©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Effect of Mersalyl on Inositol Trisphosphate Receptor Binding and Ion Channel Function (*)

(Received for publication, July 27, 1994; and in revised form, November 29, 1994)

Suresh K. Joseph (§) Sean V. Ryan Shawn Pierson Dominique Renard-Rooney Andrew P. Thomas

From the Department of Pathology and Cell Biology, Thomas Jefferson University School of Medicine, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A number of thiol-reactive agents induce repetitive Ca spiking in cells by a mechanism thought to involve sensitization of the inositol 1,4,5-trisphosphate receptor (IP(3)R). To further define the basis of this interaction, we have studied the effect of several thiol-reactive agents on [^3H]IP(3) binding, IP(3)-gated channel activity, and conformation of the IP(3)R in membranes from hepatocytes, cultured WB rat liver epithelial cells, and cerebellum microsomes. At 4 °C, the organomercurial thiol-reactive agent mersalyl markedly stimulates (3-4-fold) [^3H]IP(3) binding to permeabilized hepatocytes. The closely related molecule, thimerosal, has only a small stimulatory effect under these conditions, and GSSG or N-ethylmaleimide are without effect. The stimulatory effect of mersalyl was associated with a decrease in K of the IP(3)R with no change in B(max). Mersalyl was without effect on detergent-solubilized hepatocyte binding sites or on the [^3H]IP(3) binding activity of cerebellum microsomes. In contrast to thimerosal, which potentiates IP(3)-mediated Ca release, mersalyl blocked IP(3)-gated Ca channels. Mersalyl pretreatment of WB membranes altered the pattern of immunoreactive receptor fragments generated upon subsequent cleavage of the receptor with proteinase K. This effect was not reproduced by thimerosal and was also not observed in experiments on cerebellum microsomes. We conclude that the WB cell and brain IP(3) receptors are differently regulated by modification of thiol groups. Reaction of the WB cell IP(3) receptor with mersalyl alters its conformation and modifies the accessibility of sites on the protein that are cleaved by proteinase K. In the presence of mersalyl, the receptor has high affinity for IP(3) but is inactive as a Ca channel. This contrasts with the high affinity receptor/active Ca channel induced by thimerosal, suggesting that even closely related thiol agents may interact at different thiol groups.


INTRODUCTION

Intracellular Ca mobilization occurring in response to agonist stimulation of cells is mediated by the interaction of inositol 1,4,5-trisphosphate (IP(3)) (^1)with a specific receptor/Ca channel(1) . At least three different receptor isoforms have been identified by molecular cloning(2, 3, 4, 5) . A domain model of the receptor has been proposed in which binding of IP(3) to the N-terminal region of the receptor initiates a conformational change in the protein that gates a Ca channel comprising six-transmembrane domains located in the C-terminal region(6, 7) . It has been directly demonstrated that the purified IP(3)R is a functional Ca channel(8, 9) .

Ca transients in single cells stimulated with suboptimal concentrations of agonists occur as repetitive spikes(1, 10) . A number of models have been proposed to explain the complex behavior of Ca signals recorded from individual cells. Experimental evidence suggests that the feed-back regulation of the IP(3)R plays a central role in the initiation and propagation of Ca spikes. Small elevations of Ca above resting levels have been shown to enhance IP(3)-mediated Ca release(11, 12, 13) . and Ca sensitization of the IP(3)R to endogenous levels of IP(3) has been proposed as one mechanism of Ca wave propagation (14, 15) . The thiol-reactive agents t-butylhydroperoxide and thimerosal have also been shown to promote repetitive Ca spiking in several cell types(15, 16, 17, 18) . In both instances, it is believed that the effects of these agents are related to a sensitization of the IP(3)R to endogenous levels of IP(3). In the case of t-butylhydroperoxide, enhanced levels of oxidized glutathione (GSSG) are thought to underlie the sensitization. GSSG has been shown to decrease the half-maximal concentration of IP(3) required for Ca release from permeabilized hepatocytes(19) . Low concentrations of thimerosal have also been shown to potentiate IP(3)-mediated Ca release in several experimental systems(20, 21, 22, 23, 24) . The exact mode of action of thiol reagents on the IP(3)R has not been delineated, and it is not known if all of these agents have a common site of action. We have attempted to address this question by comparing the effects of several different thiol-reactive agents on [^3H]IP(3) binding and IP(3)-gated channel activity. In the present study we report that mersalyl and thimerosal, two structurally related organomercurial thiol-reactive agents, have different effects on the function of the isoforms of the IP(3)R in brain and liver. The data suggest the presence of several distinct reactive thiols that are important in regulating IP(3)R function.


