Isoform- and Species-specific Control of Inositol 1,4,5-Trisphosphate (IP3) Receptors by Reactive Oxygen Species*

Background: Reactive oxygen species (ROS) affect cytoplasmic calcium signaling. Results: Superoxide anion causes oxidation of the IP3 receptor and sensitization of calcium release to promote cytoplasmic calcium oscillations and mitochondrial calcium uptake. Conclusion: Physiologically relevant ROS controls cytoplasmic and mitochondrial calcium transport through IP3 receptors. Significance: Mechanisms of calcium and ROS interactions are relevant for both physiological and pathophysiological signaling. Reactive oxygen species (ROS) stimulate cytoplasmic [Ca2+] ([Ca2+]c) signaling, but the exact role of the IP3 receptors (IP3R) in this process remains unclear. IP3Rs serve as a potential target of ROS produced by both ER and mitochondrial enzymes, which might locally expose IP3Rs at the ER-mitochondrial associations. Also, IP3Rs contain multiple reactive thiols, common molecular targets of ROS. Therefore, we have examined the effect of superoxide anion (O2⨪) on IP3R-mediated Ca2+ signaling. In human HepG2, rat RBL-2H3, and chicken DT40 cells, we observed [Ca2+]c spikes and frequency-modulated oscillations evoked by a O2⨪ donor, xanthine (X) + xanthine oxidase (XO), dose-dependently. The [Ca2+]c signal was mediated by ER Ca2+ mobilization. X+XO added to permeabilized cells promoted the [Ca2+]c rise evoked by submaximal doses of IP3, indicating that O2⨪ directly sensitizes IP3R-mediated Ca2+ release. In response to X+XO, DT40 cells lacking two of three IP3R isoforms (DKO) expressing either type 1 (DKO1) or type 2 IP3Rs (DKO2) showed a [Ca2+]c signal, whereas DKO expressing type 3 IP3R (DKO3) did not. By contrast, IgM that stimulates IP3 formation, elicited a [Ca2+]c signal in every DKO. X+XO also facilitated the Ca2+ release evoked by submaximal IP3 in permeabilized DKO1 and DKO2 but was ineffective in DKO3 or in DT40 lacking every IP3R (TKO). However, X+XO could also facilitate the effect of suboptimal IP3 in TKO transfected with rat IP3R3. Although in silico studies failed to identify a thiol missing in the chicken IP3R3, an X+XO-induced redox change was documented only in the rat IP3R3. Thus, ROS seem to specifically sensitize IP3Rs through a thiol group(s) within the IP3R, which is probably inaccessible in the chicken IP3R3.

] c signal in every DKO. X؉XO also facilitated the Ca 2؉ release evoked by submaximal IP 3 in permeabilized DKO1 and DKO2 but was ineffective in DKO3 or in DT40 lacking every IP 3 R (TKO). However, X؉XO could also facilitate the effect of suboptimal IP 3 in TKO transfected with rat IP 3 R3. Although in silico studies failed to identify a thiol missing in the chicken IP 3 R3, an X؉XO-induced redox change was documented only in the rat IP 3 R3. Thus, ROS seem to specifically sensitize IP 3 Rs through a thiol group(s) within the IP 3 R, which is probably inaccessible in the chicken IP 3 R3.
Inositol 1,4,5-trisphosphate receptors (IP 3 Rs) 4 are Ca 2ϩ channels that serve to release Ca 2ϩ from the endoplasmic reticulum (ER) in response to cell stimulation by a wide array of hormones, growth factors, and neurotransmitters (1,2). Many fundamental biological processes that are activated or regulated by Ca 2ϩ signals require IP 3 R function. These include such critical functions as secretion (3), smooth muscle contraction (4), gene transcription (5), and fertilization (6). Ca 2ϩ release from IP 3 Rs localized in the vicinity of mitochondria also plays a pivotal role in propagation of Ca 2ϩ signals into the mitochondrial matrix, which, depending on the exact conditions, can lead to enhanced ATP synthesis or the initiation of apoptotic signaling (7). IP 3 R channel activity is primarily regulated by IP 3 and Ca 2ϩ concentrations, although the channel is also modulated by phosphorylation (8), ATP (9), and interaction with a large number of proteins (10).
