Depolarization-evoked secretion requires two vicinal transmembrane cysteines of syntaxin 1A

Background

The interactions of the voltage-gated Ca²⁺ channel (VGCC) with syntaxin 1A (Sx 1A), Synaptosome-associated protein of 25 kD (SNAP-25), and synaptotagmin, couple electrical excitation to evoked secretion. Two vicinal Cys residues, Cys 271 and Cys 272 in the Sx 1A transmembrane domain, are highly conserved and participate in modulating channel kinetics. Each of the Sx1A Cys mutants, differently modify the kinetics of Cav1.2, and neuronal Cav2.2 calcium channel.

Methodology/Principle Findings

We examined the effects of various Sx1A Cys mutants and the syntaxin isoforms 2, 3, and 4 each of which lack vicinal Cys residues, on evoked secretion, monitoring capacitance transients in a functional release assay. Membrane capacitance in Xenopus oocytes co-expressing Cav1.2, Sx1A, SNAP-25 and synaptotagmin, which is Bot C- and Bot A-sensitive, was elicited by a double 500 ms depolarizing pulse to 0 mV. The evoked-release was obliterated when a single Cys Sx1A mutant or either one of the Sx isoforms were substituted for Sx 1A, demonstrating the essential role of vicinal Cys residues in the depolarization mediated process. Protein expression and confocal imaging established the level of the mutated proteins in the cell and their targeting to the plasma membrane.

Conclusions/Significance

We propose a model whereby the two adjacent transmembranal Cys residues of Sx 1A, lash two calcium channels. Consistent with the necessity of a minimal fusion complex termed the excitosome, each Sx1A is in a complex with SNAP-25, Syt1, and the Ca²⁺ channel. A Hill coefficient >2 imply that at least three excitosome complexes are required for generating a secreting hetero-oligomer protein complex. This working model suggests that a fusion pore that opens during membrane depolarization could be lined by alternating transmembrane segments of Sx1A and VGCC. The functional coupling of distinct amino acids of Sx 1A with VGCC appears to be essential for depolarization-evoked secretion.     

Oxidative Inhibition of Protein Phosphatase 2A Activity: Role of Catalytic Subunit Disulfides

A molecular basis for the inhibition of brain protein phosphatase 2A (PP2A) activity by oxidative stress was examined in a high-speed supernatant (HSS) fraction from rat cerebral cortex. PP2A activity was subject to substantial disulfide reducing agent-reversible inhibition in the HSS fraction. Results of gel electrophoresis support the conclusions that inhibition of PP2A activity was associated with the both the disulfide cross-linking of the catalytic subunit (PP2A_C) of the enzyme to other brain proteins and with the formation of an apparent novel intramolecular disulfide bond in PP2A_C. Additional findings that the vicinal dithiol cross-linking reagent phenylarsine oxide (PAO) produced a potent dithiothreitol-reversible inhibition of PP2A activity suggest that the cross-linking of PP2A_C vicinal thiols to form an intramolecular disulfide bond may be sufficient to inhibit PP2A activity under oxidative stress. We propose that the dithiol–disulfide equilibrium of a vicinal thiol pair of PP2A_C may confer redox sensitivity on cellular PP2A.     

Phenylarsine Oxide Binding Reveals Redox-Active and Potential Regulatory Vicinal Thiols on the Catalytic Subunit of Protein Phosphatase 2A

Our earlier finding that the activity of protein phosphatase 2A from rat brain is inhibited by micromolar concentrations of the dithiol cross-linking reagent phenylarsine oxide (PAO) has encouraged the hypothesis that the catalytic subunit (PP2Ac) of PP2A contains one or more pairs of closely-spaced (vicinal) thiol pairs that may contribute to regulation of the enzyme. The results of the present study demonstrate using immobilized PAO-affinity chromatography that PP2Ac from rat brain formed stable DTT-sensitive adducts with PAO with or without associated regulatory subunits. In addition, a subset of the PAO-binding vicinal thiols of PP2Ac was readily oxidized to disulfide bonds in vitro. Importantly, a small fraction of PP2Ac was still found to contain disulfide bonds after applying stringent conditions designed to prevent protein disulfide bond formation during homogenization and fractionation of the brains. These findings establish the presence of potentially regulatory and redox-active PAO-binding vicinal thiols on the catalytic subunit of PP2A and suggest that a population of PP2Ac may contain disulfide bonds in vivo.     