EXPERIMENTAL PROCEDURES

Materials

Mersalyl, thimerosal, N-ethylmaleimide, p-chloromercurophenylsulfonate were from Sigma. GSSG was from Boehringer Mannheim. Unlabeled IP(3) was from Calbiochem. [^3H]IP(3) was from DuPont NEN.

[^3H]IP(3) Binding Assays

Isolated hepatocytes were prepared by collagenase digestion of perfused rat livers and were washed and stored on ice at 20-30 mg of protein/ml in Ca/Mg-free Hank's buffer as described previously(25) . For incubation with thiol-reactive agents, the cells were centrifuged (150 times g, 15 s) and resuspended in hepatocyte resuspension buffer which contained 120 mM KCl, 20 mM Tris-Hepes (pH 7.2), 2 mM HEDTA, 0.1 mM vanadate, 5 mM NaF, 4 nM okadaic acid, 1 mM PMSF, and 10 µg/ml each of aprotinin, leupeptin, and soybean trypsin inhibitor. The cells were permeabilized by addition of 40 µg of saponin/mg of cell protein. Complete permeabilization (<5 min) was monitored by trypan blue staining. The cell concentration was adjusted to 5 mg of protein/ml and 0.8 ml were incubated for 5 min at 4 °C with 0.8 ml of label medium containing 120 mM KCl, 20 mM Tris-Hepes (pH 7.2), 10 nM [^3H]IP(3) (DuPont NEN; 20 Ci/mmol) in the presence or absence of thiol-reactive agents. Triplicate 0.5-ml samples were vacuum filtered through glass-fiber filters (Gelman A/E), and the filters were washed twice with 5 ml of 50 mM Tris-HCl (pH 7.8), 1 mM EDTA, and 1 mg/ml bovine serum albumin. The filters were counted in scintillation fluid (Budget Solve, RPI Corp., Mount Prospect, IL). Permeabilized hepatocytes were solubilized at 4 °C for 30 min in hepatocyte solubilization buffer, which contained 50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1% (w/v) Triton X-100, 1 mM EDTA, 1 mM PMSF, and 5 µg/ml each of aprotinin, soybean trypsin inhibitor, and leupeptin. Insoluble material was removed by centrifugation for 10 min at 25,000 times g. Binding to hepatocyte extracts was measured using a polyethylene glycol precipitation assay as described(26) .

Measurement of IP(3)-mediated Mn Quenching of Compartmentalized Fura-2

Intact hepatocytes (4 mg of protein/ml) were loaded with 5 µM Fura-2/AM for 35 min at 37 °C in a buffer (pH 7.4) containing 10 mM Na/Hepes, 120 mM NaCl, 4.7 mM KCl, 5 mM NaHCO(3), 1.2 mM KH(2)PO(4), 1.2 mM MgSO(4), 2 mM CaCl(2), 10 mM glucose, and 2% bovine serum albumin. After loading, the cells were washed and stored at 4 °C in Na/Hepes buffer (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 20 mM Tris/Hepes, 1 mM KH(2)PO(4), and 0.2 mM MgCl(2). Prior to use, the cells were washed once in Na/Hepes buffer containing 100 µM EGTA. The cells were then permeabilized at 4 °C in a buffer (pH 7.2) containing 120 mM KCl, 10 mM NaCl, 20 mM Tris/Hepes, 1 mM KH(2)PO(4), 0.2 mM MgCl(2), 40 µg/ml digitonin, 5 µM carbonyl cyanide m-chlorophenylhydrazone, 5 µM oligomycin, 1 µM rotenone, 2 mM ATP, 5 mM phosphocreatine, 0.5 unit/ml creatine kinase, 1 µg/ml each pepstatin, antipain, and leupeptin, and 2 µM thapsigargin. Incubations were performed in the cuvette of a fluorimeter (Photon Technology Deltascan) maintained at 4 °C with continuous stirring. The excitation wavelength was 360 nM with emission at 510 nM. The quenching of the Fura-2 trapped into the organelles was monitored after addition of 40 µM MnCl(2).