Another factor that regulates IP 3 Rs is the cellular redox state, although the molecular basis for this mode of regulation is poorly understood (reviewed in Ref. 11). Various exogenously added oxidants stimulate IP 3 R-mediated Ca 2ϩ release. This includes thimerosal (12)(13)(14), t-butylhydroperoxide (15), and diamide (16,17). In the case of thimerosal, the proposed mechanism involves an increased sensitivity of the receptor to lower [IP 3 ], which in some cells is sufficient to trigger Ca 2ϩ oscillations at the ambient [IP 3 ] present in unstimulated cells (11). Although sensitization to IP 3 may be a general mechanism applicable to other oxidants, it has also been suggested that they may alter the Ca 2ϩ sensitivity of the receptor (15,16).
Three different IP 3 R isoforms are expressed in different amounts in various cells, and the different isoforms are capable of forming homo and heterotetramers (18,19). The selective localization or regulation of individual isoforms has been proposed to play a role in different biological processes. For exam-ple, the IP 3 R3 isoform has been suggested to have the predominant role in supplying Ca 2ϩ to the mitochondria in CHO cells (20). However, little is known regarding the IP 3 R isoform selectivity for regulation by redox agents. IP 3 Rs located at ER/mitochondrial junctions would be particularly prone to the reactive oxygen species (ROS) derived from both organelles. In contrast to the exogenous reagents added to manipulate the cellular redox state, the primary endogenous ROS generated as a consequence of mitochondrial respiratory chain activity are superoxide anions (

EXPERIMENTAL PROCEDURES
Cells-RBL-2H3, HepG2, and DT40 (wild type and IP 3 R knock-outs alike) cells were cultured as described previously (7,22,23). Stable colonies of DT40 IP 3 R triple knock-out cells rescued by rat IP 3 R3 were produced as described previously (24). Expression of the IP 3 R3 in each clone was assessed by Western blotting.
Measuring Changes in the Redox State of IP 3 Rs-The method employed was modified from the "thiol trapping" procedure described by Ref. 25 in which TCA is used to preserve the thiol redox state of the proteins. DT40 cells expressing the endogenous chicken IP 3 R3 or the rat IP 3 R3 were centrifuged (800 ϫ g, 5 min) and resuspended in an extracellular-like medium containing 0.25% BSA (0.25% BSA-ECM). 2.5-ml aliquots (ϳ2 ϫ 10 7 cells/ml) were treated for 5 min with 0.1 mM xanthine and 20 milliunits/ml xanthine oxidase. The samples were rapidly centrifuged (1,500 ϫ g, 1 min), resuspended in 0.5 ml of PBS, and quenched by addition of TCA to a final concentration of 10% (w/v). The TCA pellet was recovered by centrifugation (3,000 ϫ g, 5 min) and dissolved in denaturing buffer containing 6 M urea, 0.5% SDS, 200 mM Tris-HCl (pH 8.0), and 10 mM EDTA. Free thiol groups in the lysate were blocked by reaction with 10 mM iodoacetamide for 30 min followed by reprecipitation with TCA and solubilization in denaturing buffer. Modified thiol groups on the receptor were converted to the reduced form by reaction with 10 mM DTT for 30min. The lysate was again reprecipitated with TCA and resolubilized in denaturing buffer containing 20 M DTT at a protein concentration of 2-3 mg/ml. Free thiol groups present in the receptor from control and XϩXO treated cells were reacted in a final volume of 25 l with 0.5 mM PEG-maleimide (5 kDa, Fluka). Gel shifts in the IP 3 R were visualized after running the samples on 5% SDS-PAGE mini-gels and immunoblotting with a monoclonal Ab to the IP 3 R3 isoform (BD Biosciences).
