©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Thimerosal Interacts with the Ca Release Channel Ryanodine Receptor from Skeletal Muscle Sarcoplasmic Reticulum (*)

(Received for publication, September 18, 1995; and in revised form, October 20, 1995)

Jonathan J. Abramson (§) Anthony C. Zable Terence G. Favero (1) Guy Salama (2)

From the  (1)Department of Physics, Portland State University, Portland, Oregon 92707, Department of Biology, University of Portland, Portland, Oregon, and (2)Department of Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The thiol-oxidizing reagent, thimerosal, has been shown to increase the intracellular Ca concentration, to induce Ca spikes in several cell types, and to increase the sensitivity of intracellular Ca stores to inositol 1,4,5-trisphosphate. Ryanodine-sensitive stores have also been implicated in the generation of Ca oscillations induced by the addition of thimerosal. Here we report that micromolar concentrations of thimerosal stimulate Ca release from skeletal muscle sarcoplasmic reticulum vesicles, inhibit high affinity [^3H]ryanodine binding, and modify the channel activity of the reconstituted Ca release protein. Thimerosal inhibits ryanodine binding by decreasing the binding capacity (B(max)) but does not affect the binding affinity or the dissociation rate of bound ryanodine. Single channel reconstitution experiments show that thimerosal (100-200 µM) stimulates single channel activity without modifying channel conductance. The thimerosal-stimulated channel is not inhibited by heparin. Furthermore, a Ca-stimulated channel is first activated and then inhibited in a time-dependent fashion by high concentrations of thimerosal (1 mM). Once inactivated, the channel cannot be reactivated by addition of either Ca or ATP.


INTRODUCTION

Several mechanisms have been proposed to explain oscillations in the cytoplasmic Ca concentration observed during fertilization of egg cells(1, 2, 3) . Ca released from inositol 1,4,5-triphosphate (IP(3)) (^1)and/or ryanodine-sensitive Ca stores from the endoplasmic reticulum appears to be responsible for these Ca oscillations. It has been demonstrated that micromolar concentrations of the sulfhydryl reagent thimerosal (TMS) evoke similar repetitive Ca spikes. The IP(3) receptor has been strongly implicated in this process on the basis of the observations that TMS stimulates Ca release from liposomes containing the reconstituted purified IP(3) receptor(4) , increases the affinity of IP(3) for receptor binding sites(5) , and increases the potency of IP(3)-induced Ca release (4, 6) . Moreover, a monoclonal antibody raised against the IP(3) receptor blocks TMS enhanced Ca-induced Ca release and Ca oscillations in hamster eggs(7) . In contrast to these observations, the effects of ryanodine forced a reevaluation of the role of IP(3) on the release of Ca from endoplasmic reticulum of oocytes. The observation that the amplitude of Ca oscillations in response to sperm factor, IP(3), or thimerosal decreased and was eventually blocked by the addition of ryanodine suggested that ryanodine-sensitive Ca stores also play a role in generating intracellular Ca oscillations(3) .

The mechanism underlying thimerosal's effects on internal endoplasmic reticulum Ca stores appears to involve an interaction with sulfhydryl groups. The addition of reducing agents such as dithiothreitol (DTT) has been shown to inhibit TMS potentiation of IP(3)-induced Ca release in sea urchin eggs (9) and TMS-induced Ca spikes in single HeLa cells(6) . Furthermore, Ca-sensitized IP(3)-induced Ca release has been shown to be amplified by addition of oxidized glutathione (GSSG)(5, 10, 11) . These results suggest that the oxidative state of critical sulfhydryls on the IP(3) receptor (IP(3)R) modifies receptor activity and responsiveness. It has been proposed that a highly conserved sequence containing two cysteine residues located near the carboxyl terminus of all subtypes of the IP(3)R and the ryanodine receptors are likely targets for TMS regulation of channel activity(4) .

The Ca release mechanism from sarcoplasmic reticulum (SR) is also sensitive to the oxidative state of critical thiols found on the ryanodine receptor. Oxidation of sulfhydryl groups to a disulfide results in 1) stimulation of Ca release across SR vesicles, 2) contraction of skinned muscle fibers, 3) modification of the gating characteristics of single Ca channels reconstituted into a planar bilayer lipid membrane (BLM), and 4) alteration in high affinity [^3H]ryanodine binding to its receptor(12, 13, 14, 15, 16) . Using a fluorescent coumarin maleimide (cpm) at nanomolar concentrations, it has recently been demonstrated that hyperreactive thiols on the RyR are disulfide-linked into a high molecular weight complex during activation of Ca release(17, 18) . This complex dissociates via reduction upon closing of the Ca release channel. If, as recently proposed, conserved sequences found in both the IP(3)R and the RyR are targets for TMS regulation of channel activity(4) , then one would expect that the SR Ca release mechanism would also be sensitive to TMS modification. In this report, we demonstrate a direct interaction between TMS and the Ca release channel/ryanodine receptor from skeletal muscle sarcoplasmic reticulum.


