Introduction

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Quinone structures are ubiquitous in the human environment, having both natural and anthropogenic sources. Human exposure to quinones can occur clinically, e.g., the antineoplastic anthraquinones such as doxorubicin (DXR) (Olson and Mushlin, 1990) and by environmental exposure to diesel exhaust, cigarette smoke, and industrial particulate matter (Monks and Lau, 1992). In addition, a large number of environmental contaminants from industrial sources including carbamate pesticides, naphthalene, and polyaromatic hydrocarbons are metabolized via quinone intermediates. Quinones are of significant concern to human health because their intrinsic electrophilicity can induce various patterns of acute and chronic oxidative damage to biological tissues. The biological activity of quinones has been closely associated with changes in cellular Ca²⁺ regulation in a number of cell types. However, there is a critical need to identify key Ca²⁺ regulatory proteins that are the principle targets of quinone-mediated oxidative insult and to determine the exact role that these altered macromolecules play in cellular dysfunction and organ-selective toxicity (Monks et al., 1992).

Ca²⁺ channels localized to the sarcoplasmic reticulum (SR)/endoplasmic reticulum (ER) membrane including ryanodine receptors (RyRs) (Agdahsi et al., 1997a; Quinn and Ehrlich, 1997; Zable et al., 1997) and inositol 1,4,5-trisphosphate receptors (Bootman et al., 1992, Bird et al., 1993; Kaplin et al., 1994) have been shown to be extremely sensitive to oxidation-induced changes in function elicited by chemically diverse xenobiotic oxidizing agents. More recently, nitric oxide has been demonstrated to activate cardiac RyRs by poly-S-nitrosylation (Xu et al., 1998), and nitric oxide seems to confer protection against oxidation-induced Ca²⁺ release (Aghdasi et al., 1997a). The mechanism by which diverse oxidizing agents alter Ca²⁺ channel activity has remained unclear. One possible mechanism underlying the high sensitivity of microsomal Ca²⁺ channels to oxidizing agents may involve the presence of a small number of extremely reactive (hyperreactive) sulfhydryl groups which are important for regulating aspects of function (Liu et al., 1994; Liu and Pessah, 1994). The existence of a class of hyperreactive sulfhydryl moieties associated with the RyR complex, which is several orders of magnitude more reactive than other SR protein thiols, was revealed by the ability of these sulfhydryls to rapidly and selectively form Michael adducts with a limiting concentration of the fluorogenic maleimide 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM; 0.01-1 pmol CPM/µg of SR protein). A unique feature of channel-associated hyperreactive sulfhydryl moieties is that their hyperreactivity appears to be allosterically regulated by physiological ligands such as Ca²⁺ and Mg²⁺ and by pharmacological probes such as ryanodine, neomycin, and ruthenium red (RR). The RyR complex appears to possess a biochemical "sensor" which can monitor the local redox environment. Recent advances indicating microsomal Ca²⁺ channels are under strict redox control raises an important question as to whether redox active quinones can selectively target hyperreactive sulfhydryl moieties associated with ryanodine-sensitive Ca²⁺ channels (RyRs), thereby altering microsomal Ca²⁺ transport function. Fluxes of Ca²⁺ across SR/ER stores are essential for normal cellular signaling in healthy cells. The fact that oxidative metabolism of prooxidants to active quinone structures occurs principally by the cytochrome P-450 monooxygenases localized to the microsomal membrane raises the possibility that site-selective oxidation of ryanodine-sensitive calcium channels may be relevant to early mechanisms of oxidative damage. In the present article, fluorescent kinetic labeling experiments with discriminating concentrations of CPM and intact SR membranes are utilized to validate the hypothesis that the RyR complex is uniquely sensitive to local changes in redox environment induced by the presence of reactive quinones, thereby revealing an important mechanism by which quinones can alter cellular Ca²⁺ regulation.

	Materials and Methods

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Preparation of SR Membranes. SR membrane vesicles enriched in biochemical markers of the terminal cisternae were prepared from back and hind limb skeletal muscles of New Zealand White rabbits according to the method of Saito (Saito et al., 1984). Heavy SR from rat cardiac ventricles was prepared by sucrose-density gradient centrifugation, as described previously (Pessah et al., 1990). The preparations were stored in 10% sucrose, and 5 mM imidazole (pH 7.4) at 80°C until needed.