WB Cell Membranes and Cerebellum Microsomes

WB cells, a clonal cell line derived from rat liver(27) , were cultured in Richter's modified MEM containing 5% fetal bovine serum. Cells were grown in 100-mm dishes to confluence and were used between passages 25 and 30. The plates were washed twice in ice-cold phosphate-buffered saline and incubated at 4 °C for 20 min in 1 ml of hypotonic buffer containing 10 mM Tris (pH 7.2), 1 mM EDTA, 0.2 mM PMSF, and 1 µg/ml pepstatin, antipain, and leupeptin. The cells from all the plates were scraped, pooled, and homogenized 15 times in a Dounce homogenizer with a tight fitting pestle. The homogenate was centrifuged at 100 times g for 1 min to remove unbroken cells. The supernatant was spun at 100,000 times g for 30 min. The crude WB cell membrane fraction was resuspended in 320 mM sucrose, 5 mM Tris-HCl (pH 7.8), 1 mM EDTA and stored at -80 °C.

Microsomal membranes were prepared from rat cerebellum homogenates by differential centrifugation as described previously (28) and stored at -80 °C in the same buffer as WB cell membranes.

Protease Cleavage Assays

WB cell membranes or rat cerebellum microsomal membranes were incubated at 1 mg of protein/ml in buffer A containing 120 mM KCl, 20 mM Tris-HCl (pH 7.4), and 1 mM EDTA in the presence and absence of 200 µM mersalyl for 10 min on ice. The membranes were pelleted by centrifugation at 100,000 times g for 30 min and resuspended in buffer A at 1 mg of protein/ml. Proteinase K was added at a ratio of 10 µg/mg membrane protein and incubated at room temperature for different periods of time. The reaction was terminated by addition of 3 mM PMSF and the membranes reisolated by centrifugation at 100,000 times g. The membrane pellet was solubilized and denatured in SDS-PAGE sample buffer. Samples were electrophoresed on 10% SDS-PAGE and transferred to nitrocellulose. The nitrocellulose sheets were immunoblotted with antibodies raised to amino acids 401-414 in the N-terminal domain (KEEK-Ab), or to the C-terminal 19 amino acids of the rat type-I IP(3)R. The recognition properties of the antibodies and conditions for immunoblotting have been previously described(29, 30, 31) . Immunoreactive proteins were visualized using an enhanced chemiluminescence kit (Amersham Corp.)


RESULTS

The effect of several thiol-reactive agents on [^3H]IP(3) binding to saponin-permeabilized hepatocytes is shown in Fig. 1. The incubation period with thiol reagent and [^3H]IP(3) was 5 min and was performed at 4 °C in a Mg-free medium to minimize hydrolysis of [^3H]IP(3). Under these conditions, a marked stimulation (4-5-fold) of [^3H]IP(3) binding was observed in the presence of mersalyl, an organomercurial thiol-reactive agent. Two structurally related molecules, thimerosal and p-chloromercurophenylsulfonate, also stimulated [^3H]IP(3) binding, but the degree of stimulation was much lower than observed with mersalyl. Increasing the concentration or incubation time did not enhance the stimulatory effect of thimerosal (data not shown). Oxidized glutathione or the thiol-alkylating agent, N-ethylmaleimide, were without effect on [^3H]IP(3) binding at 4 °C.


Figure 1: The effect of different sulfydryl agents on IP(3) binding to permeabilized hepatocytes at 4 °C. Isolated hepatocytes were permeabilized with saponin and incubated with [^3H]IP(3) (5 nM) in the presence or absence of the given concentration of thiol-reactive reagent for 5 min at 4 °C (see ``Experimental Procedures'' for additional details). The specific IP(3) bound to the hepatocytes was measured with a membrane filtration assay. The data shown are from three to six separate experiments with each assay done in triplicate.