TMEM16A(a)/anoctamin-1 shares a homodimeric architecture with CLC chloride channels

TMEM16A/anoctamin-1 has been identified as a protein with the classic properties of a Ca(2+)-activated chloride channel. Here, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and chemical cross-linking to assess the quaternary structure of the mouse TMEM16A(a) and TMEM16A(ac) splice variants as well as a genetically concatenated TMEM16A(a) homodimer. The constructs carried hexahistidyl (His) tags to allow for their purification using a nondenaturing metal affinity resin. Neither His-tagging nor head-to-tail concatenation of two copies of TMEM16A(a) noticeably affected Ca(2+)-induced measured macroscopic Cl(-) currents compared with the wild-type TMEM16A(a) channel. The digitonin-solubilized, nondenatured TMEM16A(a) protein migrated in the BN-PAGE gel as a homodimer, as judged by comparison with the concatenated TMEM16A(a) homodimer and channel proteins of known oligomeric structures (e.g. the voltage-gated Cl(-) channel CLC-1). Cross-linking with glutaraldehyde corroborated the homodimeric structure of TMEM16A(a). The TMEM16A(a) homodimer detected in Xenopus laevis oocytes and HEK 293 cells dissociated into monomers following denaturation with SDS, and reducing versus nonreducing SDS-PAGE provided no evidence for the presence of intersubunit disulfide bonds. Together, our data demonstrate that the Ca(2+)-activated chloride channel member TMEM16A shares an obligate homodimeric architecture with the hCLC-1 channel.     

CaV2.1 (P/Q channel) interaction with synaptic proteins is essential for depolarization-evoked release

It is well established that syntaxin 1A (Sx1A), SNAP-25 and synaptotagmin (Syt1) either alone or in combination, modify the kinetic properties of voltage-gated Ca²⁺ channels (VGCCs). The interaction interface resides mainly at the cytosolic II-III domain of the alpha1 subunit of the channels, while Sx1A interacts with the channel also via two highly conserved cysteine residues at the transmembrane domain. In the present study, we characterized Ca²⁺-independent coupling of the human neuronal P/Q-type calcium channel (Ca_V2.1) with Sx1A, SNAP-25, Syt1 and synaptobrevin (VAMP) in BAPTA-injected Xenopus oocytes. The co-expression of Ca_V2.1 with Sx1A, SNAP-25 and Syt1, produced a multiprotein complex with distinctive kinetic properties analogous to the excitosome complexes generated by Ca_V1.2, Ca_V2.2, and Ca_V2.3. The distinct kinetic properties of Ca_V2.1 acquired by its close association with Syt1 and t-SNAREs suggest that the vesicle is tethered to the neuronal channel and to the exocytotic machinery independently of intracellular Ca²⁺. To explore the relevance of these interactions to secretion we exploited a BotC1-and a BotA-sensitive secretion system developed for Xenopus oocytes not buffered by BAPTA, in which depolarization-evoked secretion is monitored by a change in membrane capacitance. The reconstituted Ca_V2.1 release is consistent with the model in which the VGCC acts from within the exocytotic complex playing a signaling role in triggering release. The relevance of these results to secretion posits the role of possible rearrangements within the excitosome subsequent to Ca²⁺ entry, setting the stage for the fusion of channel-tethered-vesicles upon the arrival of an action potential.     

