Heavy metal-induced Ca2+ release from sarcoplasmic reticulum

Two distinct forms of Ca2+ release from isolated sarcoplasmic reticulum vesicles in response to additions of heavy metals (silver and mercurials) are described. One form of heavy metal-induced Ca2+ release involves the ruthenium red-sensitive Ca2+ release channel localized in terminal cisternae. The other form of heavy metal-induced Ca2+ release appears to involve all portions of the sarcoplasmic reticulum and is insensitive to ruthenium red. This latter form of Ca2+ release occurs over a similar range of heavy metal concentrations as inhibition of the sarcoplasmic reticulum Ca2+ pump but does not appear to be a result solely of such pump inhibition. Both forms of Ca2+ release are inhibited by glutathione, an endogenous constituent of muscle fibers, and by dithiothreitol, agents which prevent sulfhydryl oxidation. To assess the role of any sulfhydryl oxidation in sarcoplasmic reticulum Ca2+ release physiologically, dithiothreitol and glutathione were introduced inside muscle fibers and effects on excitation-contraction coupling examined. The results strongly suggest that sulfhydryl oxidation plays no essential role in skeletal muscle excitation-contraction coupling.     

Chloride-dependent sarcoplasmic reticulum Ca2+ release correlates with increased Ca2+ activation of ryanodine receptors.

The mechanism by which chloride increases sarcoplasmic reticulum (SR) Ca2+ permeability was investigated. In the presence of 3 microM Ca2+, Ca2+ release from 45Ca(2+)-loaded SR vesicles prepared from procine skeletal muscle was increased approximately 4-fold when the media contained 150 mM chloride versus 150 mM propionate, whereas in the presence of 30 nM Ca2+, Ca2+ release was similar in the chloride- and the propionate-containing media. Ca(2+)-activated [3H]ryanodine binding to skeletal muscle SR was also increased (2- to 10-fold) in media in which propionate or other organic anions were replaced with chloride; however, chloride had little or no effect on cardiac muscle SR 45Ca2+ release or [3H]ryanodine binding. Ca(2+)-activated [3H]ryanodine binding was increased approximately 4.5-fold after reconstitution of skeletal muscle RYR protein into liposomes, and [3H]ryanodine binding to reconstituted RYR protein was similar in chloride- and propionate-containing media, suggesting that the sensitivity of the RYR protein to changes in the anionic composition of the media may be diminished upon reconstitution. Together, our results demonstrate a close correlation between chloride-dependent increases in SR Ca2+ permeability and increased Ca2+ activation of skeletal muscle RYR channels. We postulate that media containing supraphysiological concentrations of chloride or other inorganic anions may enhance skeletal muscle RYR activity by favoring a conformational state of the channel that exhibits increased activation by Ca2+ in comparison to the Ca2+ activation exhibited by this channel in native membranes in the presence of physiological chloride (< or = 10 mM). Transitions to this putative Ca(2+)-activatable state may thus provide a mechanism for controlling the activation of RYR channels in skeletal muscle.     

Mechanism of calmodulin inhibition of cardiac sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor)

The functional effects of calmodulin (CaM) on single cardiac sarcoplasmic reticulum Ca(2+) release channels (ryanodine receptors) (RyR2s) were determined in the presence of two endogenous channel effectors, MgATP and reduced glutathione, using the planar lipid bilayer method. Single-channel activities, number of events, and open and close times were determined at varying cytosolic Ca(2+) concentrations. CaM reduced channel open probability at <10 micro M Ca(2+) by decreasing channel events and mean open times and increasing mean close times. At >10 micro M Ca(2+), CaM was less effective in inhibiting RyR2. CaM decreased mean open times but increased channel events, without significantly affecting mean close times. A series of voltage pulses was applied to the bilayer from +50 to -50 mV and from -50 mV to +50 mV to rapidly increase and decrease open channel-mediated sarcoplasmic reticulum lumenal to cytosolic Ca(2+) fluxes. CaM decreased the duration of the open events after the voltage switch from -50 mV to +50 mV. In parallel experiments, a Ca(2+)-insensitive calmodulin mutant was without effect on RyR2 activity. The results are discussed in terms of a possible role of CaM in the termination of cardiac sarcoplasmic reticulum Ca(2+) release.     