Fluorometric Measurements of [Ca 2ϩ ] c and [Ca 2ϩ ] m in Suspensions of Permeabilized Cells-Experiments with the RBL-2H3 cells were carried out as described earlier (26). Before recording, the fura2FF/AM-loaded cells (ϳ2 mg protein/1.5 ml) were permeabilized in an intracellular medium (120 mM KCl, 10 mM NaCl, 1 mM KH 2 PO 4 , 20 mM Tris-HEPES, 2 mM Magnesium-ATP, and antipain, leupeptin, and pepstatin 1 g/ml each at pH 7.2) supplemented with 25 g/ml digitonin for 5 min at 35°C, followed by washout of the released cytosolic fura2FF (125 ϫ g for 4 -5 min). Compartmentalized fura2FF has been shown to occur in the mitochondria of the RBL-2H3 cells (22). Permeabilized cells were resuspended in intracellular medium supplemented with 2 mM succinate and 0.25 M rhod2/FA and maintained in a stirred thermostatted cuvette at 35°C. rhod2/FA was added to monitor [Ca 2ϩ ] in the intracellular medium that exchanges readily with the cytosolic space, and so [Ca 2ϩ ] rhod2 was abbreviated as [Ca 2ϩ ] c .
When [Ca 2ϩ ] c was measured in permeabilized DT40 cells, the harvested cells were first preincubated in Ca 2ϩ -free extracellular buffer for 1 h at 37°C to drain Ca 2ϩ from intracellular compartments and stored on ice. Cells were permeabilized with saponin (40 g/ml) and incubated in intracellular medium and to measure [Ca 2ϩ ] c , 1.5 M fura2/FA was added.
Fluorescence was monitored in a fluorometer (Delta-RAM, PTI) using 340 nm and 380 nm excitation and 500 nm emission for fura2 or fura2FF and 540 nm excitation and 580 nm emission for rhod2. Calibration of the fura2, fura2FF, and rhod2 fluorescence was carried out at the end of each measurement as described previously (26).
Statistics-Experiments were carried out with at least three different cell preparations, and the data are shown as mean Ϯ S.E. Significance of differences from the relevant controls was calculated by Student's t test.   (33) and MitoSox (34).

RESULTS
Here, we recorded the cytoplasmic glutathione redox state simultaneously with [Ca 2ϩ ] c . Although glutathione redox might change with a slower kinetic than superoxide anion, it can be measured in a more specific and reliable manner. These measurements showed a change in the redox starting together with the XϩXO-induced first [Ca 2ϩ ] c spike (p Ͻ 0.03 at 1 min) (Fig. 2). Because the signal-to-noise ratio is much lower for the redox sensors than that for the calcium sensors it does not seem to be feasible to confirm a redox change before the first calcium spike. A recent study indicated that superoxide anion produced by XϩXO in the extracellular space traverses the plasma membrane ( (Fig. 4). These cells were also selected because RBL-2H3 cells provide a model for the quantification of both [Ca 2ϩ ] c and . . The time course shows the [Ca 2ϩ ] c spikes recorded in the individual cells of the imaging field (red) and the mean response in the GSH redox state (black). The mean response faithfully represents the kinetic of the single cell responses that were averaged because of the relatively low signal to noise ratio. B, single cell Grx1-roGFP2 ratios obtained at 1 min of stimulation were normalized to the prestimulation ratio values (90 s before stimulation), and the mean was calculated for cells treated with XϩXO and with X alone, respectively (nine measurements for each, ϳ10 cells/measurement). A significant increase was obtained for XϩXO as compared with X alone (p Ͻ 0.03). Please note that a continuous downward baseline drift caused lowering R 160s /R 10s under 1 in 150 s.  MARCH  . generation (Fig.   4, A and B). In the RBL-2H3 cells, the [Ca 2ϩ ] c spike was regularly followed by baseline-spike [Ca 2ϩ ] c oscillations (Fig. 4A), whereas in the DT40 cells, the [Ca 2ϩ ] c showed a plateau slightly above the baseline (Fig. 4B) (22). When a suboptimal dose of IP 3 was added, the IP 3 R-mediated Ca 2ϩ release was greatly enhanced by O 2 . (Fig. 5, A and B, lower panel)    and no change in the effect of maximal IP 3 (Fig. 5, A and B (Fig. 6, A and B). However, upon stimulation with IgM, an agonist that stimulates IP 3 formation every DKO, but the TKO cells showed a [Ca 2ϩ ] c rise (Fig. 6C) (Fig. 7A). Although the size of the thapsigargin-induced [Ca 2ϩ ] c increase was similar in each DT40 line, including the TKO cells (Fig. 7C), the IP 3 -sensitive increase was considerably smaller in the DKO3 cells than in the wild type or DKO1 and DKO2 cells (Fig. 7B). Furthermore, the IP 3 dose-response relationship was rightward shifted for the DKO3 cells, whereas the curves for DKO1 and DKO2 were very close to that for the wild type (Fig. 7D).