The C2A domain of synaptotagmin alters the kinetics of voltage-gated Ca2+ channels Ca(v)1.2 (Lc-type) and Ca(v)2.3 (R-type)

Biochemical and genetic studies implicate synaptotagmin (Syt 1) as a Ca2+ sensor for neuronal and neuroendocrine neurosecretion. Calcium binding to Syt 1 occurs through two cytoplasmic repeats termed the C2A and C2B domains. In addition, the C2A domain of Syt 1 has calcium-independent properties required for neurotransmitter release. For example, mutation of a polylysine motif (residues 189-192) reverses the inhibitory effect of injected recombinant Syt 1 C2A fragment on neurotransmitter release from PC12 cells. Here we examined the requirement of the C2A polylysine motif for Syt 1 interaction with the cardiac Cav1.2 (L-type) and the neuronal Cav2.3 (R-type) voltage-gated Ca2+ channels, two channels required for neurotransmission. We find that the C2A polylysine motif presents a critical interaction surface with Cav1.2 and Cav2.3 since truncated Syt 1 containing a mutated motif (Syt 1*1-264) was ineffective at modifying the channel kinetics. Mutating the polylysine motif also abolished C2A binding to Lc753-893, the cytosolic interacting domain of Syt 1 at Cav1.2 1 subunit. Syt 1 and Syt 1* harboring the mutation at the KKKK motif modified channel activation, while Syt 1* only partially reversed the syntaxin 1A effects on channel activity. This mutation would interfere with the assembly of Syt 1/channel/syntaxin into an exocytotic unit. The functional interaction of the C2A polylysine domain with Cav1.2 and Cav2.3 is consistent with tethering of the secretory vesicle to the Ca2+ channel. It indicates that calcium-independent properties of Syt 1 regulate voltage-gated Ca2+ channels and contribute to the molecular events underlying transmitter release.     

Protection of tamoxifen against oxidation of mitochondrial thiols and NAD(P)H underlying the permeability transition induced by prooxidants

The effects of tamoxifen (TAM) were studied on the mitochondrial permeability transition (MPT) induced by the prooxidant tert-butyl hydroperoxide (t-BuOOH) or the thiol cross-linker phenylarsine oxide (PhAsO), in the presence of Ca2+, in order to clarify the mechanisms involved in the MPT inhibition by this drug. The combination of Ca2+ with t-BuOOH or PhAsO induces mitochondrial swelling and depolarization of membrane potential (deltapsi). These events are inhibited by cyclosporine A (CyA), suggesting the inhibition of the MPT. The pre-incubation of mitochondria with TAM also prevents those events and induces a time-dependent reversal of deltapsi depolarization following MPT induction, similarly to CyA. Moreover, TAM inhibits the Ca2+ release and the oxidation of NAD(P)H and protein thiol (-SH) groups promoted by t-BuOOH plus Ca2+. On the other hand, the MPT induced by PhAsO plus Ca2+ does not induce -SH groups oxidation, supporting the notion that MPT induction by this compound is not mediated by the oxidation of specific membrane proteins groups. However, TAM also inhibits the PhAsO induced MPT, suggesting that this drug may inhibit this phenomenon by inhibiting PhAsO binding to -SH vicinal groups, implicated in the MPT induction. These data indicate that the MPT inhibition by TAM may be related to its antioxidant capacity in preventing the oxidation of NAD(P)H and -SH groups or by blocking these groups, since the oxidation of these groups increases the sensitivity of mitochondria to the MPT induction. Additionally, they suggest an MPT-independent pathway for TAM-induced apoptosis and a potential ER-independent mechanism for the effectiveness of this drug in the cancer therapy and prevention.     

Prm1 Functions as a Disulfide-linked Complex in Yeast Mating

Prm1 is a pheromone-induced membrane glycoprotein that promotes plasma membrane fusion in yeast mating pairs. HA-Prm1 migrates at twice its expected molecular weight on non-reducing SDS-PAGE gels and coprecipitates with Prm1-TAP, indicating that Prm1 is a disulfide-linked homodimer. The N terminus of a plasma membrane-localized GFP-Prm1 endocytic mutant projects into the cytoplasm, where it is protected from low pH quenching in live cells and from external protease in spheroplasts. In a revised topological map, Prm1 has four transmembrane domains and two large extracellular loops. Mutation of all four cysteines in the extracellular loops blocked disulfide bond formation and destabilized the Prm1 homodimer without preventing Prm1 transport to contact sites in mating pairs. Cys¹²⁰ in loop 1 and Cys⁵⁴⁵ in loop 2 form disulfide cross-links in the Prm1 homodimer and are required for fusion activity. Cys¹²⁰ lies between a hydrophobic segment formerly thought to be a transmembrane domain and an amphipathic helix. An interaction between either of these regions and the opposing membrane could promote fusion.     

