JBC Papers in Press. Published on May 16, 2008 as Manuscript M

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1 JBC Papers in Press. Published on May 6, 8 as Manuscript M855 The latest version is at COMPLEX ACTIONS OF -AMINOETHYLDIPHENYL BORATE () ON STORE- OPERATED CALCIUM ENTRY Wayne I. DeHaven, Jeremy T. Smyth, Rebecca R. Boyles, Gary S. Bird and James W. Putney Jr. From the Laboratory of Signal Transduction National Institute of Environmental Health Sciences NIH Department of Health and Human Services, PO Box Research Triangle Park, NC 779 Running Head: Complex Actions of Address correspondence to: JW Putney, NIEHS-NIH, PO Box, Research Triangle Park, NC. Tel.: 99-5-; FAX: ; putney@niehs.nih.gov Store-operated Ca + entry (SOCE) is likely the most common mode of regulated influx of Ca + into cells. However, only a limited number of pharmacological agents have been shown to modulate this process. -Aminoethyldiphenyl borate () is a widely used experimental tool that activates then inhibits SOCE and the underlying calcium-release-activated Ca + current (I CRAC ). The mechanism by which depleted stores activates SOCE involves complex cellular movements of an endoplasmic reticulum Ca + sensor, STIM, which redistributes to puncta near the plasma membrane and in some manner activates plasma membrane channels comprised of Orai, or subunits. We show here that - APB blocks puncta formation of fluorescently tagged STIM in HEK9 cells. Accordingly, - APB also inhibited SOCE and I CRAC -like currents in cells co-expressing STIM with the CRAC channel subunit, Orai, and with similar potency. However, inhibited STIM puncta formation less well in cells co-expressing Orai, indicating that the inhibitory effects of are not solely dependent upon STIM reversal. Further, only partially inhibited SOCE and current in cells coexpressing STIM and Orai, and activated sustained currents in HEK9 cells expressing Orai and STIM. Interestingly, the Orai- dependent currents activated by showed large outward currents at potentials greater than +5 mv. Finally, Orai, and to a lesser extent Orai, could be directly activated by - APB, independently of internal Ca + stores and STIM. These data reveal novel and complex actions of effects on SOCE that can be attributed to effects on both STIM, as well as Orai channel subunits. In many cell types, the activation of phospholipase C through G protein coupled receptors liberates Ca + from the lumen of the endoplasmic reticulum (ER); this depletion of ER Ca + stores results in activation of a process called capacitative or storeoperated Ca + entry (SOCE), whereby extracellular Ca + enters the cell via plasma membrane channels (;). The best characterized SOCE current is the Ca + -release-activated Ca + current (I CRAC ), first described in mast cells () and since recorded in several cell types (). Until recently, the mechanism by which I CRAC is activated by store depletion, as well as the channels themselves, was unknown. However, the discoveries of both STIM (5;6) and Orai (CRACM) (7-9) have revealed two key molecular components of the I CRAC signaling pathway. It is now clear that STIM functions as the Ca + sensor within the ER, while members of the family of Orai proteins (including Orai,, and ) function as poreforming subunits of CRAC channels in the plasma membrane. When intracellular Ca + stores are depleted, STIM rearranges from a fibrillar localization that depends on microtubules to discrete punctate structures near the plasma membrane (6;-). Remarkably, Orai channels also rearrange into punctate structures in response to store depletion that coincide with those formed by STIM (-5). Thus, highly orchestrated molecular rearrangements underlie I CRAC activation. Over expression of Orai together with STIM in HEK9 cells produces unusually large currents with biophysical properties similar to I CRAC (9;6-8), suggesting that either these two proteins are sufficient to completely recapitulate I CRAC, or any additional proteins required must be present in excess within the cell (9). Orai and Orai also Copyright 8 by The American Society for Biochemistry and Molecular Biology, Inc.