Ca has been shown to stimulate IP(3) binding to the hepatic IP(3)R(32) . It has been proposed that Ca mediates a conversion of receptors from a low affinity (active) form to a high affinity (inactive) form(33, 34) . Hilly et al.(20) have shown previously that the stimulatory effect of Ca and thimerosal on IP(3) binding to permeabilized hepatocytes are not additive. Fig. 2A demonstrates that the stimulation of [^3H]IP(3) binding by mersalyl is dose-dependent with maximal effects being observed at 100-200 µM mersalyl. Ca (buffered at 10 µM concentration) stimulated IP(3) binding to a lesser extent than a maximal concentration of mersalyl and the effect of both agents were not additive. Scatchard analysis of the binding data (Fig. 2B), indicated that mersalyl stimulated [^3H]IP(3) binding by increasing the affinity of the IP(3) receptor without altering the maximal number of binding sites. In three experiments the respective apparent K(d) and B(max) values were 27 ± 3 nM and 125 ± 6 fmol/mg protein under control conditions, and 4.7 ± 0.3 nM and 98 ± 3 fmol/mg protein in the presence of 100 µM mersalyl. The effect of mersalyl is qualitatively similar to the effect of Ca on the binding affinity of the hepatic IP(3)R(33) .


Figure 2: The effect of mersalyl on Ca sensitivity and binding affinity for IP(3). A, [^3H]IP(3) binding was measured as described in Fig. 1in the presence of increasing concentrations of mersalyl in the presence or absence of added Ca. The amount of total Ca added to the buffer containing 1 mM HEDTA was adjusted to yield a free concentration of 10 µM as measured in parallel determinations with a Ca-sensitive mini-electrode. B, the amount of [^3H]IP(3) bound to permeabilized hepatocytes was measured in the absence of Ca and in the presence and absence of 100 µM mersalyl with increasing concentrations of unlabeled IP(3) present in the incubation medium. The displacement curves obtained in the presence and absence of mersalyl are shown as a Scatchard plot.



We have shown previously that the stimulatory effect of Ca on IP(3) binding in permeabilized hepatocytes is lost after detergent solubilization of membranes(26) . The experiment in Fig. 3was carried out to determine if this was also the case with mersalyl. Permeabilized hepatocytes were treated with mersalyl and then washed. The washed mersalyl-treated hepatocytes retained an enhanced IP(3) binding activity (Fig. 3A). These hepatocytes were then solubilized with Triton X-100, and binding activity was measured in extracts exposed to increasing concentrations of mersalyl (Fig. 3B). The addition of mersalyl to Triton X-100 extracts prepared from control hepatocytes had no significant effect on [^3H]IP(3) binding. Binding activity in Triton X-100 extracts of mersalyl pretreated membranes was only slightly higher than control extracts and the further addition of mersalyl produced a dose-dependent inhibition of binding. These results indicate that, as with Ca, the stimulatory effect of mersalyl cannot be observed after detergent solubilization. The reason why mersalyl pretreatment of membranes causes mersalyl to inhibit [^3H]IP(3) binding in detergent extracts is presently not clear. A possibility is that mersalyl binding to the receptor in membranes alters the conformation of the protein (see below) in a manner that exposes an additional mersalyl-reactive thiol group after detergent solubilization that is inhibitory to ligand binding.


Figure 3: The effect of mersalyl on [^3H]IP(3) binding to detergent solubilized binding sites. Panel A, hepatocytes (5 mg of protein/ml) were permeabilized with saponin and an aliquot was incubated with [^3H]IP(3) in the absence (box) or presence () of 100 µM mersalyl for 5 min. The amount of [^3H]IP(3) bound was determined (control cells). The remaining permeabilized cells were divided into two equal portions, and one portion was incubated with 100 µM mersalyl for 5 min. Both aliquots were centrifuged (2 min, 80 times g), washed once in hepatocyte resuspension buffer-HEDTA, and resuspended in this buffer to 5 mg protein/ml. The [^3H]IP(3) binding activity of these cells were measured (washed cells). Panel B, the control (circle) and mersalyl pretreated cells (bullet) were solubilized by addition of 1% (w/v) Triton X-100. Insoluble material was removed by centrifugation (25,000 times g; 10 min), and [^3H]IP(3) binding to the solubilized extracts was measured in the presence of increasing concentrations of mersalyl as described previously(26) .