Nitroxyl triggers Ca2+ release from skeletal and cardiac sarcoplasmic reticulum by oxidizing ryanodine receptors

The biological activity of nitric oxide (NO) and NO-donors has been extensively investigated yet few studies have examined those of nitroxyl (HNO) species even though both exist in chemical equilibrium but oxidize thiols by different reaction mechanisms: S-nitrosation versus disulfide bond formation. Here, sodium trioxodinitrate (Na2N2O3; Angeli's salt; ANGS) was used as an HNO donor to investigate its effects on skeletal (RyR1) and cardiac (RyR2) ryanodine receptors. At steady-state concentrations of nanomoles/L, HNO induced a rapid Ca2+ release from sarcoplasmic reticulum (SR) vesicles then the reducing agent dithiothreitol (DTT) reversed the oxidation by HNO resulting in Ca2+ re-uptake by SR vesicles. With RyR1 channel proteins reconstituted in planar bilayers, HNO added to the cis-side increased the open probability (Po) from 0.056+/-0.026 to 0.270+/-0.102 (P<0.005, n=4) then DTT (3 mM) reduced Po to 0.096+/-0.040 (P<0.01, n=4). In parallel experiments, the time course of HNO production from ANGS was monitored by EPR and UV spectroscopy and compared with the rate of SR Ca2+ release indicating that picomolar concentrations of HNO triggered SR Ca2+ release. Controls showed that the hydroxyl radical scavenger, phenol did not alter ANGS-induced SR Ca2+ release, indicating that hydroxyl radical production from ANGS did not account for Ca2+ release from the SR. The findings indicate that HNO is a more potent activator of RyR1 than NO and that HNO activation of RyRs may contribute to NO's activation of RyRs and to the therapeutic effects of HNO-releasing prodrugs in heart failure.     

Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca2+-permeable pore of the calcium release channel

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified by immunoaffinity chromatography as a single approximately 450,000-Da polypeptide and it was shown to mediate single channel activity identical to that of the ryanodine-treated Ca2+ release channel of the sarcoplasmic reticulum. The purified receptor had a [3H]ryanodine binding capacity (Bmax) of 280 pmol/mg and a binding affinity (Kd) of 9.0 nM. [3H]Ryanodine binding to the purified receptor was stimulated by ATP and Ca2+ with a half-maximal stimulation at 1 mM and 8-9 microM, respectively. [3H]Ryanodine binding to the purified receptor was inhibited by ruthenium red and high concentrations of Ca2+ with an IC50 of 2.5 microM and greater than 1 mM, respectively. Reconstitution of the purified receptor in planar lipid bilayers revealed the Ca2+ channel activity of the purified receptor. Like the native sarcoplasmic reticulum Ca2+ channels treated with ryanodine, the purified receptor channels were characterized by (i) the predominance of long open states insensitive to Mg2+ and ruthenium red, (ii) a main slope conductance of approximately 35 pS and a less frequent 22 pS substate in 54 mM trans-Ca2+ or Ba2+, and (iii) a permeability ratio PBa or PCa/PTris = 8.7. The approximately 450,000-Da ryanodine receptor channel thus represents the long-term open "ryanodine-altered" state of the Ca2+ release channel from sarcoplasmic reticulum. We propose that the ryanodine receptor constitutes the physical pore that mediates Ca2+ release from the sarcoplasmic reticulum of skeletal muscle.     

Evidence for a Ca2+ channel within the ryanodine receptor complex from cardiac sarcoplasmic reticulum

The solubilized [3H]ryanodine receptor from cardiac sarcoplasmic reticulum was centrifuged through linear sucrose gradients. A single peak of radioactivity with apparent sedimentation coefficient of approximately 30S specifically comigrated with a high molecular weight protein of apparent relative molecular mass approximately 400,000. Incorporation of the ryanodine receptor into lipid bilayers induced single Ca2+ channel currents with conductance and kinetic behavior almost identical to that of native cardiac Ca2+ release channels. These results suggest that the cardiac ryanodine receptor comprises the Ca2+ release channel involved in excitation-contraction coupling in cardiac muscle.     