Kinetic Fluorescence Measurement of CPM-Thioether Adducts. The nonfluorescent maleimide CPM (Molecular Probes, Eugene, OR) readily undergoes Michael addition with protein thiols producing an irreversible adduct with high fluorescent yield (Sipple, 1981). Studies aimed at quantifying the kinetics of forming CPM-thioether adducts were performed with SR protein (50 µg/ml) diluted 100-fold in solution A consisting of 100 mM KCl and 20 mM 3-(Nnorpholino)propanesulfonic acid (MOPS; pH 7.0) just before initiating an experiment. The measurement and analysis of the reaction kinetics of forming CPM-thioether adducts were performed according to the protocol of Liu et al. (1994) with minor modifications. All labeling studies utilized CPM at concentrations ranging between 0.2 and 1.0 pmol/µg SR protein) such that the SR thiol concentration greatly exceeded that of CPM. Unless otherwise noted, 50 µg/ml SR protein was exposed to 10 to 50 nM CPM. The vesicles were incubated with the test quinone in solution A for 5 min before the introduction of CPM by Hamilton syringe into a cuvette whose contents were stirred constantly at 37°C. The increase in fluorescence intensity was continuously monitored by a SML 8000 spectrofluorometer (SML Instruments Inc., Urbana, IL) interfaced with an IBM computer/recording system. Excitation and emission were set at 397 nm and 465 nm (width of slit = 4 nm), respectively. The rates of increasing fluorescence were sampled at 1 Hz and analyzed by nonlinear regression analysis (ENZFITTER, Elsevier BioSoft). Each of the agents used in the study (e.g., quinone, CaCl₂, MgCl₂) were initially examined for autofluorescence or for their ability to quench CPM fluorescence in the presence of glutathione or SR vesicles (i.e., after CPM-thioether fluorescence had reached a maxima).

SDS-polyacrylamide gel electrophoresis (PAGE). Native SR protein (10-20 reactions each at 50 µg/ml) was incubated with 1 mM Mg²⁺ or EGTA in the presence or absence of quinone compound at 37°C in solution A. After exposure of SR membranes to CPM (<1.0 pmol/µg protein) for 1 min, 2 mM N-ethylmaleimide (NEM) was added to quench the reaction. The CPM-labeled SR protein was combined and pelleted by centrifugation (90 min at 200,000g). The pellets were resuspended in a small volume of buffer and denatured with an equal volume of nonreducing sample buffer consisting of 48 mM NaH₂PO₄, 170 mM Na₂HPO₄ (pH 7.4), 6 M urea, 0.02% bromophenol blue, and 1% (w/v) SDS (final concentrations). The samples were incubated at 60°C for 10 min and 30 to 80 µg of protein was loaded onto a 3 to 10% gradient SDS-polyacrylamide gel (Laemmli, 1970) and electrophoresed at constant voltage (200 V). The fluorescent protein bands on PAGE gels were visualized at 360 nm excitation using a transilluminator and the fluorescence image photographed through a 450-nm cutoff filter. The fluorescence intensity of protein bands was digitized by a video analysis system (SPSS, Chicago, IL) and integrated by computer within the linear range of protein density.

Ca²⁺ Flux Measurement. Measurement of Ca²⁺ transport across SR membranes were performed using the absorbance dye antipyrylazo III (APIII) or the fluorescent indicator fluo-3. SR membranes (50 µg/ml) were equilibrated at 37°C with transport buffer consisting of 92 mM KCl, 20 mM K-MOPS (pH 7.0), 7.5 mM Na-pyrophosphate, and 250 µM APIII or 0.5 µM fluo-3. A coupled enzyme (CE) system consisting of 1 mM MgATP, 10 µg/ml creatine phosphokinase, and 5 mM phosphocreatine was present to regenerate ATP. Ca²⁺ fluxes were monitored by measuring APIII absorbance at 710  790 nm using a diode-array spectrophotometer (model 8452A; Hewlett Packard, Palo Alto, CA). Alternately, changes in fluo-3 florescence intensity were measured at 530 nm emission (510 nm excitation) at 37°C using a SML 8000 fluorometer. To measure the influence of quinones on Ca²⁺ efflux, SR was loaded either with six sequential additions of 20 nmol of CaCl₂, allowing the extravesicular Ca²⁺ to return to baseline between additions, or one 100 nmol addition of CaCl₂. Once the loading phase was complete, quinone or dihydroquinone was added to the cuvette to assess the influence on Ca²⁺ efflux. Alternately, quinone was added just before initiating SR Ca²⁺ loading to assess influences on initial rates of uptake. In these experiments, some of the SR was incubated with 50 nM CPM for 1 min (terminated by 50 µM glutathione reduced form) at 37°C in the presence of 1 mM free Mg²⁺ (to reduce channel-open probability) to selectively react with hyperreactive sulfhydryls to form thioether adducts. Raw data were collected digitally and analyzed by nonlinear regression analysis.

