies many of the same peptides identified in unlabeled JP1 digests (not shown). However, several peaks unique to spectra derived from digests of CPM-labeled JP1 correspond to CPM adducted peptides (A). The peptide sequence derived from ESI-MS-MS sequencing of a single CPM-labeled peptide purified by HPLC is displayed to demonstrate that the CPM label can be directly observed on these peptides (B).

 
We next set out to identify the exact location of the cysteines adducted by CPM. Both the crude tryptic digest and HPLC fractions were subjected to MS analysis using the following two methods: direct MALDI-TOF MS (MALDI) and ESI MS/MS (ESI) (see "Experimental Procedures"). Analyses of 85 samples were carried out by means of 60 direct MALDI/TOF mass spectral experiments, 357 MALDI MS/MS, 28 direct ESI scans, and 3 ESI product ion experiments. In total, three CPM/Hy-CPM adducted cysteines were identified, which refer to cysteine residues 101, 402, and 627 (Table 2). Three cysteine residues (15, 29, and 318) were always identified in the carboxyamidated state (i.e. alkylated by iodoacetamide).

Digest fragments containing cysteine residues (Cys-264 and Cys-267) were difficult to detect, possibly because they reside within a tryptic peptide possessing a mass of at least 4273 Da (residues 248–286) or the region may be inaccessible to trypsin. We detected a signal at m/z 4989 Th which could represent the first order incomplete digest product of the oxidized (disulfide-bonded) form of this large tryptic fragment (residues 241–286). These two cysteines include a CXXC motif, characteristic of redox-active proteins such as thioredoxin (38). Additionally, this peptide contains the only consensus glycosylation sequence in JP1 (NST; residues 254–256); however, no glycans were detected on JP1 by SDS-PAGE based fluorescent staining (Fig. 4B). This experimental result is in good agreement with our mass spectral data that indicate that the CXXC motif resides within a highly protected region of JP1. Our findings are summarized for all JP1 individual cysteines as follows.


Figure 7
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  		FIGURE 7.
A shows the detected signals of the (M + H)+ molecular ion of digest fragment 241–286. The raw signals were processed using Sawitsky-Golay smoothing method with peak width of 0.5 Da. B shows the theoretical isotope distribution of the (M + H)+ molecular ion of digest fragment 241–286, with elementary composition of C208H311N54O81S4 with a theoretical peak width of 0.5 Da. Five of the most intense adjacent peaks were selected for error measurement; the average error measured was less than 165 ppm. For mass measurement, peak top values were used, to exclude the influence of the asymmetry of the detected peaks. The experimental signals fit well to the theoretical isotope distribution despite the fact that the intensity of the detected signals was less than 70 counts.

 
Cys-15—This cysteine residue was found in the carboxyamidated state in all cases. Peptide 6–22 was identified in multiple crude tryptic digests with high mass accuracy (m/z 1939.747 Th, {Delta}= 1.0 ppm). No CPM adducts of this residue were found.

Cys-29—This cysteine residue was found in the first order incomplete tryptic peptide 6–33 containing Cys-29 and Cys-15 with both residues carboxyamidated (Table 1). This ion was found in multiple crude tryptic digests with high mass accuracy (m/z 3055.260 Th, {Delta}= 4.5 ppm). No CPM adducts of this residue were found.

Cys-101—This cysteine residue was found in the tryptic peptide 98–105, adducted with Hy-CPM (m/z 1295.611 Th, {Delta}= 6.9 ppm). Additionally, the first order incomplete tryptic peptide 92–105 was identified with a Hy-CPM adduct. These ions were found with low intensity in crude tryptic digests but with greatly enhanced signal strength in HPLC-purified peptide fractions. This cysteine was never found in the free or alkylated state. Based on the mass spectral data, the high activity level of this cysteine residue is evident (Table 2).

Cys-264/Cys-267—The lowest molecular mass digest fragment is 4271.777 Da (monoisotopic) representing the tryptic peptide fragment of 248–286, and this fragment possesses a CXXC motif. To detect any possible fragments encompassing these residues, the mass range was extended to 6000 Da. Close inspection identified low intensity signals that were partly superposed with electronic noise that could correspond to digest fragment 241–286 (first order incomplete digest product) at m/z 4990 Th, with cysteines 264 and 267 in an oxidized (disulfide bridge) form (Fig. 7A). The experimental signals fit well to the theoretical 13C isotope distribution (Fig. 7B) despite the fact that the overall intensity of the detected signals was less than 70 counts. Five of the most intense adjacent peaks were selected for error measurement; the average error measured was less than 165 ppm (for mass measurement peak top values were used, in order to exclude the influence of the asymmetry of the detected peaks). The isotope distribution pattern mirrored the theoretical distribution and the relatively high mass accuracy supported our interpretation that this signal group was produced by the first order incomplete digest fragment 241–286 with the Cys-264/Cys-267 residues in disulfide bridge form.