High-affinity Ca2+-binding site inhibiting Ca2+ release from sarcoplasmic reticulum

Ca2+ release from sarcoplasmic reticulum membranes, activated by alkaline pH occurs only when EGTA is present in the release medium. Addition of very low concentrations of Ca2+ to the medium inhibits Ca2+ release. The concentration of free Ca2+ required for 50% inhibition ranges from between 5 and 20 nM in different experiments and/or membrane preparations, irrespective of whether the free Ca2+ concentration is controlled by EGTA or CDTA. Other divalent cations such as Mn2+, Ba2+, Cu2+, Cd2+ and Mg2+ also exert an inhibitory effect on Ca2+ release, with higher or lower potency than that of Ca2+. The inactivation of Ca2+ release by Ca2+ is reversible. We suggest the involvement of high-affinity Ca2+-binding sites in the control of Ca2+ release.     

Sulfhydryl oxidation and Ca2+ release from sarcoplasmic reticulum

Summary Our interest in the role of sulfhydryl groups (SH) in regulating or altering transport across biological membranes has focused on the significance of a critical SH group associated with the Ca²⁺-release protein from skeletal muscle sarcoplasmic reticulum (SR). We have shown that binding of heavy metals to this group or oxidation of this sulfhydryl to a disulfide induces rapid Ca²⁺ release from SR vesicles [1, 2] and induces contraction in skinned muscle fibers [3]. Several models are described in which oxidation and reduction might control the state of the Ca²⁺-release channel from SR.Abbreviations DTT Dithiothreitol, redox. - oxidation-reduction - SDS Sodium Dodecyl Sulfate - SH Sulfhydryl - SR Sarcoplasmic Reticulum - T-tubule Transverse tubule     

The action of carboxyl modifying reagents on the ryanodine receptor/Ca2+ release channel of skeletal muscle sarcoplasmic reticulum

Summary

In this work we show that ryanodine binding to junctional sarcoplasmic reticulum (SR) membranes or purified ryanodine receptor (RyR) is inhibited in a time — and concentration-dependent fashion by prior treatment with the carboxyl reagent dicyclohexylcarbodiimide (DCCD). Exposure of the membrane-bound RyR to the water soluble carboxyl reagents 1-ethyl-3 (3-(dimethylamino) propyl carbodiimide (EDC) or N-ethyl-pheny-lisoxazolium-3 -sulfonate (WRK) only slightly affects their ryanodine binding capacity. The amphipathic reagent N-ethoxy cabonyl-2-ethoxy-1, 2-dihydroquinaline (EEDQ) inhibited ryanodine binding at relatively high concentrations. DCCD-modifica-tion of the SR decreased the binding affinities of the RyR for ryanodine and Ca²⁺ by about 3- and 18-fold, respectively.

The single channel activity of SR membranes modified with DCCD and then incorporated into planar lipid bilayers is very low (5–8%) in comparison to control membranes. Application of DCCD to either the myoplasmic (c/s) or luminal (trans) side of the reconstituted unmodified channels resulted in complete inhibition of their single channel activities. Similar results were obtained with the water soluble reagent WRK applied to the myoplasmic, but not to the luminal side. The DCCD-modified non-active channel is re-activated by addition of ryanodine in the presence of 250_üM Ca²⁺ and is stabilized in a sub-conductance state. With caffeine, ryanodine re-activated the channel in the presence of 100_üM of Ca^2+. The results suggest that a carboxyl residue(s) in the RyR is involved either in the binding of Ca²⁺, or in conformational changes that are produced by Ca²⁺ binding, and are required for the binding of ryanodine and the opening of the Ca²⁺ release channel.     

Interdependence of ryanodine binding, oligomeric receptor interactions, and Ca2+ release regulation in junctional sarcoplasmic reticulum

We have examined ryanodine binding to its receptor (RR) and compared its effect on Ca2+ release to the Ca2+ release triggered by Ca2+ plus ATP, using vesicular fragments of junctional terminal cisternae (JTC) obtained from skeletal muscle. Ryanodine binding is slow (taking hours or days to complete) and is highly temperature (Q10 = 4) and Ca2+ dependent. At equilibrium, the extent of binding increases as the concentration of ryanodine is raised above 10(-9) M, exhibiting negative cooperativity and reaching the stoichiometry of the 560,000-Da RR chains near 10(-5) M ryanodine. The specificity of the high affinity binding is demonstrated by competitive binding of ryanodine analogs. Kinetic studies using rapid filtration show that, in the absence of ryanodine, rapid (k = 15 s-1) release of Ca2+ follows a triggering exposure of loaded JTC vesicles to perfusion media containing Ca2+ plus ATP. Induction of this release has no lag period and displays minimal temperature dependence. In contrast, prolonged exposure of JTC vesicles to low (10(-7) M) ryanodine concentrations changes the JTC to a state permitting slow (k = 1 s-1) release of Ca2+ even in the absence of the Ca2+ plus ATP trigger. Higher (greater than microM) concentrations of ryanodine do not allow any Ca2+ release and prevent even the release normally triggered by Ca2+ plus ATP. Our data suggest that ryanodine binds to the open state of the tetrameric RR, inducing protein conformational changes and altered oligomeric interactions. Binding of the first molecule of ryanodine to one of the four binding sites on the receptor produces a partially closed and low conductance state of the Ca2+ release channel and reduces the ryanodine binding affinity of the remaining sites. Ryanodine occupancy of all four binding sites on the receptor completes closure of the Ca2+ channel and blocks the triggering action of Ca2+ plus ATP. The tetrameric association of the RR chains is demonstrated by crosslinking with bifunctional reagents, generating crosslinked tetramers that retain ryanodine binding and Ca2+ release functions.     