Measurement of [³H]Ryanodine Binding and Data Analysis. Equilibrium and kinetic measurements of specific high-affinity [³H]ryanodine binding were determined according to the method of Pessah et al. (1987). SR vesicles (50 µg protein/ml) were incubated with quinone (10 nM to 10 µM) in assay buffer containing HEPES (20 mM, pH 7.1), KCl (250 mM), NaCl (15 mM), CaCl₂ (25 µM), MgCl₂ (1 mM), and [³H]ryanodine (1 nM). Equilibrium studies were performed by incubating the reaction at 37°C in the dark for 3 h, at which time the samples were filtered through GF/B glass-fiber filters and washed twice with ice-cold harvest buffer composed of 20 mM Tris-HCl, 250 mM KCl, 15 mM NaCl, and 50 µM CaCl₂ (pH 7.1). Apparent association kinetics were determined in the presence and absence quinone as described above except that reactions were quenched at times ranging between 5 min and 3 h. Each assay was performed in duplicate and repeated at least twice. Nonspecific binding was determined by incubating SR vesicles with the concentration of quinone that give maximum binding and 1000-fold excess unlabeled ryanodine.

Single-Channel Kinetics in Bilayer Lipid Membranes. RyR channels were reconstituted into artificial planar lipid bilayer (5:3:2 phosphatidylethanolamine/phosphatidylserine/phosphatidylcholine, 60 mg/ml in decane) by introducing SR vesicles to the cis chamber. The cis chamber contained 0.7 ml of 500 mM CsCl, 50 µM CaCl_2, and 10 mM HEPES (pH 7.4), whereas the trans side contained 100 mM CsCl, 50 µM CaCl₂, and 10 mM HEPES (pH 7.4). Upon the fusion of SR vesicle into bilayer, the cis chamber was perfused with the identical solution, except lacking CaCl₂. Single-channel activity was measured at a holding potential of +30 mV (applied cis relative to the trans ground side) using a patch clamp amplifier (model 3900A; Dagan Co., Minneapolis, MN). The data was filtered at 1 kHz before acquisition at 10 kHz by a DigiData 1200 (Axon Inst., Foster City, CA). The data were analyzed using pClamp 6 (Axon Instruments, Burlingame, CA) without additional filtering.

	Results

Top Summary Introduction Materials and Methods Results Discussion References

Quinones Decrease Kinetics of Forming CPM-Thioether Adducts. The presence of pharmacological or physiological agents that promote SR Ca²⁺ channel closure have been shown to enhance significantly the rate by which CPM forms Michael adducts with hyperreactive sulfhydryl moieties localized on RyR1 (skeletal isoform of ryanodine receptor) and channel-associated proteins found within the triad junction (Liu et al., 1994; Liu and Pessah, 1994). Figure 1, A and B (traces labeled 0), show the rapid kinetics of adduct formation between 1 pmol CPM/µg skeletal junctional SR in the presence of 7 µM Ca²⁺ and 1 mM Mg²⁺ (calculated initial rate, k = 0.0275 ± 0.0035 s¹; mean of 12 determinations). Under these conditions, the rate of CPM-thioether adduct formation was reduced in a dose-dependent manner by a 30-s pretreatment of SR membranes with 1,4-naphthoquinone (NQ) or 1,4-benzoquinone (BQ). The maximal concentration of NQ or BQ used in the present experiments (2 µM) decreased the initial rate of CPM labeling >10-fold (k = 0.0023 ± 0.0007 with NQ, mean of four determinations), when compared with rates obtained in the absence of quinone. The presence of reactive quinone when channel closure is favored (in the presence of 1 mM Mg²⁺) qualitatively and quantitatively mimics results obtained with a physiological channel activator, e.g., the presence of 100 µM Ca²⁺, in reducing CPM labeling kinetics (to k = 0.0024 s¹; Liu et al., 1994), but differs in the mechanism by which channel activation is obtained.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Quinone compounds dose-dependently diminish the kinetics of CPM-thioether adduct formation with skeletal junctional SR membrane proteins. SR vesicles (50 µg/ml) were equilibrated at 37°C in solution A containing 100 mM KCl, 20 mM MOPS (pH 7.0) and 1 mM MgCl2 for 3 min. The indicated concentration of NQ (A) or BQ (B) was added to the mixture and incubated for 30 s before CPM (50 nM, i.e., 1 pmol/µg protein) was introduced to the mixture to initiate fluorescent thiol labeling as described in Materials and Mehtods. For each control trace (labeled 0), 10 µl of solvent (dimethyl sulfoxide) was added instead of quinone. C, NQ (), BQ (×), NQS (), DXR (+), or THQ () was added and incubated for 30 s (3 min for DXR) before labeling with CPM. The experiments shown in A and B are representatives of at least three determinations and produced similar results. The data shown in C were fit with a single- or double-exponential model for the samples treated with 1 mM MgCl2 or quinone, respectively. The apparent rate constants (k) were calculated from apparent half-time (T1/2). Each datum point is the average from three experiments. The quinone compounds at concentrations used did not interfere with CPM fluorescence in the absence of SR membranes.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   NQ, BQ, and DXR dramatically reduce the rate of CPM-thioether adduct formation with cardiac junctional SR membrane proteins. Cardiac SR vesicles (50 µg/ml) were equilibrated at 37°C in solution A in the presence of 1 mM MgCl2 for 3 min. Dimethyl sulfoxide (10 µl, trace labeled "Quinone"), NQ (2 µM), or BQ (2 µM) was added to the mixture and incubated for 30 s. DXR was preincubated longer than NQ or BQ (3 min). CPM (10 nM, i.e., 0.2 pmol/µg protein) was introduced to the mixture and the increase in CPM fluorescence was monitored. The experiment was repeated with two independent preparations.