EXPERIMENTAL PROCEDURES

Preparation of SR Vesicles

Sarcoplasmic reticulum vesicles were prepared from rabbit hind leg and back white skeletal muscle according to the method of MacLennan(19) . The protein concentration was determined by absorption spectroscopy(20) .

Measurement of Ca Fluxes

Ca fluxes across SR vesicles were monitored using a dual wavelength spectrophotometer (12) by measuring the differential absorption changes of antipyrylazo III at 720-790 nm.

Ca uptake into SR vesicles (0.5 mg/ml) was carried out in a buffer containing 100 mM KCl, 20 mM HEPES, 1 mM MgCl(2), 15 mM creatine phosphate, 5 units of creatine phosphokinase, 50 µM CaCl(2), and 100 µM antipyrylazo III. Uptake was initiated by the addition of 0.2 mM MgATP. Upon achieving steady state Ca uptake, release was initiated by the addition of TMS, and the free extravesicular Ca concentration was recorded as a function of time.

[^3H]Ryanodine Binding

Ryanodine binding measurements were conducted according to the methods of Pessah et al.(21) . Briefly, SR membranes (100 µg/ml) were incubated at 37 °C for 3 h in a medium containing 250 mM KCl, 15 mM NaCl, 15 nM [^3H]ryanodine, 20 mM HEPES at pH 7.1. Depending on the conditions of the assay, various concentrations of Ca (100 µM EGTA + CaCl(2)), TMS, or ryanodine (for Scatchard analysis) were present during the incubation period. The binding reaction was quenched by rapid filtration through Whatman GF/B glass fiber filters, which were then rinsed twice with 5 ml of ice-cold buffer containing 50 µM Ca. The filters were placed in polytubes (Fisher), filled with 3 ml of scintillation mixture (Beckman, ReadySafe), shaken overnight, and counted the following day. The experiments were repeated at least twice on two different SR preparations with essentially identical results. Nonspecific binding was measured in the presence of a 100-fold excess of unlabeled ryanodine. For details of individual experiments refer to figure captions.

Measurement of Dissociation Kinetics

Dissociation of [^3H]ryanodine from the equilibrium complex followed equilibration of 1 nM [^3H]ryanodine with SR membranes for 3 h at 37 °C. The SR was then diluted into a 100-fold excess of the assay medium or the assay medium containing 50 or 200 µM TMS. Determinations of residual specific binding were made at times ranging from 5 to 150 min.

Equilibrium Binding Analysis

Equilibrium binding data from saturation analysis were fitted to a one-site model. The dissociation constant, K(d), and the maximal binding capacity, B(max), were derived from linear regression analysis of Scatchard plots.

Single Ca Channel Analysis

BLM, made with a 5:3 mixture of phosphatidylethanolamine and phosphatidylserine at 50 mg/ml suspended in decane, were formed across a 150-µm hole drilled in a polystyrene cup separating two chambers of 0.7 ml each. The cis chamber contained 500 mM CsCl, 200 µM CaCl(2), 10 mM HEPES, pH 7.2, while the trans side contained 100 mM CsCl, 10 mM HEPES, pH 7.2. SR vesicles, suspended in 0.3 M sucrose were added to the cis side. Following the fusion of a single vesicle, 200 µM EGTA, pH 7.0, was added to the cis chamber to stop further fusions. The cis chamber was then perfused with an identical buffer containing no added Ca or EGTA. Single channel activity was then measured at a holding potential of +25 mV with respect to the trans (ground) side. A Warner Instruments bilayer clamp amplifier (model BC-525A) was used to amplify picoampere currents. The data were processed with an Instratech digital data recorder (model VR-10), stored unfiltered on a VCR tape, and subsequently analyzed for channel activity. All single channel analyses were performed using the pClamp 5.0 software (Axon Instruments, Burlingame, CA).

Materials

All reagents were analytical grade. HEPES was obtained from Research Organics (Cincinnati, OH). [^3H]Ryanodine was purchased from Du Pont NEN and ryanodine-dehydroryanodine was purchased from Agrisystems International (Wind Gap, PA). All other chemicals were obtained from Sigma.