We have examined the functional effect of mersalyl and thimerosal on IP(3)-mediated Ca channel function in Fig. 4and Fig. 5. In order to avoid the known inhibitory effect of thiol-reactive agents on Ca-ATPase (16, 22, 35) , we have utilized an assay method based on the ability of Mn to traverse the IP(3)-activated Ca channel in a retrograde manner and quench the fluorescence of Fura-2 compartmentalized in intracellular stores(36) . All the fluorescence measurements were carried out at 4 °C in order to permit comparison to the ligand binding data. The addition of Mn to permeabilized Fura-2-loaded hepatocytes, pretreated with the Ca pump inhibitor thapsigargin, produced a rapid quenching of cytosolic Fura-2 released from the permeabilized cells. The subsequent addition of 1 µM IP(3) (a maximal dose) produced an additional quench corresponding to the entry of Mn into the IP(3)-sensitive compartment (Fig. 4). Further entry of Mn into the IP(3)-insensitive compartment could be observed after addition of ionomycin. When the permeabilized hepatocytes were pretreated with mersalyl a complete inhibition of the IP(3)-mediated quench was observed (Fig. 4). The total pool size of the intracellular stores was not altered by mersalyl pretreatment. Greater than 95% inhibition of IP(3)-mediated Mn quench was observed with IP(3) concentrations in the range 0.1-10 µM (data not shown). The inhibitory effects of mersalyl were also noted at 37 °C, although the effects were less marked than at 4 °C. (^2)Previous studies have shown that the potentiating effect of thimerosal on IP(3)-induced Ca release is seen only at suboptimal IP(3) concentrations(22, 23) . In agreement with these studies, thimerosal at 4 °C markedly potentiated the Mn quench mediated by 10 nM IP(3) (Fig. 5, lower panel) with a much smaller effect on the responsiveness to 500 nM IP(3) (Fig. 5, upper panel).


Figure 4: The effect of mersalyl on IP(3)-mediated Mn quenching of compartmentalized Fura-2 in intracellular stores. Hepatocytes were loaded with Fura-2/AM, washed, and permeabilized at 4 °C as described under ``Experimental Procedures.'' The permeabilized cells were pretreated with mersalyl (100 µM) for 5 min before addition of Mn. All recordings were made at 4 °C.




Figure 5: The effect of thimerosal on IP(3)-mediated Mn quenching of compartmentalized Fura-2 in intracellular stores. Hepatocytes were loaded with Fura-2/AM, washed, and permeabilized at 4 °C as described under ``Experimental Procedures.'' The permeabilized cells were pretreated with thimerosal (100 µM) for 5 min before addition of Mn. All recordings were made at 4 °C.



A possible mechanism of action of mersalyl is that binding of this agent to a free thiol group on the IP(3)R alters the conformation of the protein, resulting in a form of the receptor with an inactive Ca channel and a high affinity for ligand. To try to detect a conformational change in the protein in its native membrane environment, we have looked for changes in the pattern of immunoreactive fragments generated after addition of proteases. Such experiments are facilitated by using membranes that contain higher levels of immunoreactive IP(3)R than found in hepatocytes. Fig. 6shows the effect of mersalyl on [^3H]IP(3) binding to membranes prepared from WB rat liver epithelial cells and rat cerebellum. Both membranes are known to contain relatively high levels of immunoreactive type-I IP(3)R(31, 37) . Only the receptor in WB membranes showed a stimulation of [^3H]IP(3) binding by mersalyl, and these were used in subsequent studies.


Figure 6: The effect of mersalyl on IP(3) binding to WB-cell and rat cerebellum membranes A comparison of the effect of mersalyl on [^3H]IP(3) binding to WB and rat cerebellum microsomes is shown. Assay conditions were as described for Fig. 1except that the final assay volume was reduced to 250 µl and triplicate 75-µl samples were removed for filtration. The results are the mean ± S.E. of three observations.