Activation of the ryanodine receptor Ca2+ release channel of sarcoplasmic reticulum by a novel scorpion venom.

We identified a peptide fraction from the venom of the scorpion Buthotus hottentota that stimulated binding of [3H]ryanodine to ryanodine receptors of skeletal and cardiac sarcoplasmic reticulum and brain microsomes in a highly specific manner. Activity was concentrated in a peptide fraction of Mr 5,000-8,000. Assuming a single active peptide in this fraction, we estimated a dissociation constant of 20-30 nM for the interaction of the peptide with the ryanodine receptor. The whole venom and the purified fraction activated skeletal ryanodine receptor Ca2+ release channels incorporated into planar lipid bilayers. The venom produced a 10-fold increase in the mean open time and induced the appearance of a long lasting subconductance state not seen in controls. Changes were reversible and could be induced by the partially purified venom fraction. This novel scorpion venom should be helpful in establishing the role of ryanodine receptors in the initiation of intracellular Ca2+ release in striated muscle and in nonmuscle cells containing functional ryanodine receptors such as neurons and secretory cells.     

Inhibitors of Ca2+ release from the isolated sarcoplasmic reticulum. I. Ca2+ channel blockers

The effects of Ruthenium red and tetracaine, which inhibit Ca2+-induced Ca2+ release from the isolated sarcoplasmic reticulum (e.g., Ohnishi, S.T. (1979) J. Biochem. (Tokyo) 86, 1147-1150), on several types of Ca2+ release in vitro were investigated. Ca2+ release was triggered by several methods: (1) addition of quercetin or caffeine, (2) Ca2+ jump, and (3) replacement of potassium gluconate with choline chloride to produce membrane depolarization. The time-course of Ca2+ release was monitored using stopped-flow spectrophotometry and arsenazo III as a Ca2+ indicator. Ruthenium red inhibited all of these types of Ca2+ release with the same concentration for half-inhibition C1/2 = 0.08-0.10 microM. Similarly, tetracaine inhibited these types of Ca2+ release with C1/2 = 0.07-0.11 mM. Procaine also inhibits both types of Ca2+ release induced by method 2 and 3 with C1/2 = 0.67-1.00 mM. These results suggest that Ruthenium red, tetracaine and procaine interfere with a common mechanism of the different types of Ca2+ release. On the basis of several pieces of evidence we propose that Ruthenium red and tetracaine block the Ca2+ channel of sarcoplasmic reticulum.     

Drug-induced Ca2+ release from isolated sarcoplasmic reticulum. I. Use of pyrophosphate to study caffeine-induced Ca2+ release

A demonstration is made of pyrophosphate's use as a precipitating anion in studies of Ca2+ release from isolated sarcoplasmic reticulum (SR). Not only does pyrophosphate speed up the rate at which Ca2+ can be preloaded into SR, but it also allows the accumulated Ca2+ to be released in response to agents such as caffeine. Because so much Ca2+ can be preloaded into SR with pyrophosphate present, more experiments can be performed with a given amount of SR material, and even rapid Ca2+ release rates (greater than 1 mumol/mg X min) are maintained for many seconds. These rates can easily be quantified using conventional spectrophotometric and isotopic methods, without the need for expensive rapid mixing equipment. Caffeine-induced Ca2+ release is exhibited by triadic and terminal cisterna SR subfractions but not by light SR. Caffeine specifically increases the rate of unidirectional 45Ca2+ efflux. This increased efflux is blocked by ruthenium red at submicromolar concentrations and by tetracaine, 9-aminoacridine, or Ba2+ at submillimolar concentrations.     