2 produce highly calcium selective when coexpressed with STIM, although the currents are somewhat smaller and the biophysical and pharmacological properties differ somewhat (;). Knowledge of key molecular players in the SOCE pathway allows for the first time investigation of the cellular and molecular mechanisms of pharmacological modulators. Of the limited number of SOCE inhibitors that have been described, perhaps -aminoethyldiphenylborate (- APB) has been the most extensively utilized. This drug was originally described as a membrane permeant inhibitor of the IP receptor, and the inhibition of SOCE by was taken as evidence for direct IP receptor activation of CRAC channels () in a process called conformational coupling (). However, more recent work has shown that inhibition of I CRAC is independent of IP receptors (-). It has been reported that Orai, Orai and Orai differ in their responses to (). However, the cellular or molecular mechanism underlying these differing effects has not been investigated. Ideally, one would like to investigate the actions of pharmacological agents on ion channels expressed in their native environments. However, to date, only functional expression of native Orai channels has been documented, whereas functions of native Orai or Orai channels have not been clearly demonstrated in any specific cell type. We have thus investigated the actions of on HEK9 cells transiently transfected with plasmids encoding Orai, or, N-terminally tagged with a cyan fluorescent protein (CFP- Orai, and ), in most instances together with an eyfp-stim construct. Our results indicate a complex array of effects of that differ among the three Orai subtypes. We also describe for the first time striking effects of on the function of the Ca + sensor, STIM. Thus, effects on STIM and Orai proteins are extremely complex, and may at some point reveal details of the molecular actions of these important proteins. However, the complexity of s actions also brings into question the utility of this drug as a specific modulator of SOCE. Materials and Methods Cell Culture. HEK9 cells were obtained from ATCC and cultured in Dulbecco s Modified Eagle s Medium (DMEM) supplemented with % heat inactivated fetal bovine serum and mm glutamine. The HEK9 cells were maintained at 7 C in a humidified incubator set at 5% CO. All experiments were carried out on HEK9 cells plated onto mm round glass coverslips mounted in a Teflon chamber. Plasmids and transfections. The N-terminus tagged eyfp-stim was obtained from the laboratory of Tobias Meyer at Stanford University. CFP-tagged Orai plasmids were constructed using human Orai, -, and - purchased from Invitrogen in the pentr TM vector and the Gateway system LR reaction (Invitrogen) using the destination vector pdest5 (). Cells were transfected with Lipofectamine ( μl/well; Invitrogen), as previously described (8). Briefly, HEK9 cells were plated in a 6-well plate. On the following day, cells were transfected with CFP- Orai ( μg/well), CFP-Orai ( μg/well; in some experiments in Figures 5 and 6, an untagged version was used), CFP-Orai ( μg/well), or eyfp-stim (μg/well) cdna. Six hours later the medium bathing the cells was replaced with complete DMEM and maintained in culture overnight. The next morning transfected cells were transferred to -mm glass coverslips in preparation for experiments. In some experiments, STIM expression was reduced by RNAi, as previously described (). Intracellular Ca + measurements. Intracellular Ca + measurements were carried out as previously described (8). Briefly, cells were loaded with μm Fura-5F/AM (Invitrogen) for 5 min at 7 C. Fura-5F fluorescence was measured when cells were excited alternately at nm and 8 nm, and Ca + concentrations are reported as the ratio of fluorescence emission at the two excitation wavelengths. Cells transfected with eyfp-stim were chosen based on their fluorescence when excited at 77 nm. Generally, - eyfp positive cells were measured on a single coverslip in each experiment. Ratio values were corrected for contributions by autofluorescence, which was

3 measured after treating cells with μm ionomycin and mm MnCl after the experiments had ended. Live cell confocal and TIRFM imaging. Cells were maintained in HEPES-buffered saline solution (HBSS; in mm: NaCl, 5. KCl,.8 CaCl,.8 MgCl, glucose, and HEPES, ph 7.) at room temperature. Confocal imaging was carried out using a Zeiss LSM 5 laser scanning system, and either a x water-immersion (N.A..) or a 6x oil-immersion (N.A..) objective was used. All confocal images were collected with the pinhole set at Airy Unit. For eyfp-stim, 88 nm or 5 nm illumination was provided by an Argon laser and emission was selected with a 5-6 nm bandpass filter. TIRFM was carried out essentially as previously described (). For fluorescence intensity profiles, data are represented as the fluorescence intensity at each time point divided by the fluorescence intensity at the start of the experiment (F/F). Fluorescence intensities were collected from regions of interest encompassing the visible footprints of single cells and were background subtracted. Electrophysiology. Whole-cell currents were measured at room temperature in HEK9 cells using the patch-clamp technique in the whole-cell configuration. The standard HEPES buffered saline solution for these experiments contained (mm): 5 NaCl, KCl, CsCl,. MgCl,. CaCl, glucose, and HEPES (ph to 7. with NaOH). Fire-polished pipettes fabricated from borosilicate glass capillaries (WPI, Sarasota, FL) with -5 MΩ resistance were filled with (in mm): 5 Cs-methanesulfonate, BAPTA, HEPES, and 8 MgCl (ph to 7. with CsOH). For experiments in Figures 8 and 9, Ca + was added to the internal solution to yield a final concentration