Fig. 7A shows the pattern of fragments observed after proteinase K digestion of WB cell membranes, as visualized with an antibody raised to amino acids 401-414 in the N-terminal region of the type-I IP(3)R. In addition to several intermediate digestion products, a prominent proteolytic product of 37.1 ± 2.2 kDa (n = 4) was formed with progressive proteinase K digestion under control conditions. This band was not observed after proteinase K treatment of WB cell membranes that had first been treated with mersalyl and reisolated by centrifugation. The protease fragmentation pattern of cerebellum IP(3)R was substantially different from that observed with WB cell membranes, although a 35.4 ± 0.4 kDa (n = 3) polypeptide was also formed in cerebellar membranes (Fig. 7B). In agreement with the results of binding data, pretreatment of cerebellum membranes with mersalyl had no effect on the protease cleavage pattern.


Figure 7: The effect of mersalyl pretreatment on protease susceptibility of IP(3) receptors in WB and cerebellum membranes. A, WB cell membranes were prepared, preincubated with mersalyl (200 µM), and subjected to cleavage with proteinase K as described under ``Experimental Procedures.'' The membranes treated with protease for the indicated periods of time were reisolated by centrifugation and denatured with SDS-PAGE sample buffer, and approximately 80 µg of protein was electrophoresed on a 10% gel. Immunoblotting was carried out with an affinity-purified IP(3)R antibody raised to amino acids 401-414 of the rat type-I IP(3)R. The arrows indicate the position of the intact IP(3)R in WB membranes and a major 37-kDa fragment that forms after proteinase K digestion. B, the same experiment shown in panel A was repeated using rat cerebellum microsomes.



Fig. 8A shows that thimerosal, at an equivalent concentration, does not mimic the action of mersalyl. The effect of mersalyl on the protease cleavage pattern of the WB IP(3)R was completely prevented by inclusion of an excess of dithiothreitol. The 37-kDa immunoreactive fragment in both WB cell and cerebellum was associated with the membrane fraction. This reflected a peripheral association with the membrane, since we were able to remove this fragment by washing the membranes with 0.1 M Na(2)CO(3), pH 11.0 (data not shown). The supernatant fractions obtained after proteinase K digestion of control and mersalyl-treated WB membranes were analyzed for the presence of the 37-kDa KEEK-reactive fragment (Fig. 8B). The absence of the 37-kDa cleavage product in WB membranes after mersalyl pretreatment was not the result of a selective loss of this fragment into the soluble fraction. The combination of mersalyl and thimerosal together produces a pattern of protease digestion which is the same as obtained with mersalyl alone (Fig. 8C). This observation supports the conclusion that the two sulfydryl reagents interact with different thiol groups.


Figure 8: The effect of thimerosal and dithiothreitol on proteinase K generation of 37-kDa fragment. A, WB cell membranes (1 mg of protein/ml) were incubated in Buffer A under the following conditions: lane 1, control; lane 2, + mersalyl (200 µM); lane 3, + thimerosal (200 µM); lane 4, + mersalyl + dithiothreitol (20 mM). After 10 min of incubation at 4 °C, the membranes were pelleted by centrifugation (100,000 times g; 20 min), resuspended to 1 mg of protein/ml, and treated with proteinase K (10 µg/mg protein) for 15 min at room temperature. The protease-treated membranes were again pelleted by centrifugation, quenched in SDS-PAGE sample buffer, and immunoblotted as described for Fig. 5A. B, WB membranes were treated with and without mersalyl and then digested with proteinase K as described for panel A. After centrifugation the supernatants were precipitated with 10% trichloroacetic acid. The protease-digested membrane fraction and the trichloroacetic acid-precipitated supernatant fractions were analyzed on 10% SDS-PAGE and Western-blotted with KEEK antibody. C, WB cell membranes were treated as in A under the following conditions: lane 1, control; lane 2, + mersalyl (200 µM); lane 3, + thimerosal (200 µM); lane 4, + mersalyl + thimerosal. Only the lower part of the immunoblots are shown. The arrow indicates the location of the 37-kDa immunoreactive fragment.