Cys-318—This cysteine residue was found in the carboxyamidated state in all cases. Ions were identified corresponding to tryptic peptides 314–338 at m/z 1867.831 Th, {Delta}= 1.6ppm, and the third order incomplete peptide fragment 304–338 at m/z 2868.388 Th, {Delta}= 7.9 ppm (Table 2). No CPM adducts of this residue were found.

Cys-402—This cysteine was identified in tryptic peptide 400–406 adducted with Hy-CPM (m/z 1254.560 Th, {Delta}= 11.1 ppm) in HPLC-purified peptide fractions. Additionally, we identified tryptic peptide 386–406 with Hy-CPM adducts (m/z 2578.189 Th, {Delta}= 18.2 ppm) (Table 2).

Cys-627—This cysteine residue was identified in tryptic peptide 620–632 as an Hy-CPM adduct (m/z 1827.731 Th, {Delta}= 1.7 ppm). The triple-charged molecular ion (m/z 609.92 Th) was subjected for MS/MS experiment in product mode (Fig. 6B), giving the expected sequence along with the Hy-CPM adduct on the cysteine residue. The result of the spectrum transformation to first charge state is given in Table 2.

CPM adducts were almost exclusively detected in the CPM-hydrolyzed state, suggesting that the CPM molecule itself may suppress ionization of CPM-tagged peptides. A possible mechanism for ion suppression is through charge delocalization phenomena related to coumarin ring conjugation; conversion to the CPM-hydrolyzed state disrupts the conjugation. In tests where CPM adducts of glutathione were directly infused for ESI-MS analysis, the predominant signal detected was for hydrolyzed CPM-GSH, with little intact CPM mass signal even though the samples were highly fluorescent and therefore containing the intact CPM molecule.


Figure 8
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  		FIGURE 8.
JSR or CPM-labeled JSR were lysed under channel (closed) (100 nM Ca2+) or (open) (100 µM Ca2+/2 mM caffeine) condition with 1% Triton X-100 in the absence (A) or presence (B) of 1,4-NQ. Using anti-RyR1 antibody (34C), RyR1 was immunoprecipitated (IP), and the precipitate was separated by SDS-PAGE to look for co-immunoprecipitated proteins. Blots of gels were probed with antibodies against RyR1 (1:2,000), JP1 (1:3,000), JP2 (1:50,000), triadin (1:1,000), and calsequestrin (CSQ, 1:2,500). IB, immunoblot.

 
JP1 Associates with RyR1 in a Conformationally Sensitive Manner
By utilizing 34C monoclonal antibody against RyR1, we were able to demonstrate that JP1 (but not JP2) physically associates with RyR1 (Fig. 8A). RyR1 has been shown previously to associate with triadin and calsequestrin to form a trimeric protein complex (18, 19, 39), and our current results confirm these previous observations. A new finding is that significantly higher levels of JP1 and triadin are found in the co-immunoprecipitates under a channel open buffer conditions (100 µM Ca2+ + 20 mM caffeine) compared with co-immunoprecipitates obtained under a channel closed (100 nM Ca2+) buffer condition (Fig. 8A). Considering that no observable differences were seen in the amount of RyR1 and calsequestrin co-immunoprecipitated under either buffer condition, these results indicate that JP1 and triadin form more stable associations with RyR1 when it assumes an open conformation. Interestingly, labeling of JSR hyper-reactive thiols with CPM appears to selectively stabilize the JP1-RyR1 and triadin-RyR1 interactions regardless of the buffer condition used in the co-immunoprecipitation step. Although JP2 is expressed in skeletal muscle, attempts to co-immunoprecipitate JP2 with RyR1 antibody 34C failed under any of the conditions attempted. The maleimide moiety of CPM forms CPM-thioether adducts by Michael addition to reactive Cys. We therefore explored the possible influence of 1,4-NQ, which is known to redox cycle and arylate biomolecules, on modifying the stability of the JP1-RyR1 complex. Fig. 8B shows that in the presence of 500 nM 1,4-NQ, more JP1 co-immunoprecipitated with anti-RyR1 antibody 34C, regardless of whether the experiment was performed under channel closed or channel open buffer conditions. These result indicate that changes in redox potential can influence the stability of the JP1-RyR1 complex.