Control of IP 3 Receptors by Reactive Oxygen Species
In wild type DT40 cells, the O 2 .
-generating system promoted the [Ca 2ϩ ] c rise induced by a suboptimal IP 3 dose and failed to alter the effect of maximal IP 3 (Fig. 8A). Furthermore, DTT, a thiol protecting agent, slightly attenuated the [Ca 2ϩ ] c rise evoked by suboptimal IP 3 but did not change the response to maximal IP 3 (Fig. 8A). Thus, thiol oxidation controlled IP 3 sensitivity in DT40 cells expressing three IP 3 R isoforms. DTT-induced desensitization was also observed in DKO2, whereas the desensitization was not significant in DKO1 (Fig. 8B). Furthermore, the IP 3 sensitivity of DKO3 was not affected by O 2 . or DTT (Fig. 8B). These results suggest that differential sensitization of IP 3 (Fig. 9). First, quantification of IP 3 R3 Western blots of cell lysates was used to select four clones that showed a 10-fold range in IP 3 R3 expression level (100, 30, 17, and 12%, normalized to the highest expressing clone). The highest IP 3 sensitivity was indeed associated with the highest IP 3 R3 expression and the IP 3 -releasable fraction of the ER Ca 2ϩ store was consistently higher in every rat IP 3 R3 expressing clones than in the DKO3 (Fig. 9, A and B). Strikingly, every rat IP 3 R3 expressing TKO showed an apparent sensitization in the presence of O 2 . generating system (Fig. 9C). Collectively, these results indicate that O 2 . sensitizes IP 3 R regardless of their expression level. Surprisingly, the rat IP 3 R3 is similar to chicken IP 3 R1 and IP 3 R2 in its sensitivity to O 2 . .

Sequence Heterogeneity between Chicken IP 3 R3 and Other
. likely affects the IP 3 R function through a reactive Cys residue(s) within the IP 3 R or in a protein that interacts with and controls the IP 3 R. Because the latter group includes many proteins, we focused on studying the presence of Cys thiol groups in various IP 3 R isoforms. We searched for a Cys that is present it rat but is absent in chicken IP 3 R3. We found that three of 51 Cys present in rat IP 3 R3 were absent in chicken IP 3 R3 (Table 1). However, none of these Cys was also present in IP 3 R1 and IP 3 R2. Thus, these Cys groups are unlikely to confer O 2 . sensitivity to the IP 3 R.
To determine whether there are differences in the redox responses of the chicken and rat IP 3 R3s, we measured the redox state of the receptors expressed in DT40 cells using a modification of the thiol trapping procedure (Fig. 10) (25). In this method, TCA is used to deproteinize the cells and prevent thiol transformations. The precipitated protein is solubilized under denaturing conditions (SDS/urea) and successively treated with iodoacetamide and DTT to block free thiol groups and to make available oxidized residues for subsequent reaction with maleimide conjugated to a 5-kDa methoxy polyethylene glycol. The magnitude of the gel shift in immunoreactive IP 3 R is proportional to the number of available oxidized residues in the protein. The minimal shift observed for the chicken and rat IP 3 R3 is an indication that very few of the thiol residues in the receptor are oxidized under control conditions. The addition of XϩXO to the cells expressing the rat IP 3 R3 isoform resulted in an enhanced reactivity of the receptor for methoxy polyethylene glycol indicating the oxidation of additional thiols. In contrast, the chicken isoform did not show an enhanced methoxy polyethylene glycol shift. A similar difference was also noted in response to 0.2 mM H 2 O 2 (data not shown). Thus, some evolutionary conserved Cys are likely to be modified by O 2 . only in the rat IP 3 R3 and are candidates to mediate sensitization of the IP 3 R to IP 3 . . caused sensitization of IP 3 R1 and IP 3 R2 (p Ͻ 0.01) but failed to affect IP 3 R3. ctrl, control.