Quinones Alter Hyperreactive Sulfhydryls on RyR1 and Triadin. The identity of protein(s) labeled by CPM in the presence and absence of quinone was determined by visualizing fluorescent labeled bands after SDS-PAGE as described in Materials and Methods. Consistent with previous findings, SR labeled for 1 min in a medium containing 10 nM CPM and 1 mM Mg²⁺, but lacking quinone, revealed CPM fluorescence was predominantly localized to the RyR1 protomer of M_r 565,000, a major proteolytic fragment of RyR1 of M_r 150,000 (Meissner et al., 1989), and triadin of M_r 95,000 (Fig. 3A, lane 1 labeled Mg). A 30-s preincubation of SR with NQ (2 µM), BQ (2 µM), or NQS (10 µM) before labeling with CPM for 1 min revealed a selective loss of fluorescence associated with RyR1 and triadin protein bands (Fig. 3A, lanes 2-4 labeled NQ, BQ, and NQS, respectively). Digital imaging of the fluorescent bands on gels revealed a >98% decreased in the CPM fluorescence intensity associated with the RyR1 protomer and triadin in SR-pretreated with quinone compared to control SR treated with Mg²⁺ alone (Fig. 3A, left panel). However, no significant change in the pattern of CPM labeling was detected with SR pretreated with fully reduced THQ (50 µM for 30 min; Fig. 3B, lane labeled THQ) when compared with the control SR (lane labeled ). Consistent with the behavior of DXR in CPM kinetic labeling experiments, a higher concentration and longer pretreatment time were needed for anthraquinone to alter the pattern of fluorescent labeling on SDS-PAGE. The degree to which DXR (50 µM) decreased CPM labeling on RyR1 and triadin protomers by a detectable level was dependent on the length of time SR was exposed to the drug. SR protein pretreated with DXR for 3, 10, and 30 min largely eliminated detectable fluorescence associated with these bands (Fig. 3A, DXR lanes 5, 6, and 7, respectively). Importantly, 5-iminodaunorubicin (IDAU; 50 µM), which lacks redox activity, fails to alter the pattern of CPM labeling even with several hours of incubation (Fig. 3B, lane labeled IDAU).

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Quinones selectively influence CPM labeling on RyR and triadin protomers in skeletal SR as revealed by CPM fluoresce on SDS-PAGE. SR protein (50 µg/ml; total 1 mg) in solution A was preincubated under varied conditions. A, in the presence of 1 mM MgCl2 alone (lane 1), or 1 mM MgCl2 plus 2 µM NQ (lane 2), 2 µM BQ (lane 3), or 10 µM NQS (lane 4) for 30 s, respectively at 37°C; or 50 µM DXR for 3, 10, and 30 min (lanes 5, 6, 7, respectively) at 37°C. Note the change in CPM labeling from RyR protomer (Mr ~565,000), a band at Mr ~160,000 (possibly a major proteolytic fragment of RyR) and triadin (Mr ~ 5,000) to the abundant Ca2+ ATPase (Mr ~110,000). B, in the absence or presence of redox-inactive THQ (50 µM) or IDAU (50 µM) for 30 min or 16 h, respectively, does not alter the pattern of CPM fluorescence. C, SR was treated in the presence of 0.2 mM EGTA and 0, 10, 20, 30, or 40 pmol of NQ/µg of protein for 30 s at 37°C. In each case CPM (1 pmol/µg protein) was added to the mixture, incubated for 1 min, and the reaction was quenched with 2 mM NEM. The CPM-labeled protein was denatured in SDS sample buffer in the absence of reducing agents. Thirty micrograms of protein from each sample was loaded onto a 3 to 10% gradient gel and electrophoresed at 200 V. The fluorescent proteins on the gels were visualized on a transilluminator, photographed through a filter, and digitized as described in Materials and Methods.