Figure 9
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  		FIGURE 9.
Schematic of the primary structure of rabbit JP1 and JP2. Indicated in the structure are the 8 MORN motifs, the C-terminal ER membrane anchor, and the cysteine residues (eight in JP1 and four in JP2). JP2 lacks the hyper-reactive Cys (101, 402, and 627) identified in this paper as well as the CXXC motif. The cysteines are labeled as SH (nonreactive residue), CPM (hyper-reactive residues), or C–C (disulfide bonded) based on the data from Table 2. The sections colored gray in the upper panel correspond to the areas of sequence identified by MS analysis of JP1.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Two new and related findings are reported in this study. First, JP1 is unambiguously identified to contain highly reactive thiols at positions 101, 402, and 627, whereas the remaining five cysteines were nonreactive toward the fluorogenic maleimide CPM (Fig. 9). The reactivity of JP1 Cys-101, -402, and -627 thiols toward CPM is highly dependent on the conformational state of the RyR1 channel complex in a manner previously reported for a small number of "hyper-reactive" thiols on RyR1 itself (18, 19). The apparent reactivity of hyper-reactive thiols on both RyR1 and JP1 are coincidentally regulated. Buffers that promote the closed (nonconducting) channel state, e.g. in the presence of 10 mM Mg2+ and/or 10 nM Ca2+, greatly enhance the rates of forming CPM-thioether adducts on these proteins, whereas buffer conditions that stabilize an open state of the channel, e.g. 100 µM Ca2+, 10 mM caffeine, and 5 mM ATP, reduce the rate of adduct formation 10–12-fold. Here we extend our measurements to two known thiol-active modulators of RyR1 activity, NO (26–28) and H2O2 (36). Both spermineNONOate (which efficiently generates NO) and H2O2 are found to reduce the rates of forming CPM-thioether adducts in the same respective range of concentrations previously reported for their activity in regulating RyR1 channel function. It is important to note that the coordinated regulation of thiol reactivity on JP1 and RyR1 by physiological and pharmacological ligands may reflect precise communication between these two proteins that, at least in part, involve changes in sulfhydryl chemistry leading to changes in oxidation state, S-nitrosylation, and S-glutathionylation.

The second new observation reported here, that of a conformationally sensitive interaction between JP1 and RyR1, supports this conclusion. Co-immunoprecipitation experiments with RyR1 monoclonal antibody 34C and solubilized JSR have been shown previously to influence the interaction of triadin and RyR1 (19). These data were based on probing the immunoprecipitates with the same anti-triadin used in this study. Although the present study re-affirms the conformationally sensitive nature of the triadin-RyR1 interaction, here we provide direct and unambiguous evidence that the hyper-reactive thiols at Mr 90,000 belong to JP1 and not triadin. The stability of RyR1 interactions with triadin and JP1 depends on buffer constituents that promote specific conformational states of the channel complex, and intra- or intermolecular changes in thiol status appear to be essential. Support for this conclusion comes from results showing that formation of CPM thioether adducts on the most reactive JSR cysteines selectively stabilizes RyR1 interactions with JP1 and triadin, whereas its interactions with calsequestrin or JP2 are unchanged by buffer conditions (i.e. open versus closed). The stability of the JP1-RyR1 complex is also influenced by the presence of 1,4-NQ (whose redox potential is 484 mV), indicating changes in redox potential can influence the stability of the JP1-RyR1 complex. 1,4-NQ has been shown previously to enhance and then inhibit RyR1 channel activity in a concentration- and time-dependent manner (21). The complex activity of 1,4-NQ toward RyR1 is likely because of its ability to redox cycle and arylate target proteins. The conditions used in this study (100–500 nM) have been shown to promote the open state of the channel (21) and are consistent with the interpretation that a change in the redox environment to a more oxidizing potential enhances the stability of the JP1-RyR1 complex. RyR1 has been shown to physically interact with several accessory proteins, including calmodulin (40, 41), FKBP12 (42), calsequestrin (39), and Homer (43). In this regard, S-glutathionylation and S-nitrosylation have been also shown to influence calmodulin and FKBP12 binding to RyR1 (44). It has also been suggested that hyper-reactive thiols in key proteins of the triad junction provide a mechanism for responding to oxidative stress by modulating Ca2+ release (20). Collectively, these results also suggest a role for hyper-reactive sulfhydryls in influencing the stability of conformationally driven protein-protein interactions within the triad junction.