C-terminal dimerization activates the nociceptive transduction channel transient receptor potential vanilloid 1

Covalent modification of the specific cysteine residue(s) by oxidative stress robustly potentiates transient receptor potential vanilloid 1 (TRPV1) and sensitizes nociception. Here we provide biochemical evidence of dimerization of TRPV1 subunits upon exposure to phenylarsine oxide and hydrogen peroxide (H(2)O(2)), two chemical surrogates of oxidative stress. A disulfide bond formed between apposing cysteines ligates two C termini, serving as the structural basis of channel sensitization by oxidative covalent C-terminal modification. Systematic cysteine scanning of the C terminus of a cysteineless TRPV1 channel revealed a critical region within which any cysteine introduced phenylarsine oxide activation to mutant TRPV1. Oxidative sensitization persisted even when this region is substituted with a random peptide linker containing a single cysteine. So did insertion of this region to TRPV3, a homolog lacking the corresponding region and resistant to oxidative challenge. These results suggest that the non-conserved linker in the TRPV1 C terminus senses environmental oxidative stress and adjusts channel activity during cumulative oxidative damage by lowering the activation threshold of gating elements shared by TRPV channels.     

Cysteine-3 and cysteine-4 are essential for the thioredoxin-like oxidoreductase and antioxidant activities of Plasmodium falciparum macrophage migration inhibitory factor

Plasmodium falciparum macrophage migration inhibitory factor (PfMIF) exhibits thioredoxin (Trx)-like oxidoreductase activity but the active site for this activity and its function have not been evaluated. A bioinformatics search revealed that the conserved CXXC motif, which is responsible for Trx-like oxidoreductase activity, is absent from PfMIF. In contrast, the adjacent N-terminal Cys-3 and Cys-4 are conserved in MIF across species of malarial parasites. Mutation of either vicinal Cys-3 or Cys-4 of PfMIF abolished the Trx-like activity, whereas the mutation of the remaining Cys-59 or Cys-103 did not affect it. PfMIF has an antioxidant function. It prevents reactive oxygen species-mediated lipid peroxidation and oxidative damage of DNA as evident from DNA nicking assay. Interestingly, chemical modification of the vicinal cysteines by phenylarsine oxide (PAO), a specific vicinal thiol modifier, significantly prevented this antioxidant activity. Modification of Cys-3 and Cys-4 was confirmed by MALDI-TOF mass spectroscopy of peptide fragments obtained after cyanogen bromide digestion of PAO-modified PfMIF. Furthermore, mutation of either Cys-3 or Cys-4 of PfMIF resulted in the loss of both Trx-like oxidoreductase and antioxidant activities of PfMIF. Altogether, our results suggest that the vicinal Cys-3 and Cys-4 play a critical role in the Trx-like oxidoreductase activity and antioxidant property of PfMIF.     

SNAP-25 Contains Non-Acylated Thiol Pairs that can Form Intrachain Disulfide Bonds: Possible Sites for Redox Modulation of Neurotransmission

Intrachain disulfide bond formation among the cysteine thiols of SNAP-25, a component of the SNARE protein complex required for neurotransmitter release, has been hypothesized to link oxidative stress and inhibition of synaptic transmission. However, neither the availability in vivo of SNAP-25 thiols, which are known targets of S-palmitoylation, nor the tendency of these thiols to form intrachain disulfide bonds is known. We have examined, in rat brain extracts, both the availability of closely spaced, or vicinal, thiol pairs in SNAP-25 and the propensity of these dithiols toward disulfide bond formation using a method improved by us recently that exploits the high chemoselectivity of phenylarsine oxide (PAO) for vicinal thiols. The results show for the first time that a substantial fraction of soluble and, to a lesser extent, particulate SNAP-25 contain non-acylated PAO-binding thiol pairs and that these thiols in soluble SNAP-25 in particular have a high propensity toward disulfide bond formation. Indeed, disulfide bonds were detected in a small fraction of soluble SNAP-25 even under conditions designed to prevent or greatly limit protein thiol oxidation during experimental procedures. These results provide direct experimental support for the availability, in a subpopulation of SNAP-25, of vicinal thiols that may confer on one or more isoforms of this family of proteins a sensitivity to oxidative stress.     