DISCUSSION

Several recent studies have examined the effect of thimerosal on IP(3) binding and IP(3)-dependent Ca release. Low concentrations of thimerosal were found to potentiate IP(3)-mediated Ca release from adrenal cortex microsomes(21) , permeabilized hepatocytes(20) , cerebellum microsomes (20, 22) , and permeabilized A7r5 smooth-muscle cells(23) . In adrenal cortex and hepatocytes, the potentiation by thimerosal was accompanied by a large stimulation of IP(3) binding reflecting an increased affinity of the IP(3)R. On the basis of these data, it has been concluded that the high affinity form of the receptor induced by thimerosal is functionally active and is distinct from the high affinity form of the receptor induced by Ca, which is functionally less active as a Ca channel(33) . Our data show that the related thiol-reagent, mersalyl, has a marked stimulatory effect on IP(3) binding and behaves more like Ca, in that it generates a high affinity form of the receptor that is functionally inactive. These studies reinforce the idea that the IP(3)R can exist in several non-equivalent high affinity states that may have high or low Ca conductance (20, 38) . It should be noted that the stimulatory effect of thimerosal on IP(3) binding has not been observed in all studies (e.g.(22) and (23) ), and only a modest stimulation of binding was observed in permeabilized hepatocytes under our assay conditions at 4 °C. We attribute this difference to reaction of the sulfydryl reagents with separate thiol groups on the receptor rather than to differences of reactivity with the same thiol group for several reasons. First, incubation of permeabilized hepatocytes at 4 °C for longer periods with higher concentrations of thimerosal did not enhance the effect on [^3H]IP(3) binding. Second, the effects of mersalyl and thimerosal on channel function and protease digestion of the receptor are clearly different. Third, the combination of mersalyl and thimerosal together produces a pattern of protease digestion which is the same as obtained with mersalyl alone (Fig. 8C).

Other factors, such as temperature, may modify the reactivity of individual thiol groups. For example, we have found that the effect of thimerosal on ligand binding could be enhanced, and the effect of mersalyl diminished, by preincubating the permeabilized hepatocytes at 37 °C with the thiol agents prior to measurement of binding at 4 °C. (^3)The elevation of temperature does not, however, qualitatively modify the effect of the sulfydryl agents on channel function (Footnote 2 and data not shown). A temperature-dependent alteration in the accessibility of N-ethylmaleimide-reactive thiol groups on the hepatic IP(3)R has also been documented previously(39) . Whereas low concentrations of thimerosal stimulate IP(3)-mediated Ca release, higher concentrations (>10 µM) have been found to inhibit the release process in some systems(22, 23) . In part, this is due to an inhibition of the Ca pump and to an increase in the passive Ca permeability of the endoplasmic reticulum membrane(22, 23) . Using the Mn quench assay, a biphasic dependence on thimerosal concentrations up to 100 µM was not observed in the present study.

Mersalyl stimulates IP(3) binding to liver and WB cell membranes but is without effect on cerebellum microsomes. The full spectrum of IP(3)R isoforms in each of these tissues has not been established unequivocally. However, it is known that the cerebellum contains predominantly type-I IP(3)R (3, 40) and that non-neuronal cells contain an alternative transcript of the type-I isoform that has a 40-amino acid deletion(41, 42) . These ``long'' and ``short'' forms of the type-I IP(3)R have been shown to differ in their regulation by Ca and cAMP-dependent phosphorylation(41) . It has recently been reported that the liver expresses low amounts of type-III IP(3)R mRNA and much higher amounts of type-I and type-II IP(3)R mRNA(43) . By contrast, the WB cell contains predominantly types I and III IP(3)R and very little of any other isoform, as judged by the 90% immunodepletion of [^3H]IP(3) binding sites from WB cell extracts by a combination of type-I and type-III specific antibodies. (^4)Thus the difference in reactivity toward mersalyl of the hepatocyte/WB cell and cerebellum IP(3)R could be accounted for by the selective insensitivity of the type-I (long form) to the sulfydryl agent or alternatively, to tissue-specific differences in ancillary sulfydryl reagent-sensitive regulatory proteins.