What possible mechanisms could coordinate sulfhydryl reactivity and protein-protein interactions between JP1 and RyR1 within the triad junction? JP1 contains a CXXC motif (Cys-264 and Cys-267) that has been shown to be the target of thiol-dependent enzymes and to participate in disulfide bond formation, reduction, and isomerization (38). CXXC serves as a reaction center for the formation of intramolecular and intermolecular disulfides at reactive cysteines that in turn shuttle reducing equivalents to closely associated proteins such as membrane-bound oxidoreductases. Reduction of the CXXC disulfide bond is apparently difficult to achieve in vitro in the presence of oxygen (46). It is interesting to note that our present spectral analysis of reduced, iodoacetylated JP1 tryptic digests failed to identify first or second order fragments containing alkylated Cys-264/Cys-267, indicating that this CXXC may also be resistant to reduction. In support of this interpretation, we identified signals that are consistent with digest fragment 241–286 (first order incomplete digest product) at m/z 4990 Th, with Cys-264/Cys-267 in an oxidized (disulfide bridge) form. All three RyR isoforms possess five invariant CXXC motifs widely spaced in their primary sequence. This observation may help explain why our previous attempts to identify tryptic fragments of RyR1 containing the CXXC motif by mass spectrometry failed, as they may also be resistant to reduction in vitro. The present results suggest that the hyper-reactive cysteines at positions 101, 402, and 627 could coordinate shuttling of reducing equivalents with Cys-264/Cys-267 in a manner similar to that described for thioredoxin with its redox partners. The data presented show that the stability of the interaction between JP1 (but not JP2) and RyR1 is largely regulated by small ligands that stabilize closed or open conformations of the channel complex. It is important to note that formation of CPM-thioether adducts on JP1 Cys-101, Cys-402, and Cys-627 and hyper-reactive Cys previously identified within RyR1 (29) stabilizes the interaction of these proteins regardless of the conformational state set experimentally (Fig. 8). In other words, capping the hyper-reactive Cys of JP1 and RyR1 essentially stabilizes the complex regardless of the conformational state of the channel (Fig. 8) and disables the ability of RyR1 to respond to local shifts in redox potential (24, 25). These data implicate a role for hyper-reactive thiols present in JP1 and RyR1 in stabilizing/destabilizing their interactions in response to conformational changes induced by physiological ligands. Although both JP1 and JP2 are expressed in skeletal muscle, JP2 possess neither a CXXC motif nor does it possess the three analogous cysteines identified in JP1 as hyper-reactive (Cys-101, Cys-402, and Cys-627) (Fig. 9). The fact that JP2 fails to interact with RyR1 in a conformation-sensitive manner further supports a unique role for JP1 in conferring redox sensing properties to the RyR1 channel complex and stabilizing/destabilizing the interaction. In this regard, recent evidence has been published showing that, upon receptor binding, the membrane fusion protein of murine leukemia virus rearranges an intersubunit disulfide bond into a disulfide bond isomer within the CXXC motif thereby changing the stability of protein-protein interactions and activation (47). Whether a similar mechanism operates between JP1 and RyR1 remains to be explored. NADH oxidoreductase activities have been associated with both RyR1 (48, 49) and RyR2 (50) and regulate their channel activities. Recently the Mu glutathione transferases were shown to regulate RyR1 and RyR2 in an isoform-specific manner (35). Moreover the invariant C-terminal CXXC motif of RyR1 was shown essential for channel function because mutations C4958S and/or C4961S disabled Ca2+ release even though RyR1 is properly targeted to junctions and engages retrograde signaling (45). Collectively these data suggest the possible participation of one or more catalytic partners in regulating redox sensing within RyR complexes that involve reactive cysteines and CXXC motifs that may shuttle reducing equivalents within the complex to stabilize/destabilize specific conformational states.

In conclusion, the present results provide the first evidence that JP1 and RyR1 interact in a conformation-sensitive manner that involves changes in the reactivity of specific cysteines unique to JP1.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants 2RO1-AR43140, 2PO1-AR17605 (to P. D. A. and I. N. P.), and 1PO1-ES11269 (to I. N. P. and J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Novartis Pharmaceuticals, 4560 Horton St., Emeryville, CA 94608. Back

2 To whom correspondence should be addressed. Tel.: 530-752-6696; Fax: 530-752-4698; E-mail: inpessah{at}ucdavis.edu.

3 The abbreviations used are: JP, junctophilin; TT, T-tubules; SR, sarcoplasmic reticulum; JSR, junctional SR; MORN, membrane occupation and recognition nexus; RyR, ryanodine receptor; RyR1, skeletal type ryanodine receptor; CPM, 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin; Hy-CPM, hydrolyzed CPM; MOPS, 4-morpholinepropanesulfonic acid; HPLC, high performance liquid chromatography; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; ESI, electrospray ionization; GSH, reduced glutathione; GSSG, GSH disulfide; 1,4-NQ, 1,4-naphthoquinone; ESI-MS, electrospray ionization-mass spectrometry; MS/MS, tandem mass spectrometry; SERCA, sarcoplasmic reticulum Ca2+-ATPase; Th, Thomsons. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Andrew Voss and Dexter Morin for their technical advice. Mass spectrometry support was provided by NIEHS Grant P42-ES04699 from the National Institutes of Health (Bruce Hammock, Director) and the Nevada Proteomics Center Grant P20 RR16464.



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 DISCUSSION
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