of nm free Ca +, as determined using Maxchelator software. This prevents passive store depletion of the internal Ca + stores. In indicated experiments, the pipette also contained μm inositol,,5-trisphosphate (IP, Sigma) to actively deplete intracellular Ca + pools. Voltage ramps (- mv to + mv) of 5 ms were recorded every two seconds immediately after gaining access to the cell from a holding potential of mv, and the currents were normalized based on cell capacitance. Leak currents were subtracted by taking an initial ramp current before I CRAC developed and subtracting this from all subsequent ramp currents. Access resistance was typically between 5- MΩ. The currents were acquired with pclamp- (Axon Instruments) and analyzed with Clampfit (Axon Instruments) and Origin 6 (Microcal) software. All solutions were applied by means of a gravity based multi-barrel local perfusion system with an extremely low dead volume common delivery port (Perfusion Pencil, Automate Scientific, Inc.). Results dose-dependently activates and inhibits SOCE. Figure shows Fura-5F mediated calcium imaging experiments on wild-type (WT) HEK9 cells (Figure A) or in cells expressing eyfp- STIM (Figure C). Under nominally Ca + free conditions (HBSS), internal Ca + stores were depleted with the SERCA pump inhibitor, thapsigargin (), and extracellular Ca + was restored revealing SOCE. Five minutes after the addition of Ca +, various concentrations (- 5 µm) of the drug were added in the continued presence of external Ca +. In wild-type HEK9 cells, SOCE was nearly completely blocked by at concentrations greater than μm (Figure B). However, lower concentrations ( and µm shown, and up to μm not shown) appeared to potentiate SOCE. Often inhibition by high concentrations was preceded by a brief potentiation, as evident for μm in Figure A. Expression of eyfp-stim (Figure C, D), did not appear to influence the potentiating or inhibitory effects of on SOCE. reverses or prevents STIM localization in near plasma membrane puncta. Upon store depletion, STIM rearranges into puncta in close proximity to the plasma membrane, and this rearrangement of STIM appears necessary for SOCE and I CRAC development. Thus, we considered whether the bimodal actions of on SOCE could result from enhancement or disruption of STIM puncta formation. Figure A is a representative experiment using confocal microscopy and HEK9 cells expressing eyfp- STIM. Shown is the distribution of eyfp-stim before and after treatment confirming that

4 STIM clearly rearranges into puncta in response to ER Ca + store depletion. After the addition of 5 μm, the punctate structures were largely dispersed, and the eyfp-stim arrangement more closely resembles that seen when Ca + stores are filled (before ) than when stores are depleted (after ). In order to more quantitatively assess the effect of on STIM, TIRFM experiments were carried out using the eyfp-stim construct. Figure B shows that treatment caused a marked increase in near-plasma membrane eyfp- STIM fluorescence. However, after the addition of 5 μm, eyfp-stim near-membrane fluorescence intensity was significantly reversed (Figure B and C). Furthermore, as shown in Figure D and E, pretreatment of cells with completely prevented eyfp-stim from migrating to near-membrane regions in response to store depletion. Note that the application of prior to store depletion generally decreased the TIRF intensity, as shown by the small decrease in F/F in Figure D after the application of. Finally, at lower micromolar concentrations ( µm) of, where potentiation is seen in the Ca + imaging (Figure ) and electrophysiological (see below) experiments, no enhancement of near plasma membrane eyfp-stim was detected (Data not shown). Note that 5 μm Gd +, a specific channel inhibitor of SOCE and I CRAC () had no effect on the rearrangement of eyfp-stim measured by TIRFM, despite significant inhibition of SOCE. Thus, channel block alone is not sufficient to reverse the punctate, near plasma membrane localization of STIM. These data suggest that the inhibition of SOCE by may, at least partly, be mediated by the reversal of STIM puncta formation. However, the potentiation appears to be independent of STIM relocalization, at least within the resolution of our methods. activates and inhibits I CRAC in HEK9 cells co-expressing STIM and Orai. Figure shows recordings of I CRAC from HEK9 cells coexpressing CFP-Orai with eyfp-stim. In these cells, increased I CRAC in a sustained manner at concentrations less than or equal to μm, while higher concentrations of (> μm) strongly inhibited the currents (Figure A, B and not shown). Shown in Figure A is focal application of μm, then 5 μm onto the same cell after active store depletion with IP and BAPTA in the patch pipette. Currents were recorded from voltage ramps ( mv holding potential; - mv to + mv) applied every two seconds. Interestingly, the peak transient currents seen when 5 μm was applied on its own (Figure A inset) were similar in amplitude to the sustained currents seen with μm ( μm : ±.59 pa/pf sustained increase in I CRAC, n=6; 5 μm : ±.96 pa/pf transient increase in I CRAC, n=). Figure C shows the current-voltage relationships taken from the voltage ramps for the currents recorded in eyfp-stim plus CFP-Orai positive cells (shown in Figure A) after store depletion and in the presence of μm and 5 μm. I CRAC potentiated by was inwardly rectifying, with an indistinguishable current-voltage relationship compared to the current recorded in the absence of. Orai expression reduces inhibition of STIM rearrangement. The results of Figure indicate that the effects of on I CRAC in Orai and STIM co-expressing cells is similar to the effects of on SOCE in WT cells or in cells overexpressing