Nanomolar Quinone Alters Ca²⁺ Transport across Actively Loaded SR Vesicles. Figure 4A shows that NQ mobilizes Ca²⁺ from actively loaded SR in a dose-dependent manner that quantitatively parallels its ability to diminish labeling of hyperreactive SR thiols with CPM. In the presence of 50 µg/ml SR protein and transport buffer containing ATP and CE, the Ca²⁺-sensitive dye APIII responded to addition of 100 µM Ca²⁺ with an abrupt rise in absorbance which was followed by a rapid decrease that stemmed from the uptake of Ca²⁺ into SR vesicles. Addition of 300 nM to 2 µM NQ induced a net efflux of Ca²⁺ from SR attributable to activation of the RyR1 complex. As expected, addition of 2 µM RR during the release phase blocks the channel and results in reaccumulation of Ca²⁺ despite the presence of NQ. The threshold for NQ-induced Ca²⁺ release ranged between 50 and 100 nM (n = 12 determinations). NQ was not found to interfere with the APIII dye signal at the concentrations used in these experiments by final addition of ionophore A23187 to calibrate the signal (Fig. 4A). Similar effects on Ca²⁺ transport were observed with BQ (Fig. 4B). After the Ca²⁺ loading phase in which six additions of 20 µM CaCl₂ were made to the SR mixture, addition of BQ (300 nM to 2 µM) induced a dose-dependent release of accumulated Ca²⁺ which could largely be inhibited by prior addition of 2 µM RR (Fig. 4B, lowest trace). Consistent with findings obtained from CPM-labeling kinetics, 5- to 6-fold higher concentrations of NQS were required to produce release rates comparable to NQ and BQ (not shown).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   NQ and BQ induce Ca2+ release from actively loaded skeletal SR vesicles in a dose-dependent and ruthenium-sensitive manner. SR vesicles (50 µg/ml) were added to Ca2+ transport assay buffer. MgATP (1 mM) and CE were added (first arrow). CaCl2 (100 µM) was added to the mixture (second arrow) and absorbance was monitored as described in Materials and Methods. A, when Ca2+ uptake was complete, the indicated concentration of NQ was added (third arrow). Once release proceeded for 10 min, 2 µM RR (fourth arrow) was introduced to block RyR. Ionophore A23187 (2 µg) was added at the end of each assay to calibrate the signal and assure equal Ca2+ loading of the vesicles. B, Ca2+ uptake was carried out by six sequential additions of 20 µM CaCl2 to the vesicle mixture. BQ potently induced Ca2+ release from SR and its actions could be blocked by preincubation with RR (2 µM). These experiments were repeated three times with similar results.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Quinone-mediated reduction of active Ca2+ accumulation by SR is selectively eliminated by formation of CPM adducts with hyperreactive SR sulfhydryl moieties. SR membranes (50 µg/ml) were equilibrated in transport buffer at 37°C containing 1 mM MgCl2, 1.4 mM MgATP, and CE with either APIII (A and B) or fluo-3 (C) as a Ca2+ indicator. Reaction mixtures were then exposed for 1 min to either 1% dimethyl sulfoxide as vehicle control (A) or 50 nM CPM (B) and terminated by 50 µM reduced glutathione. One of the following was subsequently added to each reaction mixture: 1 µM TG (trace 1), 2 µM NQ (trace 2), 1% dimethyl sulfoxide (trace 3), or 10 µM RR and 2 µM NQ (trace 4). After 15 min of additional incubation, uptake was initiated by addition of Ca2+ (90 µM), and APIII absorbance was monitored as described in Materials and Methods. After the Ca2+-loading phase was complete, TG (1 µM) was added to each cuvette to induce efflux of accumulated Ca2+ from the vesicles and to calibrate the dye. C, SR was pretreated in the presence of 1 mM MgCl2 and the indicated concentration of CPM for 1 min. DXR (30 µM) or its solvent [trace labeled DXR ()] was added into the reaction mixture and100 µM CaCl2 (Ca) (formed free Ca2+ ~ 10 µM in the presence of pyrophosphate) and 0.5 µM fluo-3 were subsequently introduced. Fluorescence measurement of Ca2+ uptake was initiated by the addition of 1 mM MgATP and CE. At the end of each experiment, 1 µM Ca2+ ionophore 4-Br-A23187 was added to the cuvette to determine the amount of Ca2+ loaded into the vesicles, and 1 mM EGTA was added to chelate Ca2+ and to calibrate the dye. The experiments were repeated with three different independent preparations and similar results were obtained.

Concentration- and Time-Dependent Mechanism by Which NQ Modifies RyR Function. To further elucidate the mechanism underlying NQ-mediated effects on vesicular Ca²⁺ transport, the actions of NQ on the binding of [³H]ryanodine to SR membranes were examined under equilibrium and kinetic conditions. Figure 6A reveals that the ability of NQ to modify equilibrium binding of [³H]ryanodine to SR (12.5 µg of protein) was highly dependent on concentration. Under assay conditions which were less than optimally favorable for the binding of [³H]ryanodine (25 µM Ca²⁺, 1 mM Mg²⁺), nanomolar NQ enhanced occupancy of [³H]ryanodine to SR membranes nearly 3-fold, with an EC₅₀ = 123 nM (2.46 pmol NQ/µg SR; Fig. 6A). By contrast, low micromolar NQ inhibited the binding of [³H]ryanodine to high-affinity sites with an IC₅₀ = 1.2 µM (24 pmol NQ/µg SR; Fig. 6A).