Conformational changes of the Ca2+-ATPase as early events of Ca2+ release from sarcoplasmic reticulum

Rabbit skeletal sarcoplasmic reticulum vesicles were loaded with Ca2+ by ATP-dependent Ca2+ accumulation in the presence of low [Mg2+] (0.2-0.5 mM), and Ca2+ release was induced by addition of caffeine or ADP or by means of a Ca2+ jump. The levels of the phosphorylated intermediate (EP) and the tryptophan fluorescence of the Ca2+-ATPase were monitored during both the Ca2+ accumulation and the induced Ca2+ release using fast kinetic techniques. During Ca2+ uptake, both the EP level and the tryptophan fluorescence gradually decreased following a time course similar to that of the Ca2+ accumulation. Upon inducing Ca2+ release by addition of either caffeine or ADP, there was a transient increase of the EP level (from 0.3-0.5 to 1-2 nmol/mg protein) preceding the release of Ca2+. Similarly, a transient increase of the tryptophan fluorescence prior to Ca2+ release produced by the application of a Ca2+ jump was also found. These results indicate that the Ca2+-ATPase enzyme undergoes a rapid conformational change in response to triggering of Ca2+ release.     

The luminal Ca2+ transient controls Ca2+ release/re-uptake of sarcoplasmic reticulum

Our recent study (Saiki, Y., and Ikemoto, N., Biochemistry 38, 3112-3119, 1999) suggests that Ca2+ release and re-uptake of the released Ca2+ are coordinated. The following results suggest that the coordination is mediated by the luminal Ca2+ ([Ca2+]lum) transient. Upon inducing the release of the passively loaded Ca2+ from the SR with polylysine, the luminal Ca2+ ([Ca2+]lum) first increased then decreased ([Ca2+]lum transient). The activity of the SR Ca2+ ATPase was monitored at different times after inducing Ca2+ release. The phosphoenzyme (EP) formation as determined by the MANT-fluorescence increased concurrently with the initial rapid increase in the [Ca2+]lum. EP decay (pumping turnover) was accelerated concurrently with a decrease of the [Ca2+]lum. The results suggest that the [Ca2+]lum transient serves as a mediator for the acceleration of the Ca2+ re-uptake occurring soon after the induction of Ca2+ release.     

Ca2+-induced Ca2+ release from fragmented sarcoplasmic reticulum: Ca2+-dependent passive Ca2+ efflux

Characterization of the putative Ca2+-gated Ca2+ channel of sarcoplasmic reticulum, which is thought to mediate Ca2+-induced Ca2+ release, was carried out in order to elucidate the mechanism of Ca2+-induced Ca2+ release. Heavy and light fractions of fragmented sarcoplasmic reticulum isolated from rabbit skeletal muscle were loaded passively with Ca2+, and then passive Ca2+ efflux was measured under various conditions. The fast phase of the Ca2+ efflux depended on the extravesicular free Ca2+ concentration and was assigned to the Ca2+ efflux through the Ca2+-gated Ca2+ channel. Vesicles with the Ca2+-gated Ca2+ channels comprised about 85% of the heavy fraction and about 40% of the light fraction. The amount of Ca2+ loaded in FSR was found to be much larger than that estimated on the basis of vesicle inner volume and the equilibration of intravesicular with extravesicular Ca2+, indicating Ca2+ binding inside FSR. Taking this fact into account, the Ca2+ efflux curve was quantitatively analyzed and the dependence of the Ca2+ efflux rate constant on the extravesicular free Ca2+ concentration was determined. The Ca2+ efflux was maximal, with the rate constant of 0.75 s-1, when the extravesicular free Ca2+ was at 3 microM. Caffeine increased the affinity for Ca2+ of Ca2+-binding sites for opening the channel with only a slight change in the maximum rate of Ca2+ efflux. Mg2+ inhibited the Ca2+ binding to the sites for opening the channel while procaine seemed to inhibit the Ca2+ efflux by blocking the ionophore moiety of the channel.     