only STIM. Thus, we assessed the effects of on SOCE and STIM movements in TIRFM experiments with CFP-Orai expressed in conjunction with eyfp- STIM. Parallel experiments were carried out using single cell Ca + imaging in order to track effects of on SOCE under the same conditions. Figures A and B show TIRFM and Ca + imaging experiments, respectively, in which 5 μm much less effectively (compared to eyfp-stim alone, Figure ) reversed store depletion-dependent eyfp-stim rearrangement (panels A and C), yet nearly completely blocked SOCE (panels B and D). Increasing the concentration of to μm did not cause any significantly additional block (F/Fo = % of control, n=9, as compared to %, n=6, for 5 μm). Furthermore, unlike for HEK9 cells expressing eyfp-stim alone, cells co-expressing eyfp-stim with CFP-Orai and pretreated with 5 μm for 5 minutes showed significant TIRFM responses after

5 treatment (Figure E and G); however, pretreatment with completely blocked SOCE from developing (Figure F and H). Thus, while expression of Orai in combination with STIM does not disrupt s abilities to inhibit I CRAC and SOCE, it does significantly reduce the ability of to interrupt STIM reorganization in response to store depletion. This indicates that the inhibitory effects of must at least in part represent additional actions on the Orai channel or the activation mechanism. We suspect that the effect of overexpressed Orai to reduce the effects of on STIM movements may result from stabilization of STIM at plasma membrane punctae when Orai is present in stoichiometrically similar amounts. Thus, knowing that under our conditions, Orai and express at considerably lower levels than Orai (), we examined their effects on the reversal and inhibition of STIM movements. As predicted, Orai and were indeed considerably less effective in either inhibiting or reversing STIM movements than was Orai (Supplemental Figure ). Responses to differ in cells co-expressing STIM with Orai and Orai. It was previously reported that Orai, Orai and Orai, when coexpressed with STIM, differ in their responses to (). Figures 5A and B show that inhibited store-operated currents in Orai and eyfp-stim expressing cells in a manner qualitatively different from its action in cells expressing Orai and eyfp-stim (see Figure A). μm caused a transient activation in some, but not all cells, followed by a slow decline in current. Increasing the concentration to 5 μm caused a small, abrupt drop in current, but did not appear to hasten the slow decline in current. Although these experiments were not carried out for longer times, we assume the inhibition by - APB would eventually be complete because we previously showed that pretreatment with completely blocks SOCE in Orai and STIM expressing cells (8). In all instances the currentvoltage relationships were inwardly rectifying, reminiscent of I CRAC (Figure 5B). Even more interesting were the results obtained with cells expressing CFP-Orai plus eyfp- STIM. While μm caused a small potentiation of Orai current, the focal application of 5 μm caused the activation of a biphasic current that initially resembles I CRAC ; however, longer exposure to caused much larger currents to develop which no longer were solely inwardly rectifying (Figure 5C and D). Over a period of minutes, the initially inwardly rectifying current changed to one that appeared doubly rectifying, with large inward and outward currents at the most negative and positive potentials. The inward currents recorded at - mv were 8 times larger after prolonged exposure when compared to the Ca + currents after store depletion (Figure 5F). A larger overall current with the development of an outward current suggests that causes changes in the Ca + selectivity of channels formed by ectopically expressed Orai. Similar currents were not seen in cells expressing eyfp or eyfp-stim alone, or in conjunction with CFP-Orai or CFP-Orai in response to these concentrations of (Data not shown). Direct activation of Orai by. Data in Figure 5 show that following Ca + store depletion, increases whole-cell currents in cells expressing STIM plus Orai. In order to determine whether the potentiating effects of - APB depend on store depletion and activation by STIM, single-cell calcium imaging experiments were carried out using HEK9 cells overexpressing CFP-Orai, - or - without exogenous STIM expression or after RNAi knockdown of endogenous STIM (as in ()). In HEK9 cells that were not transfected with any of the three Orai, 5 μm had no effect on the basal cytoplasmic Ca + concentration (not shown). In cells expressing CFP-Orai, a small, transient but reproducible Ca + response was detected, which was identical in cells in which STIM had been reduced by RNAi (Figure 6A, D). No such responses were seen in Orai-expressing cells (Figure 6B, D). However, in Orai-expressing cells activated considerable Ca + influx, and as for Orai, this entry was unaffected by knockdown of endogenous STIM (Figure 6C, D). Figures 6E and F show the concentration dependent activation of Orai by, from which the half-maximal concentration was calculated to be.5 ±.6 μm using a 5