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Concentration- and time-dependent activation and inactivation of [3H]ryanodine-binding activity of SR. SR membranes (50 µg/ml) were incubated without or with NQ in a buffer containing HEPES (20 mM), pH 7.1, KCl (250 mM), NaCl (15 mM), CaCl2 (25 µM), MgCl2 (1 mM), and [3H]ryanodine (1 nM). A, equilibrium-binding experiments showing activation (EC50 = 123 nM) and inhibition (IC50 = 1.2 µM) of the [3H]ryanodine binding by NQ assayed at 37°C in the dark for 3 h. Control activity in the absence of NQ was 0.12 pmol/mg. Optimal activation was 0.30 pmol/mg. B, time-dependent binding assay was carried out in the absence () and presence of 500 nM () or 5 µM () NQ for 5 to 180 min at 37°C in the dark. Reactions were quenched by filtering samples through GF/B glass filters and washing the samples with ice-cold harvest buffer as described in Materials and Methods. Data presented are the mean of two independent measurements, each performed in duplicate.

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View larger version (55K): [in this window] [in a new window]   Fig. 7.   Single-channel kinetics in BLMs. Single channel of RyR was reconstituted into planar lipid bilayer following addition of SR vesicles to the cis side of the bilayer. For all experiments, the cis chamber contained 500 mM CsCl, 50 µM CaCl2, and 10 mM HEPES (pH 7.4), and trans contained 100 mM CsCl, 50 µM CaCl2, and 10 mM HEPES (pH 7.4). After fusion of SR into the bilayer, the cis chamber was perfused with the same solution lacking Ca2+. The Ca2+ was then adjusted to a concentration indicated. After acquiring a period of control data without and with 1 mM MgCl2, 200 nM (A) or 2 µM (B) NQ was added cis. Currents through the single channel were recorded at a holding potential of +30 mV for 30 to 40 min. The o and c indicate current levels of fully open and closed channels, respectively. Each experiment is a representative of 6 and 12 channels, respectively. C and D, a diary plot of results obtained from experiments at 200 nM and 2 µM NQ.

View larger version (42K): [in this window] [in a new window]   Fig. 8.   Reversibility of the actions of NQ on single-channel gating kinetics. After the fusion of a RyR single channel into BLM, the channel activity was continuously recorded through sequential changes in cis solution as indicated below the graph. To test reversibility, cis perfusion was performed with 15 ml of identical buffer containing no NQ. The times denoted in the graph indicates the length of the time the channel was exposed to 200 nM NQ (6 min) or 2 µM NQ (2 min or 15 min) before perfusion. The channel-open probability was determined by measuring >20 s of recording at + 30 mV holding potential. This is representative of three independent experiments with similar results.

	Discussion

Top Summary Introduction Materials and Methods Results Discussion References

The present results reveal that the RyR1 complex represents one of the most sensitive biological targets yet described for reactive quinones. Utilizing three different measures of channel function (analysis of [³H]ryanodine-binding, macroscopic SR Ca²⁺ transport, and single channels in BLM), nanomolar quinone is found to promote channel activation by a mechanism which modifies a very small number of hyperreactive cysteine residues localized primarily on the RyR and triadin. In this respect, the intact quinone moiety is essential for activity toward the channel since reduced forms such as THQ and IDAU have no significant effect on CPM-labeling kinetics, localization of fluorescence, or SR function (Pessah et al., 1990). This observation suggests that reactive quinones enhance channel-open state by a mechanism which alters the oxidation state of hyperreactive cysteines, a mechanism which is apparently conserved between skeletal and cardiac RyR isoforms. Previously, we showed that the rate constant (k) for CPM-thioether adduct formation is proportional to the number of free sulfhydryl groups which are available for CPM labeling, (i.e., k = K_m [SH]_t) (Liu et al., 1994). The present results suggest that nanomolar naphtho- or benzoquinone cause a quantitative diminution in the total number of hyperreactive thiol groups associated with SR membranes, as revealed by the dose-dependent slowing of CPM-labeling kinetics. Comparing Figs. 1 and 3 reveals that the slower kinetics of CPM labeling of SR induced by quinones is associated with a selective disappearance of CPM labeling from channel-associated protein thiols. These data can be explained by one of three mechanisms (schemes 1-3, Fig. 9). Common to each mechanism is the presence of a nucleophilic domain within the RyR-triadin complex which renders a small number of cysteines hyperreactive (Fig. 9, shaded regions of schemes 1-3).

View larger version (18K): [in this window] [in a new window]   Fig. 9.   Three possible mechanisms by which quinones modulate channel function. Scheme 1, redox cycling model for oxidation-induced channel activation by quinones. Scheme 2, nucleophilic arylation-induced channel activation by quinones. Scheme 3, redox-sensing model for quinone-induced channel activation. See text for details.