DISCUSSION
Our  (38). S-Glutathionylation of the IP 3 R1 has been demonstrated during diamide sensitization of the IP 3 R in cultured aortic endothelial cells (16,17). Whether this is a general modification occurring with other oxidants, IP 3 R isoforms and cell types remain to be determined.    induced thiol oxidation is absent in chicken IP 3 R3 but is present in rat IP 3 R3. Trichloroacetic acid and a strongly denaturing buffer (SDS/urea) was used to prepare lysates from control and XϩXO-treated DT40 cells expressing rat IP 3 R3 (TKO rescued with rat IP 3 R3) or chicken IP 3 R3 (DKO3) as described in "Experimental Procedures." After initially blocking all free thiol groups with iodoacetamide, the remaining modified thiol residues were reduced with DTT and then reacted with methoxy polyethylene glycol (MPEG-5). The presence of oxidized thiol residues in the receptor is indicated by a gel shift reaction detected by immunoblotting on 5% SDS-PAGE. The data shown indicate that the thiols in the endogenous rat or chicken IP 3 R3 receptor are almost entirely in the reduced state under control conditions and only the rat isoform shows an oxidation response with XϩXO. Because of differences in the expression levels of the chicken and rat isoforms the amount of protein loaded for the two isoforms was different (2 g of rat; 20 g of chicken). The data shown are representative of three experiments.
Redox regulation of ryanodine receptor channels share several common features with IP 3 Rs. Ryanodine receptors show enhanced activity in response to exogenous oxidants as well as endogenously produced ROS in both heart and skeletal muscle (39,40). Attempts to identify the redox-sensitive, "hyper-reactive" thiols by mass spectrometry indicate the involvement of multiple thiols dispersed throughout the linear sequence (41,42). Mutagenesis of multiple residues did not entirely eliminate the functional effects of the redox agents (43). In addition, the findings in the present paper indicate that redox sensitivity may not solely be determined by thiols on the IP 3 R but could also involve other factors such as associated proteins or the local environment. This suggests that unraveling the molecular basis of redox sensing in these intracellular Ca 2ϩ release channels will be a challenging task.
Recent studies indicate broad physiological and pathophysiological relevance of ROS (44,45). The present results suggest that O 2 . produced by multiple intracellular enzymes might utilize IP 3 R-mediated Ca 2ϩ mobilization to make a contribution to cell signaling. Because DTT that reduces disulfide bonds in proteins had some desensitizing effects on the IP 3 R activity under resting conditions, low levels of ROS continually produced inside the cells might be relevant for IP 3 R function. However, the large effect of the O 2 . generating system indicates that increased endogenous ROS production has the potential to enhance IP 3 R-linked calcium signaling. Our studies primarily focused on the effects of O 2 . however, its breakdown product, H 2 O 2 also seems to have sensitizing effect on the IP 3 R ( (46,47) and this work). ROS can also be converted to reactive nitrogen species, and reactive nitrogen species-mediated nitrosylation affects some components of calcium signaling, but its relevance for the IP 3 R is unclear. . Notably, our results support that extracellular superoxide anion increases cytoplasmic ROS, which can directly control IP 3 -induced Ca 2ϩ release. It remains possible that a component of the calcium signaling response observed in intact cells is also due to enhanced IP 3 formation, which could also be secondary to elevated [Ca 2ϩ ] c . The IP 3 Rs represent an intriguing target of ROS owing to their localization close to main ROS producing organelles (50). Both the ER that hosts IP 3 Rs and the mitochondria that are closely associated and physically coupled to the ER are central to cellular ROS production. It has been speculated that ROS produced by these organelles can locally expose the IP 3 Rs and ryanodine receptors (36,50). However, these ideas remain to be tested by direct measurements of ROS at cellular subdomains. Our demonstration of the potential functional relevance of ROS in both ER Ca 2ϩ mobilization and local Ca 2ϩ transfer to the mitochondria should stimulate further studies of ROS at the surface and interface of ER and mitochondria.