6 least squares fit. Interestingly, the concentration range of for inhibition of entry in WT HEK9 cells, or in WT HEK9 cells expressing STIM alone or in conjunction with Orai (see Figures and ), is similar to the concentration range at which activates Orai. Single-cell Ca + imaging experiments were also carried out on HEK9 cells expressing CFP- Orai alone, in which stores were depleted first with. Ca + was added back to assess SOCE, and then 5 μm was applied. The purpose of these experiments was to further assess whether endogenous STIM or store depletion might play a role in the activation of Orai by. Figures 7B and C show that store depletion and endogenous STIM had little to no effect on - APB activation of Orai, when compared to the effects of on Orai without store depletion (see Figure 6). Although not directly relevant to this study, it should be noted that this is the first time in which it has been shown that exogenously expressed Orai also suppresses SOCE (panel B) in a similar manner as Orai and Orai (8) when compared to control WT HEK9 cells (panel A), when not expressed with STIM. In order to determine the biophysical properties of the calcium entry seen after application in Orai and Orai expressing cells, whole-cell electrophysiological experiments were carried out in which the internal calcium was clamped to nm to prevent store depletion. In the presence of mm external calcium, 5 μm was applied and whole-cell inward currents were recorded in the same fashion as in previous figures ( mv holding potential; - mv to + mv ramps every sec). Figure 8A shows a representative recording in which directly activated Orai-dependent currents, independently of STIM or store depletion. Interestingly, the maximal currents recorded from CFP-Orai expressing cells lacking exogenous STIM expression appeared identical to the maximal currents recorded when exogenous STIM was present and stores were depleted, both in terms of magnitude and current-voltage relationship (Figures 8B and D). In a manner similar to the Ca + imaging experiments shown in Figure 6, cells expressing CFP-Orai also developed transient inward currents in response to 5 μm, independently of STIM or store depletion (Figure 8A). Unlike Orai currents, the currents seen in these cells exhibited inward rectification, reminiscent of I CRAC (Figure 8B). In Orai expressing cells, no direct activation of current by focal application of was ever detected. These results are summarized in the bar graph shown in Figure 8, panel C. Taken together, we feel the Ca + imaging and electrophysiological data suggest, at least in the case for Orai and to a lesser degree Orai, directly activates these channels, independently of store depletion and STIM. increases Cs + permeability through Orai channels. The presence of the outward currents seen in the Orai expressing cells after 5 μm - APB application suggested the compound altered the channel properties, now allowing Cs + to permeate. We previously reported that similar to Orai, Orai (and Orai) does not permeate Cs + well (). The outward currents also suggest Ca + and/or Mg + is less effective at blocking monovalent conductance through the channels when is present. In order to further investigate these possibilities, whole cell-patch clamp experiments were carried out on HEK9 cells expressing CFP-Orai. Cells were selected based on CFP fluorescence. Figure 9A shows a representative recording depicting the effects of extracellular Ca + on the activated Orai whole-cell currents. As seen in panels A and B, increasing extracellular Ca + from mm to mm reduces the outward currents recorded at + mv, shifts the reversal potential rightward, and initially decreases, then slightly increases the inward currents recorded at - mv. Like endogenous I CRAC, Na + permeated through the - APB activated Orai channels, and the inward Na + conductance was significantly inhibited by mm extracellular Mg + in the nominally Ca + free (NCF) external solution (Figure 9A and C). However, this block is not complete (Figure 9C), whereas for store-operated I CRAC and for storeoperated Orai channels (), the block is complete. There was a leftward shift in the reversal potential seen in the peak Na + -DVF currents (panel C) when compared to the Na + currents seen in Orai channels activated by STIM and store-depletion (panel G). Thus, the Ca + selectivity of Orai channels is also reduced 6

7 by. An additional major difference in pore properties of -activated Orai channels was revealed by experiments examining Cs + conductance through activated Orai channels (Figure 9D and E). After activating Orai by under mm Ca + conditions, switching to a Cs + -DVF extracellular solution reveals a large inward Cs + current in these cells which is not present in Orai plus STIM coexpressing, store-depleted controls (panels F and G). Taken together, these results indicate that - APB directly activates Orai and alters Orai channel properties, reducing the ability of extracellular divalent cations to block monovalent permeation and increasing the Cs + /Na + permeability ratio (Figure 9, panels E and G). Discussion was originally described as an IP receptor antagonist, and was later shown to dosedependently potentiate and inhibit SOCE and I CRAC, independently of IP receptor inhibition. With the recent discoveries of the STIM and Orai proteins, we sought to investigate the bimodal effects of on store-operated currents and Ca + entry in cells expressing these proteins to gain insight into the molecular mechanism of action of this extensively employed pharmacological tool. We show here that prevents, as well as reverses eyfp-stim reorganization in response to ER Ca + store depletion, independently of store refilling. It has previously been shown that the rearrangement of STIM in the ER is essential for activation of SOCE (6;;8). Thus, this effect on STIM localization could account for the inhibitory effects of on SOCE and I CRAC development. However, co-expression of STIM with the CRAC channel pore forming subunit, Orai, significantly reduced the ability of to prevent or reverse store depletion-dependent STIM movements, while having no effect on its ability to inhibit SOCE or current. The Orai homologs, Orai and Orai, respond differently to inhibitory concentrations of, yet the activation of all three depends on STIM. While