It is somewhat surprising that two such closely related organomercurial agents should have different effects on the hepatic IP(3)R. Both molecules are anionic and, in the case of mersalyl, known not to penetrate mitochondrial membranes(44) . The inhibitory effects of mersalyl are believed to be the result of the formation of a mercaptide bond with free thiol groups in a protein(45) . With thimerosal, the mercury is already linked to sulfur, and it is therefore possible that reaction with protein thiols may generate mixed disulfides. In studies on the rate of reaction of the free thiols of ovalbumin with several mercaptide forming agents, it was observed that mersalyl was more reactive than other agents such as p-chloromercurobenzoate (46) . These variations in reactivity were attributed to differences in the degree of steric hindrance around the reactive mercury in these molecules(46) . Therefore, differences in the chemistry of reaction and/or accessibility to reactive thiol groups may underlie the distinctive effects of the two agents.

The current model of IP(3) gating of the Ca channel in the IP(3)R proposes that ligand binding is associated with a large conformational change in the protein(6) . In the present study, we have shown that mersalyl pretreatment of WB cell membranes effectively eliminates the appearance of a 37-kDa immunoreactive receptor fragment generated by proteinase K cleavage. We interpret these data to indicate that reaction of a thiol group on the receptor induced a conformational change in the protein that exposed the region of the receptor containing the antibody epitope (amino acids 401-414) to cleavage by proteinase K. Two findings suggest that this conformational change is linked to the effect of mersalyl on [^3H]IP(3) binding. First, mersalyl does not affect [^3H]IP(3) binding in cerebellum microsomes and also does not alter the formation of any of the proteolytic fragments in this system. Second, thimerosal does not mimic the effect of mersalyl on [^3H]IP(3) binding or the appearance of 37-kDa proteinase K fragment. The antibody epitope falls within the region thought to be involved in ligand binding(2) . However, IP(3) itself had no effect on the pattern of proteinase K digestion fragments and did not prevent the effect of mersalyl (data not shown).

[^3H]IP(3) binding to Triton X-100 solubilized extracts (Fig. 3B) or heparin-agarose column eluates (data not shown) are not affected by mersalyl (data not shown). Mersalyl may continue to react with thiol groups on the receptor under these conditions and still have no effect on binding, since removal of the protein from its membrane environment may grossly alter regulation of the binding site(26) . However, the present data do not exclude the possibility that mersalyl or other sulfydryl agents interact with ancillary regulatory proteins rather than reacting directly with the receptor. Despite this, it is clear that reaction of specific thiol groups on the receptor, or a regulatory protein, has profound consequences on the function of the IP(3) receptor/ion channel. These thiols have varied sensitivity to sulfydryl reagents, and reactivity is also different between different IP(3)R isoforms. Identification of these functionally important cysteine residues and characterization of their role in ligand binding/ion channel function of the receptor remains a challenging objective.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK-34804 (to S. K. J.), DK-38422 (to A. P. T.), and AA-07186. 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: Dept. of Pathology and Cell Biology, Thomas Jefferson University, Rm. 230A JAH, 1020 Locust St., Philadelphia, PA 19107. Tel.: 215-955-1221; Fax: 215-923-6813; josephs{at}jeflin.tju.edu.

(^1)
The abbreviations and trivial names used are: IP(3), myo-inositol 1,4,5-trisphosphate; IP(3)R, IP(3) receptor; HEDTA, N-hydroxyethylethylenediaminetriacetic acid; PMSF, phenylmethylsulfonyl fluoride; mersalyl, O-(3-hydroxymercuri-2-methoxypropyl)carbamylphenoxyacetate; thimerosal, [(O-carboxyphenyl)thio]ethylmercury.

(^2)
The initial rate of IP(3) (1 µM) induced Fura-2 quenching at 37 °C expressed as percentage of total fluorescence/s was 2.02 ± 0.35 in control cells and 0.35 ± 0.07 in mersalyl-treated cells. Under these conditions, the total quench mediated by IP(3) expressed as a percentage of the ionomycin quenchable pool was 37.5 ± 2.7% in control cells and 19.4 ± 3.4% in mersalyl-treated cells (mean ± S.E., n = 4).

(^3)
D. Renard-Rooney, S. K. Joseph, M. Seitz, and A. P. Thomas, unpublished observations.

(^4)
S. K. Joseph, S. Pierson, and A. P. Maranto, unpublished observations.


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