A putative two pore channel AtTPC1 mediates Ca(2+) flux in Arabidopsis leaf cells

The gene encoding voltage-gated channel with high affinity for Ca(2+) permeation has not been cloned from plants. In the present study, we isolated a full-length cDNA encoding a putative Ca(2+ )channel (AtTPC1) from Arabidopsis. AtTPC1 has two conserved homologous domains, both of which contain six transmembrane segments (S1-S6) and a pore loop (P) between S5 and S6 in each domain, and has the highest homology with the two pore channel TPC1 recently cloned from rat. The overall structure is similar to the half of the general structure of alpha-subunits of voltage-activated Ca(2+) channels from animals. AtTPC1 rescued the Ca(2+) uptake activity of a yeast mutant cch1. Sucrose-induced luminescence, which reflects a cytosolic free Ca(2+) increase in aequorin-expressing Arabidopsis leaves, was enhanced by overexpression of AtTPC1 and suppressed by antisense expression of it. Sucrose-H(+) symporters AtSUC1 and 2, which depolarize membrane potential of cells receiving sucrose, also depressed a Ca(2+) increase by their antisense expression. These results suggest that AtTPC1 mediates a voltage-activated Ca(2+ )influx in Arabidopsis leaf cells.     

A structural model of pestivirus E(rns) based on disulfide bond connectivity and homology modeling reveals an extremely rare vicinal disulfide

E(rns) is a pestivirus envelope glycoprotein and is the only known viral surface protein with RNase activity. E(rns) is a disulfide-linked homodimer of 100 kDa; it is found on the surface of pestivirus-infected cells and is secreted into the medium. In this study, the disulfide arrangement of the nine cysteines present in the mature dimer was established by analysis of the proteolytically cleaved protein. Fragments were obtained after digestion with multiple proteolytic enzymes and subsequently analyzed by liquid chromatography-electrospray ionization mass spectrometry. The analysis demonstrates which cysteine is involved in dimerization and reveals an extremely rare vicinal disulfide bridge of unknown function. With the assistance of the disulfide arrangement, a three-dimensional model was built by homology modeling based on the alignment with members of the Rh/T2/S RNase family. Compared to these other RNase family members, E(rns) shows an N-terminal truncation, a large insertion of a cystine-rich region, and a C-terminal extension responsible for membrane translocation. The homology to mammalian RNase 6 supports a possible role of E(rns) in B-cell depletion.     

Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide. Evidence for a dithiol-disulfide equilibrium and implications for redox regulation.

Calcineurin (CaN) is a Ca2+-and calmodulin (CaM)-dependent serine/threonine phosphatase containing a dinuclear Fe-Zn center in the active site. Recent studies have indicated that CaN is a possible candidate for redox regulation. The inactivation of bovine brain CaN and of the catalytic CaN A-subunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO) and melarsen oxide (MEL) and by H2O2 was investigated. PAO and MEL inhibited CaN with an IC50 of 3-8 microM and the inactivation was reversed by 2, 3-dimercapto-1-propane sulfonic acid. The treatment of isolated CaN with hydrogen peroxide resulted in a concentration-dependent inactivation. Analysis of the free thiol content performed on the H2O2 inactivated enzyme demonstrated that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H2O2 could be reversed with 1,4-dithiothreitol and with the dithiol oxidoreductase thioredoxin. We propose that a bridging of two closely spaced Cys residues in the catalytic CaN A-subunit by PAO/MEL or the oxidative formation of a disulfide bridge by H2O2 involving the same Cys residues causes the inactivation. Our data implicate a possible involvement of thioredoxin in the redox control of CaN activity under physiological conditions. The low temperature EPR spectrum of the native enzyme was consistent with a Fe3+-Zn2+ dinuclear centre. Upon H2O2-mediated inactivation of the enzyme no significant changes in the EPR spectrum were observed ruling out that Fe2+ is present in the active enzyme and that the dinuclear metal centre is the target for the oxidative inactivation of CaN.     