Orai-mediated current was only slowly inhibited compared to Orai, Orai was activated by and the Orai forming channels became permeable to monovalent ions. Finally, we show that exogenously expressed Orai, and to a lesser extent Orai, can be directly activated by, independently of STIM and store depletion. Taken together, these data reveal a complex mechanism of action of on SOCE and store-operated currents, in which influences both STIM and Orai proteins located in the ER and plasma membranes, respectively. Given that at least some of the inhibitory effects on Orai channels occur downstream of STIM movements, and that the degree of inhibition of STIM movements depends on the relative levels of STIM and Orai, it is not possible to ascertain the degree to which inhibition of STIM movement contributes to channel inhibition in native cells. Nonetheless, these findings are significant given that is widely used in the investigation of SOCE and I CRAC. A better understanding of the effects of on STIM movements and SOCE can be gained by comparing its effects with another inhibitor of SOCE and I CRAC, ML-9. We recently reported that ML-9 completely inhibits endogenous SOCE and I CRAC by inhibiting store depletion-induced reorganization of STIM into near plasma membrane puncta, a similar action to that reported here for (5). However, in the case of ML- 9, exogenous expression of STIM caused a rightward shift of the inhibition curve when compared to WT HEK9 cells. This partial rescue of the ML-9 inhibition effect suggested that the primary inhibitory action of ML-9 on SOCE is mediated through STIM. Unlike ML-9, expression of STIM did not influence the concentration of required to block SOCE. Furthermore, in HEK9 cells co-expressing STIM and Orai, ML-9 is much less potent at preventing store-depletion dependent STIM puncta formation, a finding similar to what we show here for (Supplemental Figure, panel A). However, in these co-expressing cells ML-9 also is much less effective at blocking SOCE and store-operated currents (panel B), again consistent with the notion that ML-9 inhibition of SOCE and I CRAC reflects inhibition of STIM puncta formation. Again, this is unlike which blocks I CRAC in WT and Orai plus STIM- expressing cells with essentially equal potency despite differences in its inhibition of STIM 7

8 puncta formation. Although not yet fully understood, we find it intriguing that expression of Orai can significantly reduce the reversal of eyfp-stim puncta by and ML-9, and we are currently investigating why this is the case. While Orai and Orai (albeit less than Orai) were both inhibited by high concentrations of - APB, Orai was activated by the compound. Initially the currents that developed in these cells resembled I CRAC ; however, large outward currents that were permeable to Cs + at potentials greater than +5 mv subsequently developed. These currents reached a peak after several minutes and were sustained in the continued presence of - APB. The outwardly rectifying -activated currents were only present in Orai expressing cells, and do not appear to result from the activation of a secondary Ca + -dependent conductance. To our knowledge, this is the first report of a pharmacological agent that can alter the selectivity of an I crac -like store-operated channel. Site-directed mutagenesis directed towards an acidic residue on TM (E9Q) of Orai alters the selectivity of Orai-mediated I CRAC and the resultant current-voltage relationship recorded from HEK9 expressing this mutated Orai looks strikingly similar to the current-voltage relationship seen in Orai expressing cells after - APB () (but see (5)). At sub-inhibitory concentrations, strongly potentiated I CRAC in HEK9 cells co-expressing Orai and STIM by up to 5%. Similar results have been described for endogenous I CRAC in which low concentrations of have increased current amplitude by up to five-fold (6). Previous results (6;6) suggest that potentiates I CRAC only after stores are depleted, and does not directly activate the currents independently of STIM and store depletion. Nonetheless, results presented here using exogenously expressed Orai and Orai suggest that can elicit direct activation of the CRAC or CRAC-like channels. The reason for the distinct findings is not clear, but may reflect differences in the stoichiometry of Orai and STIM proteins to one another, or to other proteins, in cells transfected with cdnas driven by strong viral promoters. In summary, the results presented here indicate that has complex inhibitory and activating mechanisms on Orai channels. The inhibitory action appears to involve at least two effects, one involving alteration of movement of STIM into puncta, and one at a downstream site, involving either a direct effect on the channel, or on the ability of STIM to activate the channels. The inhibitory actions of differed markedly both quantitatively and qualitatively among the three Orai species, consistent with, but not proving a direct effect on the channels themselves. Earlier findings from a number of laboratories suggested an extracellular site of action of on I CRAC (;6;7) which is further consistent with a direct action on the channel. Both Orai and could also be activated by, and Orai channels additionally showed altered ion selectivity when activated by. These different effects of - APB on Orai, Orai and Orai could possibly be used as diagnostic criteria for detecting the expression patterns of Orai isoforms in various tissues. However, as pointed out in the Introduction, the lack of known models for studying native Orai or channels has necessitated the use of exogenously expressed proteins, usually supported by increased expression levels of STIM. Thus, it cannot be assured that these channels will behave identically at more physiological levels of expression. It is encouraging that the actions of on exogenously expressed Orai match well those previously described for effects on I CRAC in hematopoetic cells, where Orai is apparently expressed in its native environment. Future work should focus on identifying physiological sites of expression of all three channel proteins in order to further evaluate the similarities and differences in their biophysical and pharmacological properties. 8