In Fig. 9, scheme 1, reactive quinones preferentially oxidize hyperreactive thiols to intramolecular or intermolecular disulfide bonds. Such a mechanism would be consistent with the hypothesis of oxidation-induced Ca²⁺ release as proposed by Abramson and Salama (1989) in which one or more intramolecular oxidations of critical thiols on the channel complex to disulfides (possibly as a result of redox cycling with quinone) are coupled to channel activation. Implicit in this mechanism is the requisite oxidation and reduction of critical thiols coincident with channel opening and closing. In scheme 1 (Fig. 9), naphtho- and anthraquinones accept one electron from hyperreactive thiols, thereby enhancing channel activation as a direct result of oxidizing "critical" channel thiols to disulfides. Whether oxidation/reduction of critical thiols is rapid enough to account for rapid channel transitions characteristic of RyR remains unproved. However, it is unlikely that the stimulatory actions of nanomolar quinone can be attributed to oxidation to intramolecular or intermolecular disulfides because: 1) the BQ semiquinone is extremely electrophilic, making it more likely that BQ will undergo arylation than redox-cycling reactions; 2) the in vitro conditions used in the present study lack reducing cofactor to drive redox cycling; and 3) channel activation induced by reactive quinones is readily reversible in the absence of reducing agent.

In Fig. 9, scheme 2, quinones undergo nucleophilic addition to hyperreactive thiols, resulting in an arylated channel complex. This mechanism implies that normal channel gating does not proceed with a requisite change in oxidation of critical receptor thiols to disulfides per se. Alternately, the formation of arylated thio- adducts induces allosterism which promotes channel activation. Again, this mechanism is less plausible considering the reversible nature of quinone-mediated channel activation. Furthermore, NQ is a better redox cycler than it is an arylator (Monks et al., 1992) and at low concentration (nanomolar) enhances channel activation in a manner indistinguishable from DXR, a pure redox cycler. Finally, this mechanism seems untenable when one considers that nucleophilic addition of CPM to hyperreactive thiols does not itself alter Ca²⁺ uptake but rather removes the ability of reactive quinones from affecting changes in Ca²⁺ uptake (Fig. 5).

In Fig. 9, scheme 3, agents that enhance channel-open probability (Ca²⁺, adenine nucleotides, caffeine, etc.) influence a conformational transition to the open state of the channel that masks the nucleophilic domain and dramatically reduces the reactivity of functionally critical cysteines. In scheme 3 (Fig. 9), the formation and elimination of a nucleophilic domain with native channel transitions in conformation corresponds to the appearance and disappearance of hyperreactive thiols detected by CPM fluorescence. Reactive quinones such as NQ, by virtue of their electrophilic redox potentials (NQ E_redox = +36 mV; Clark, 1960) would be expected to perturb the redox microenvironment within the nucleophilic domain wherein hyperreactive thiols reside. The presence of low concentrations (nanomolar) of quinone would further enhance the nucleophilicity of hyperreactive thiols which could aid in promoting the deprotonation of R-SH to R-S' + H⁺. In the deprotonated state, the hyperreactive thiols may contribute significantly to decrease the stability of the closed state through disruption of key noncovalent interactions. Although the present study does not directly prove the mechanism proposed in scheme 3 of Fig. 9, several experimental observations are consistent with a redox-sensing model. The potency and rapidity with which channel activation occurs appears to follow the standard redox potential of the quinone. BQ (E_redox +293 mV; Clark, 1960) and NQ (E_redox +36 mV; Fig. 4) were found to be substantially more potent and rapid than anthraquinones (typical E_redox < 150 mV) in releasing SR Ca²⁺ (Abramson et al., 1988; Pessah et al., 1990). Consistent with the model, NQ and BQ were also significantly more potent than DXR toward decreasing the rate of CPM labeling of hyperreactive thiols. The apparently higher potency of NQ compared to that of BQ in the CPM assay probably stems from the extreme nucleophilicity of the latter, which is expected to decrease the actual free concentration of quinone in aqueous solution. The concept of redox sensing by the Ca²⁺ channel complex is supported by the observation that pretreatment of SR with a concentration of CPM known to derivatize a large fraction of the channel-associated hyperreactive thiols dramatically reduces the sensitivity of the channel to activation by NQ and DXR. By destabilizing the closed state, the redox-sensing hypothesis (Fig. 9, scheme 3) could account for why anthraquinones can so effectively sensitize the channel to activation by Ca²⁺ (Abramson et al., 1988; and Pessah et al., 1990).