RESULTS AND DISCUSSION

Following active accumulation of Ca, the addition of micromolar concentrations of TMS induced Ca release from SR vesicles (Fig. 1). Ca release induced by TMS was not affected by the known channel inhibitors ruthenium red (50 µM) and Mg (10 mM) (not shown). When the reducing agent DTT (1 mM) was added following TMS-stimulated release of Ca (Fig. 1A), the effect was partially reversed, and Ca was reaccumulated by the SR vesicles. Moreover, if TMS was added prior to activation of the Ca pump by ATP, Ca uptake was significantly diminished. Subsequent addition of DTT significantly increased the amount of Ca accumulated by these vesicles (Fig. 1B). Furthermore, as the TMS concentration was increased, the tdecreased (corresponding to faster release rates), and the amount of released Ca saturates at nearly 100% (Fig. 1C).


Figure 1: TMS-induced Ca release was inhibited by DTT. SR vesicles were incubated in 100 mM KCl, 20 mM HEPES, 1 mM MgCl(2), 15 mM creatine phosphate (CP), 5 units of creatine phosphokinase (CK), and 100 µM antipyrylazo III (AP III) at pH 7.0 to monitor extravesicular Ca. In A, after two additions of Ca (25 µM), ATP (0.2 mM) was added to initiate Ca uptake. The subsequent addition of TMS elicited Ca release. Addition of DTT (1 mM) reversed Ca release induced by TMS and resulted in the active reaccumulation of Ca by the SR. In B, TMS was added before ATP. Ca uptake was significantly reduced. Subsequent addition of DTT reversed the effect of TMS and promoted further uptake of Ca into the SR. In C, the half-time for Ca release (t) and the percent of the total releasable Ca (% Ca release) were plotted as a function of the added [TMS]. Abs., absorbance.



On the basis of the data presented in Fig. 1, release of Ca by TMS could be caused by a specific interaction with the Ca release mechanism of the SR, by modification of the activity of the Ca pump(22) , by a nonspecific leakage, or by a combination of these effects. A direct interaction with the Ca release protein/ryanodine receptor is demonstrated in Fig. 2. As shown in Fig. 2A, TMS inhibited high affinity ryanodine binding in a concentration-dependent manner, with an IC of 50 µM. A Scatchard plot (Fig. 2B) was fit to a one-site model using linear regression analysis. This analysis indicated that inhibition of ryanodine binding was caused by a decrease in the maximal number of binding sites (B(max) decreased by 57% (2.54 pmol/mg for the control versus 1.09 pmol/mg in the presence of 50 µM TMS)), with no apparent modification in the Ca dependence of activation or inhibition of the receptor (Fig. 2C). Moreover, the equilibrium dissociation constant for ryanodine binding, K(d), was unmodified by TMS treatment (12.2 nM for the control and 10.4 nM with TMS) (Fig. 2B). The rate of dissociation of bound ryanodine (k) was also unaffected by the presence of TMS in the dilution buffer (Fig. 2D), and it therefore can be concluded that the association rate constant (k) for ryanodine binding is also independent of TMS concentration. In contrast to these observations, Hilly et al.(5) has shown that TMS (100 µM) decreased the K(d) for IP(3) binding to permeabilized hepatocytes and cerebellar membranes without affecting the maximal binding capacity (B(max)). Under similar conditions, Renard-Rooney et al.(8) observed a decreased K(d) for IP(3) binding with an increased receptor occupancy (B(max)).


Figure 2: Thimerosal modified high affinity [^3H]ryanodine binding to SR vesicles. SR vesicles (0.1 mg/ml) were incubated at 37 °C for 3 h (A-C) in a medium containing 250 mM NaCl, 15 mM KCl, 15 nM [^3H]ryanodine, 20 mM HEPES, pH 7.1. In A, B, and D, the assay buffer contained 50 µM Ca. In C, the free Ca concentration was calculated after the addition of varying amounts of Ca and EGTA. In B, 1 nM [^3H]ryanodine and varying concentrations of unlabeled ryanodine (0.5-64 nM) were incubated with SR vesicles in the presence or absence of TMS (50 µM). In D, SR was incubated with 1 nM [^3H]ryanodine for 3 h. Dissociation of bound ryanodine was initiated by a 100-fold dilution into a binding medium without ryanodine containing the indicated concentration of TMS. Dissociation was quenched by rapid filtration at the indicated times. In all assays, the binding reaction was quenched by rapid filtration through Whatman GF/B glass fiber filters and rinsed twice with 5 ml of buffer. The data shown are the average of representative experiments performed in duplicate and repeated at least two times.