Regulation of CA(v)3.2 Ca2+ channel activity by protein tyrosine phosphorylation

Calcium entry through Cav3.2 Ca2+ channels plays essential roles for various physiological events including thalamic oscillation, muscle contraction, hormone secretion, and sperm acrosomal reaction. In this study, we examined how protein tyrosine phosphatases or protein tyrosine kinases affect Cav3.2 Ca2+ channels reconstituted in Xenopus oocytes. We found that Cav3.2 channel activity was reduced by 25% in response to phenylarsine oxide (tyrosine phosphatase inhibitor), whereas it was augmented by 19% in response to Tyr A47 or herbimycin A (tyrosine kinase inhibitors). However, other biophysical properties of Cav3.2 currents were not significantly changed by the drugs. These results imply that Cav3.2 channel activity is capable of being increased by activation of tyrosine phosphatases, but is decreased by activation of tyrosine kinases.     

An Improved Phenylarsine Oxide-Affinity Method Identifies Triose Phosphate Isomerase as a Candidate Redox Receptor Protein

Reversible oxidation on proteins of vicinal thiols to form intraprotein disulfides is believed to be an important means by which redox sensitivity is conferred on cellular signaling and metabolism. Affinity chromatography using immobilized phenylarsine oxide (PAO), which binds preferentially to vicinal thiols over monothiols, has been used in very limited studies to isolate the fraction of cellular proteins that exhibit reversible oxidation of vicinal thiols to presumed disulfide bonds. A challenge to the use of PAO-affinity chromatography for isolation of readily oxidizable vicinal thiol proteins (VTPs) has been the lack of a disulfide reducing agent that reverses oxidation of the PAO-binding protein thiols and maintains these in the reduced state necessary to bind PAO but does not also compete with the VTPs for binding to the immobilized PAO. The present study demonstrates that the capture from a detergent-soluble rat brain extract of VTPs by PAO-affinity chromatography was improved greatly by use of the reducing agent tris(2-carboxyethyl)-phosphine which, unlike more traditional disulfide-reducing agents, does not contain a thiol group. Moreover, we show that, while a substantial fraction of total brain proteins contain PAO-binding thiols, only a fraction of these were readily and reversibly oxidized. The two most abundant of these redox-active proteins were identified as albumin and triose phosphate isomerase (TPI). We propose that TPI is a candidate intracellular redox receptor protein. The improved PAO-affinity method detailed here should enable the discovery of lower abundance novel redox-active regulatory proteins.     

Nitric oxide block of outward-rectifying K+ channels indicates direct control by protein nitrosylation in guard cells

Recent work has indicated that nitric oxide (NO) and its synthesis are important elements of signal cascades in plant pathogen defense and are a prerequisite for drought and abscisic acid responses in Arabidopsis (Arabidopsis thaliana) and Vicia faba guard cells. Nonetheless, its mechanism(s) of action has not been well defined. NO regulates inward-rectifying K+ channels of Vicia guard cells through its action on Ca2+ release from intercellular Ca2+ stores, but alternative pathways are indicated for its action on the outward-rectifying K+ channels (I(K,out)), which are Ca2+ insensitive. We report here that NO affects I(K,out) when NO is elevated above approximately 10 to 20 nm. NO action on I(K,out) was consistent with oxidative stress and was suppressed by several reducing agents, the most effective being British anti-Lewisite (2,3-dimercapto-1-propanol). The effect of NO on the K+ channel was mimicked by phenylarsine oxide, an oxidizing agent that cross-links vicinal thiols. Neither intracellular pH buffering nor the phosphotyrosine kinase antagonist genistein affected NO action on I(K,out), indicating that changes in cytosolic pH and tyrosine phosphorylation are unlikely to contribute to NO or phenylarsine oxide action in this instance. Instead, our results strongly suggest that NO directly modifies the K+ channel or a closely associated regulatory protein, probably by nitrosylation of cysteine sulfhydryl groups.     

Conformation-dependent stability of junctophilin 1 (JP1) and ryanodine receptor type 1 (RyR1) channel complex is mediated by their hyper-reactive thiols

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 microM 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.