Nanomolar NQ and BQ induce rapid and selective loss of hyperreactive thiol groups on RyR1 and triadin protomers, and the immediate functional consequence is enhanced channel activity and net SR Ca²⁺ efflux. These results are in agreement with those of Aghdasi et al. (1997b) who found that channels incubated with a high concentration of NEM for increasing periods of time display three distinct phases of functional effects. However, the experimental design of Aghdasi et al al. (1997b) did not account for the conformational state of the channel before addition of sulfhydryl reagent nor was the molar ratio of sulfhydryl reagent relative to SR protein adjusted to <1 pmol/µg protein. For these reasons, labeling was not limited to the most reactive channel thiols and comparisons about the functional consequence of sulfhyrdryl oxidation cannot be directly compared with the present study which addresses the functional ramifications of site-selective modification of the most reactive channel-associated thiols. The ability of NQ at a higher concentration to produce biphasic actions on both channel function and [³H]ryanodine-binding kinetics support this interpretation since under these conditions it would be expected to 1) arylate protein thiols and 2) oxidize hyperreactive and less reactive but more abundant channel-associated thiols to disulfides. Experiments with additional quinone structures which exclusively arylate or redox cycle should clarify the relationship between chemical mechanism at the Ca²⁺ channel complex and functional response.

Micromolar NQ clearly shows biphasic actions on the binding of [³H]ryanodine, first enhancing occupancy followed by inhibition (Fig. 6), whereas anthraquinones only enhance the binding of [³H]ryanodine to SR across their dose-response range (1-200 µM) (Abramson et al., 1988; Pessah et al., 1990). Channel inactivation at high concentrations and longer exposure of the RyR complex to NQ appears to proceed by a mechanism different from that seen with nanomolar NQ. The irreversible mechanism could stem from 1) oxidation of critical thiols or disulfides; 2) oxidation of another, less reactive, class of channel thiols to disulfides; or (3) arylation of the channel complex. In this respect, the actions of anthraquinones, which are poor arylators, have been shown to activate the gating of single RyR channels reconstituted in BLM in a persistent manner without a subsequent phase of inhibition (Holmberg and Williams, 1990; Buck and Pessah, 1995). Ondrias et al. (1990) have, however, reported that DXR exhibits biphasic actions in channels reconstituted from cardiac muscle. Despite the apparent discrepancy in the reported effects of DXR between laboratories (monophasic versus biphasic), it is unlikely anthraquinones promote channel inactivation. Indeed, radioligand-binding experiments with [³H]ryanodine and skeletal (Abramson et al., 1988) or cardiac (Pessah et al., 1990) SR demonstrated only DXR-induced activation of ligand binding, even after several hours of incubation in the presence of anthraquinone.

We provide the first direct evidence for a molecular mechanism by which quinones of toxicological concern selectively target a microsomal Ca²⁺ channel. Importantly, the present results raise the possibility that microsomal Ca²⁺ channels may actually utilize hyperreactive sulfhydryl chemistry in "sensing" localized changes in the redox environment. In this respect, the injurious effects of quinones have been attributed to their ability to 1) undergo redox cycling, thereby generating reactive oxygen species; and 2) directly arylate biological macromolecules (Monks et al., 1992). In both muscle and nonmuscle cells, the acute and chronic toxicity mediated by quinones or their precursor molecules are known to be closely associated with a rise in cellular Ca²⁺ that initiates functional and structural changes which eventually lead to cell death (Farber, 1990; Reed, 1990; Nicoterra et al., 1992). Increased intracellular Ca²⁺ is known to activate proteases (Nicoterra et al., 1986; Lee et al., 1991), endonucleases (McConkey et al., 1988), phospholipases C (Berridge et al., 1987) and A₂ (Exton, 1990), and kinases (Shulman and Lou, 1989). Quinones that alter normal Ca²⁺ signaling can be expected to alter Ca^2+-dependent biochemical cascades responsible for maintenance of cellular homeostasis and function. The hypothesis that nonselective peroxidation of membrane lipids can fully account for the loss of ion barriers and the cytotoxicity of quinonoids has been questioned in recent years. Although disagreement exists concerning the sequence of events leading from quinone-mediated disruption of Ca²⁺ regulation to cell death (Herman et al., 1990), intense interest is now focused on the identity of specific cellular macromolecules which are primary targets of oxidative damage and on assessing their exact role in toxicity (Monks et al., 1992; Hinson and Roberts 1992). To date, most studies aimed at elucidating the molecular mechanisms underlying the cytotoxicity of anthraquinones (Olson and Mushlin, 1990), naphthoquinones (Frei et al., 1986), and benzoquinones (Moore et al., 1988) in a variety of cell types have examined loss of mitochondrial integrity. An added significance of the mechanism revealed in the present study is that RyRs represent a key Ca²⁺ regulatory channel that is widely expressed within microsomal membrane of a wide variety of cells where most quinone precursor molecules are metabolized to bioactive quinones by the cytochrome P-450 system. Colocalization of ryanodine-sensitive Ca²⁺ channels and cytochrome P-450 enzyme, which catalyze formation of quinone-containing compounds, could provide a fundamental mechanism by which localized oxidative stress is "sensed" by the major intracellular Ca²⁺ store. This mechanism may have both physiological and toxicological significance.