Direct modification of the Ca release mechanism was also demonstrated following reconstitution of the release mechanism into a planar BLM. As shown in Fig. 3A, in the absence of added cis Ca, 100-200 µM TMS stimulated single channel activity. With a 5:1 CsCl gradient, the unitary conductance (453 picosiemens) and selectivity (P/P 32) of the single channel activated by TMS (not shown) were the same as previously reported(16) . The TMS-activated channel was not affected by the IP(3)R inhibitor heparin (200-400 µg/ml) (not shown). Moreover, as in the case of vesicle flux measurements (Fig. 1), activation of channel activity stimulated by TMS (200 µM) was reversed by the addition of a reducing agent (5 mM DTT) (Fig. 3B). Under conditions of channel activation by TMS, treatment with DTT appeared to restore the channel to its native configuration, and subsequent addition of 50 µM Ca increased the channel open probability (Fig. 3B, trace d). In contrast to this behavior, in the presence of activating concentrations of Ca (50 µM), high concentrations of TMS (1 mM) not only activated single channel activity but also inhibited channel activity in a time-dependent manner (Fig. 3, C and D). Once inhibited by high concentrations of TMS, single channel activity was not reactivated by subsequent addition of 100 µM Ca or 1 mM ATP (not shown).


Figure 3: Thimerosal modified the single channel characteristics of the SR Ca release protein. Following fusion of an SR vesicle to a planar BLM, single channel current was recorded as a function of time. In the absence of added Ca (Ca 5 µM) (A, trace a), a control recording was followed by two consecutive additions of 100 µM TMS (A, traces b and c). The channel open probabilities for each trace are as follows: A (a), P = 0.02; A (b), P = 0.15; A (c), P = 0.40. In B, DTT reversed channel activation induced by TMS. Trace B (a) shows a control channel in the presence of 5 µM Ca (P = 0.01). Addition of 200 µM TMS (trace B (b)) activated channel activity (P = 0.25), while 5 mM DTT (trace B (c)) decreased the activity of the channel (P = 0.05). Subsequent addition of 50 µM Ca (trace B (d)) reactivated channel activity (P = 0.30). In the presence of 50 µM Ca, high concentrations of TMS modified single channel activity in a time-dependent manner (C). To a control membrane (trace C (a) (P = 0.03)), 50 µM Ca was added (trace C (b) (P = 0.55)). One minute following the addition of 1 mM TMS (trace C (c)), the open probability increased (P = 0.95). Traces C (d) and C (e) illustrate inhibition of single channel activity 80 and 100 s after the initial exposure to TMS (P = 0.55 and P = 0.10, respectively). In D, the time dependence of the open probability for traces displayed in C are shown. At t = 0 s, 1 mM TMS was added to the cis chamber, and the solution was stirred for 60 s. P was measured in 5-s intervals. For all traces shown (A--D), the holding potential was +25 mV. The solid lines and arrows represent the closed and open states of the channel, respectively. Each experiment was repeated with similar results at least 4 times.



On the basis of vesicle flux, high affinity [^3H]ryanodine binding, and single channel measurements, it is clear that TMS directly interacts with the Ca release protein/ryanodine receptor from skeletal muscle sarcoplasmic reticulum. TMS appears to be interacting with a thiol group(s) associated with the Ca release mechanism of skeletal muscle SR(12, 13, 14, 15, 16) . Oxidation and reduction of critical sulfhydryl groups have been postulated to be the mechanism underlying the gating of the Ca release protein(13, 17, 18) . The oxidative state of thiols present on both the RyR and the IP(3)R may be critical in the regulation of channel gating. However, the changes in receptor binding induced by TMS are somewhat different for the RyR and the IP(3)R. Independent of the mechanism by which these channels gate, TMS should not be used as a specific probe for interaction with the IP(3) receptor. TMS has previously been shown to affect the activity of the Ca pump from rabbit skeletal muscle SR and from rat cerebellar microsomes (22) and was shown in this report to modify the gating characteristics and receptor occupancy of the RyR/Ca release protein from skeletal muscle sarcoplasmic reticulum.


FOOTNOTES

*
This work was supported by grants-in-aid from the Oregon Affiliate of the American Heart Association (to J. J. A.) and from the Western Pennsylvania Affiliate of the American Heart Association (to G. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Physics Dept., P. O. Box 751, Portland State University, Portland, OR 97207. Tel.: 503-725-3014; Fax: 503-725-3864; John@science1.sb2.pdx.edu.

(^1)
The abbreviations used are: IP(3), inositol 1,4,5-triphosphate; TMS, thimerosal; DTT, dithiothreitol; IP(3)R, IP(3) receptor; SR, sarcoplasmic reticulum; BLM, bilayer lipid membrane; RyR, ryanodine receptor.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.