Regulation of Ca2+ release from sarcoplasmic reticulum in skeletal muscles

Ca²⁺ release from skeletal sarcoplasmic reticulum (SR) could be regulated by at least three mechanisms: 1) Ca²⁺, 2) calmodulin, and 3) Ca²⁺/calmodulin-dependent phosphorylation. Bell-shaped Ca²⁺-dependence, of Ca²⁺ release from both actively- and passively-loaded SR vesicles suggest that opening and closing of the Ca²⁺ release channel could be regulated by [Ca²⁺ _o] . The time- and concentration-dependent inhibition of Ca ²⁺ release from skeletal SR by calmodulin was also studied using passively-Ca²⁺ loaded SR vesicles. Up to 50% of Ca ²⁺ release was inhibited by calmodulin (0.01–0.5 µM); this inhibition required 5–15 min preincubation time. The hypothesis that Ca²⁺/calmodulin-dependent phosphorylation of a 60 kDa protein regulates Ca²⁺ release from skeletal SR was tested by stopped-flow fluorometry using passively-Ca²⁺-loaded SR vesicles. Approximately 80% of the initial rates of Ca²⁺-induced Ca²⁺ release was inhibited by the phosphorylation within 2 min of incubation of the SR with Mg·ATP and calmodulin. We identified two types of 60 kDa phosphoproteins in the rabbit skeletal SR, which was distinguished by solubility of the protein in CHAPS. The CHAPS-soluble 60 kDa phosphoprotein was purified by column chromatography on DEAE-Sephacel, heparin-agarose, and hydroxylapatite. Analyses of the purified protein indicate that the CHAPS-soluble 60 kDa protein is an isoform of phosphoglucomutase (PGM). cDNAs encoding isoforms of PGM were cloned and sequenced using synthetic oligonucleotides. Two types of PGM isoforms (Type I and Type 11) were identified. The translated amino acid sequences show that Type II isoform is SR-form. Our results are significant in terms of understanding evidence of an association of glycolytic and glycogenolytic enzymes with SR and a role in the regulation of SR functions. (Mol Cell Biochem 114: 105-108, 1992)     

Reactive disulfide compounds induce Ca2+ release from cardiac sarcoplasmic reticulum

Reactive disulfide compounds (RDSs) with a pyridyl ring adjacent to a disulfide bond, 2,2'dithiodipyridine (2,2' DTDP) and 4,4' dithiodipyridine (4,4' DTDP), induce Ca2+ release from isolated canine cardiac sarcoplasmic reticulum (SR) vesicles. RDSs are absolutely specific to free sulfhydryl (SH) groups and oxidize SH sites of low pKa via a thiol-disulfide exchange reaction, with the stoichiometric production of thiopyridone in the medium. As in skeletal SR, this reaction caused large increases in the Ca2+ permeability of cardiac SR and the number of SH sites oxidized by RDSs was kinetically and quantitatively measured through the absorption of thiopyridone. RDS-induced Ca2+ release from cardiac SR was characterized and compared to the action of RDSs on skeletal SR and to Ca2(+)-induced Ca2+ release. (i) RDS-induced Ca2+ release from cardiac SR was dependent on ionized Mg2+, with maximum rates of release occurring at 0.5 and 1 mM Mg2+free for 2,2' DTDP and 4,4' DTDP, respectively. (ii) In the presence of adenine nucleotides (0.1-1 mM), the oxidation of SH sites in cardiac SR by exogenously added RDS was inhibited, which, in turn, inhibited Ca2+ release induced by RDSs. (iii) Conversely, when the oxidation reaction between RDSs and cardiac SR was completed and Ca2+ release pathways were opened, subsequent additions of adenine nucleotides stimulated Ca2+ efflux induced by RDSs. (iv) Sulfhydryl reducing agents (e.g., dithiothreitol, DTT, 1-5 mM) inhibited RDS-induced Ca2+ efflux in a concentration-dependent manner. (v) RDSs elicited Ca2+ efflux from passively loaded cardiac SR vesicles (i.e., with nonfunctional Ca2+ pumps in the absence of Mg-ATP) and stimulated Ca2(+)-dependent ATPase activity, which indicated that RDS uncoupled Ca2+ uptake and did not act at the Ca2+, Mg2(+)-ATPase. These results indicate that RDSs selectively oxidize critical sulfhydryl site(s) on or adjacent to a Ca2+ release channel protein channel and thereby trigger Ca2+ release. Conversely, reduction of these sites reverses the effects of RDSs by closing Ca2+ release channels, which results in active Ca2+ reuptake by Ca2+, Mg2(+)-ATPase. These compounds can thus provide a method to covalently label and identify the protein involved in Ca2+ release from cardiac SR.