Control of IP 3 receptors by reactive oxygen species 1 Isoform-and species-specific control of IP 3 receptors by reactive oxygen species *

Reactive oxygen species (ROS) stimulate cytoplasmic [Ca] ([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 Ca 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 Ca 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 Ca release. In response to X+XO, DT40 cells lacking two out 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 [Ca 2+]c signal in every DKO. X+XO also facilitated the Ca 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 unaccessible in the chicken IP3R3. Control of IP3 receptors by reactive oxygen species 2 Introduction Inositol 1,4,5-trisphosphate receptors (IP3R) are Ca channels that serve to release Ca 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 signals require IP3R function. These include such critical functions as secretion (3), smooth muscle contraction (4), gene transcription (5) and fertilization (6). Ca release from IP3Rs localized in the vicinity of mitochondria also plays a pivotal role in propagation of Ca signals into the mitochondrial matrix which, depending on the exact conditions, can lead to enhanced ATP synthesis or the initiation of apoptotic signaling (7). IP3R channel activity is primarily regulated by IP3 and Ca 2+ 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 IP3Rs is the cellular redox state although the molecular basis for this mode of regulation is poorly understood (reviewed in (11)). Various exogenously added oxidants stimulate IP3R-mediated Ca release. This includes thimerosal (12-14), tbutylhydroperoxide (15) and diamide (16,17). In the case of thimerosal the proposed mechanism involves an increased sensitivity of the receptor to lower [IP3] which in some cells is sufficient to trigger Ca oscillations at the ambient [IP3] present in unstimulated cells (11). While sensitization to IP3 may be a general mechanism applicable to other oxidants, it has also been suggested that they may alter the Ca sensitivity of the receptor (15,16). Three different IP3R 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 have been proposed to play a role in different biological processes. For example the IP3R3 isoform has been suggested to have the predominant role in supplying Ca to the mitochondria in CHO cells (20). However, little is known regarding the IP3R isoform selectivity for regulation by redox agents. IP3Rs 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 (O2 ) which are dismutated to form H2O2. Similarly, the ER can generate substantial amounts of H2O2 from multiple sources (21). In the present study we have evaluated the effects on IP3Rmediated release of a physiological oxidant, O2 .generated from xanthine by xanthine oxidase. The experiments have been carried out using different experimental models which express individual isoforms of IP3Rs. Our data show that responsiveness to an endogenously produced ROS is dependent on the exact IP3R isoform and species variant examined. Experimental Procedures Cells: RBL-2H3, HepG2 and DT40 (wildtype and IP3R knockouts alike) cells were cultured as described previously (7,22,23). Stable colonies of DT40 IP3R triple knockout cells rescued by rat IP3R3 were produced as described previously (24). Expression of the IP3R3 in each clone was assessed by western blotting. Measuring changes in the redox state of IP3Rs: The method employed was modified from the “thiol trapping” procedure described by (25) in which TCA is used to preserve the thiol redox state of the proteins. DT40 cells expressing the endogenous chicken IP3R3 or the rat IP3R3 were centrifuged (800 g, 5 min) and resuspended in an extracellular like medium containing 0.25 % BSA (0.25 % BSA-ECM). Aliquots 2.5 ml (~2 x 10 cells/ml) were treated for 5 min with 0.1 mM Xanthine and 20 mU/ml Xanthine oxidase. The samples were rapidly centrifuged (1,500 g, 1min), resuspended in 0.5 ml PBS and quenched by addition of TCA to a final concentration of 10% (w/v). The TCA pellet was recovered by centrifugation (3,000 g, 5min) and dissolved in denaturing buffer (DB) containing 6 M Urea, 0.5 % SDS, 200 mM TrisHCl (pH 8.0) and 10mM EDTA. Free thiol groups in the lysate were blocked by reaction with 10 mM iodoacetamide for 30 min followed by re-precipitation with TCA and solubilization in DB 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 Control of IP3 receptors by reactive oxygen species 3 TCA and re-solubilized in DB buffer containing 20 μM DTT at a protein concentration of 2-3 mg/ml. Free thiol groups present in the receptor from control and X+XO treated cells were reacted in a final volume of 25 μl with 0.5 mM PEGmaleimide (5 kDa, Fluka). Gel shifts in the IP3R were visualized after running the samples on 5 % SDS PAGE mini-gels and immunoblotting with a monoclonal Ab to the IP3R3 isoform (BD Biosciences). Fluorescence imaging of [Ca 2+ ] in single cells: To monitor [Ca2+]c, cells were loaded with 5 μM fura2/AM for 20 min in the presence of 100 μM sulfinpyrazone and 0.003% (wt/v) pluronic acid in 2 % BSA-ECM at room temperature. Cells attached to coverslips were placed in 1 ml buffer to the heated stage (35 Co) of an inverted epifluorescence microscope (40X oil objective) connected to a cooled CCD camera (PXL, Photometrics). Ratiometric imaging of fura2 was used to monitor [Ca] as described previously (7,26,27). Simultaneous imaging of cytoplasmic [Ca] and GSH/GSSG was performed in cells transfected with plasmid DNA encoding RCaMP (28) and Grx1-roGFP2 (29,30) using a ProEM1024 EM-CCD (Princeton Instruments), fitted to Leica DMI 6000B inverted epifluorescence microscope (31). Two different filter sets (for RCaMP: ex:580/20 nm, bs595 nm, em: 630/60 nm and for Grx1-roGFP2: ex: 415/20 nm and 490/20 nm excitation filters and a 500 nm long-pass bs and ex:520/40nm) were alternated by a motorized turret. Fluorometric measurements of [Ca 2+ ]c and [Ca 2+ ]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 (approx. 2 mg protein/1.5 ml) were permeabilized in an intracellular medium (ICM: KCl 120 mM, NaCl 10 mM, KH2PO4 1 mM, Tris-HEPES 20 mM, MgATP 2 mM, and antipain, leupeptin and pepstatin 1 μg/ml each at pH 7.2) supplemented with 25 μg/ml digitonin for 5 min at 35 ̊C, followed by washout of the released cytosolic fura2FF (125 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 ICM supplemented with succinate 2 mM and rhod2/FA 0.25 μM and maintained in a stirred thermostated cuvette at 35 ̊C. Rhod2/FA was added to monitor [Ca] in the intracellular medium that exchanges readily with the cytosolic space and so [Ca2+]rhod2 was abbreviated as [Ca2+]c. When [Ca2+]c was measured in permeabilized DT40 cells, the harvested cells were first preincubated in Ca-free extracellular buffer for 1hr at 37°C to drain Ca from intracellular compartments and stored on ice. Cells were permeabilized with saponin (40 μg/ml) and incubated in ICM and to measure [Ca2+]c fura2/FA 1.5 μM was added. Fluorescence was monitored in a fluorometer (Delta-RAM, PTI) using 340 nm, 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 3 different cell preparations and the data are shown as mean+SE. Significance of differences from the relevant controls was calculated by Student’s t test.