9 Acknowledgements We are grateful to Drs David Armstrong and Steve Shears who read the manuscript and provided helpful comments. This research was supported in part by the Intramural Program, National Institutes of Health, NIEHS. While this paper was under revision, two reports appeared with findings similar to some of those reported here (7;8). Footnote to first page The abbreviations are: SOCE, store-operated calcium entry; STIM, stromal interacting molecule; I CRAC, calcium-release-activated calcium current;, -aminoethyldiphenyl borate; ER, endoplasmic reticulum; SERCA, sarco-endoplasmic reticulum calcium ATPase; IP, inositol trisphosphate; DMEM, Dulbecco s modified Eagle s medium; eyfp, enhanced yellow fluorescent protein; CFP, cyan fluorescent protein; TIRFM, total internally reflected fluorescence microscopy; HEK, human embryonic kidney Figure Legends Figure : dose-dependently activates and inhibits SOCE in WT HEK9 cells or in cells expressing eyfp-stim. (A) SOCE was measured in HEK9 cells using the SERCA pump inhibitor, thapsigargin () ( μm). was applied under nominally Ca + -free conditions for 5 minutes to deplete ER Ca + stores, followed by the addition of mm Ca + (5 minutes) to assess SOCE. After 5 min, different concentrations of (-5 μm) were applied, while maintaining the extracellular Ca + concentration constant. (B) Mean SOCE responses above baseline from experiments carried out as in (A) for μm (DMSO) (n=), μm (n=), μm (n=), μm (n=), and 5 μm (n=). (C) Same as (A), except in HEK9 cells transiently expressing eyfp-stim. (D) Same as in (B), except in the eyfp-stim cells. (DMSO: n=; μm: n=; μm: n=; μm: n=; 5 μm: n=). Data are represented as mean ± SEM, and each n represents the mean of a single coverslip containing at least 5 cells. Figure : reverses near-plasma membrane punctate STIM localization. (A) Live eyfp- STIM expressing HEK9 cells were imaged by confocal microscopy with replete Ca + stores (left panel), following store depletion with μm (middle panel), and 5 minutes following treatment with 5 μm (right panel). (B) TIRFM measurements were carried out on cells expressing eyfp- STIM. Shown is an average ( cells) fluorescence intensity plot from an individual experiment. As indicated, cells were treated with μm followed 5 minutes later by application of 5 μm in the continuous presence of mm extracellular Ca +. (C) Bar graph showing the percent change in TIRFM fluorescence intensities from three (6 cells total) independent experiments shown in (B). To calculate the percent change in fluorescence intensities, the average baseline-subtracted TIRFM fluorescence intensity 5 minutes after addition was divided by that just prior to application of. (D) TIRFM measurements were also carried out in experiments in which cells expressing eyfp-stim were pretreated with 5 μm for 5 minutes prior to store depletion with. (E) The fold increase in TIRFM fluorescence intensity in response to store depletion with was calculated for cells pretreated for 5 minutes with 5 μm and for untreated control cells. Figure : activates and inhibits I CRAC in HEK9 cells co-expressing STIM and Orai. (A) Current recording from a HEK9 cell co-expressing eyfp-stim with CFP-Orai, in which stores were depleted with μm IP and mm BAPTA in the pipette. The current was recorded from voltage ramps

10 (- mv to + mv) taken every seconds from a mv holding potential. At μm, consistently increased I CRAC in a sustained fashion, while 5 μm strongly inhibited the currents. Inset graph shows transient increase followed by near complete block of I CRAC by the focal application of 5 μm. (B) Bar graph of data shown in (A) represented as the current developed under different concentrations (,, 5 μm) minus the leak current, divided by the Ca + current (same as μm ) minus the leak current (ΔI X / ΔI Ca+ ). Data are represented as means ± SEM ( μm: n=6; 5 μm: n=). (C) Current-voltage (I-V) relationship from recording shown in (A). Figure : Orai expression reduces inhibition of STIM rearrangement. (A) TIRFM measurements similar to Figure were carried out on cells expressing eyfp-stim with CFP-Orai. - APB was added after Ca + store depletion with. (B) Single cell Ca + imaging experiment using the same protocol seen in panel (A). was added under the presence of mm extracellular Ca +, followed by the addition of 5 μm to assess its effects on SOCE. (C) Bar graph showing the percent changes in TIRFM fluorescence intensities from three independent experiments (6 cells total) shown in (A). (D) Bar graph showing a near complete block of SOCE by in experiments carried out similar to panel (B) (n = coverslips). (E) TIRFM experiment in HEK9 cells co-expressing eyfp-stim and CFP-Orai in which 5 μm is added 5 min prior to store depletion with. (F) Similar experiment as Panel (E); however, using Ca + imaging technique instead of TIRFM. (G) Bar graph depicting the increase in TIRFM fluorescence intensity in response to store depletion with for cells pretreated with 5 μm ( n = 6 cells from experiments) and for untreated control cells ( n = 8 cells from experiments). (H) Bar graph showing the lack of SOCE development in cells pretreated with ( n = coverslips), compared to untreated controls (n = coverslips). Data are represented as mean ± SEM, and in single cells Ca + imaging experiments each n represents the mean of a single coverslip containing at least 5 cells. Figure 5: Responses to differ in cells co-expressing STIM with Orai or Orai. (A) I CRAC recordings from HEK9 cells co-expressing eyfp-stim with Orai. Stores were actively depleted with IP and BAPTA in the pipette. ( μm and 5 μm) was focally applied through extracellular bath exchange as indicated (black trace). The control recording (gray trace) shows no slow inactivation development in I CRAC recorded from Orai + Stim expressing cells, as previously described (8;). (B) Current-voltage (I-V) relationship from recording shown in (A). In this recording, the initial sweep, used for leak subtraction, showed some residual outward current likely due to incomplete block of outward K + current by Cs +. (C) Similar experiment as (A), except in a HEK9 cell co-expressing eyfp-stim with CFP-Orai. (D) I-V relationship from experiment shown in (C), showing the biphasic currents that develop under the presence of 5 μm. The current was initially inwardly rectifying (i), followed by the development of an outward current (ii) at potentials greater than +5 mv. Figure 6: Activation of Orai and Orai by does not require STIM or store depletion. (A- D) Ca + imaging experiments showing the effects of 5 μm and then μm on cytoplasmic Ca + levels in control cells (black) or STIM sirna treated cells (gray) transfected with Orai (A), Orai (B) or Orai (C). Results similar to those seen in Orai expressing cells (Panel D) were also seen in YFP alone expressing control and STIM sirna treated cells (Data not shown). (D) Peak changes in cytoplasmic Ca + detected in Control (black) or STIM sirna treated (gray) HEK9 cells expressing either Orai, Orai or Orai and treated with 5 μm. In all conditions, the data are the mean ± S.E.M. of coverslips and each coverslip is the mean of at least 5 cells. (E) Mean of four independent Ca + imaging experiments (> 5 cells per experiment) looking at the effects of several concentrations of (-5 μm) on cytoplasmic Ca + concentration in cells expressing CFP-Orai. (F) Concentrationresponse curve showing the direct activation of Orai by.

11 Figure 7: Influence of endogenous STIM and store depletion on the activation of Orai by. Mean single cell Ca + imaging results in which stores were depleted with μm under the presence of nominally free extracellular Ca + conditions for 5 min, followed by the addition of mm Ca + to assess SOCE in WT HEK9 cells (A), or cells expressing CFP-Orai (B). Five minutes after addition of Ca +, 5 μm was applied. (C) Change in fluorescence ratio seen after 5 min in the presence of mm Ca + and 5 μm in cells expressing Orai alone without ER Ca + store depletion (replete; from Figure 6; n = 5 coverslips), or after store depletion with μm (deplete; Panel B n = coverslips). Each n represents the mean of a single coverslip containing at least 5 cells. Figure 8: activates Orai and Orai independently of STIM and internal Ca + store depletion. (A) Whole-cell currents recorded from HEK9 cells expressing Orai, Orai or Orai before, during, and after focal application of 5 μm. Cytoplasmic Ca + concentrations were buffered using BAPTA and millimolar concentrations of Ca + (calculated using Maxchelator software) in order to avoid passive store depletion. mm extracellular Ca + was present throughout these experiments. (B) Current-voltage relationships from the recordings shown in (A). (C) Bar graph showing the change in current densities (peak current minus leak current) recorded from voltage ramps in cells expressing Orai, Orai and Orai. Data were collected in studies identical to those shown in panel (A) (Orai: n = ; Orai: n = ; Orai: n = 6). (D) Comparison of the peak currents recorded in HEK9 cells expressing Orai alone (stores replete; n = 6; panel A), or Orai expressed with STIM and stores actively depleted with IP and BAPTA in the pipette (Figure 6; n = 7). Figure 9: activated Orai currents have increased Cs + permeability compared to Orai- mediated CRAC currents activated by store depletion. (A) Whole-cell patch-clamp recording taken at - mv and + mv from a HEK9 cell expressing Orai. The internal pipette solution contained Ca + clamped to nm Ca +. Voltage ramps (- to + mv) were applied every two seconds from a holding potential of mv. External solutions were applied as indicated from the bars above the recording. NCF stands for nominally Ca + free external solution, and DVF stands for divalent free external solution. (B) Current-voltage relationships taken from the recoding shown in panel A, when either mm (black trace) or mm (gray trace) extracellular Ca + was present in the bathing solution. (C) Current-voltage relationships also taken from panel A, showing that mm Mg + present in the nominally Ca + free (NCF; gray trace) external solution is sufficient enough to block nearly all Na + current through the activated Orai channels. Black arrows indicate the time points of where the I-V traces were taken. (D) Representative recording taken under similar conditions to those in panel A; however, a Cs-DVF solution was applied after activation of Orai with. (E) Current-voltage relationships of the Ca + and Cs + currents seen in these HEK9 cells expressing Orai and activated directly with. (F) Representative whole-cell recording taken from a HEK9 cell co-expressing Orai and STIM, in which ER Ca + stores were actively depleted with μm IP and mm BAPTA in the patch pipette. The extracellular bathing solution was switched from a mm Ca + containing solution to one lacking all divalent cations and containing either the monovalent cation Na + (black) or Cs + (gray) as the charge carrier. Unlike in activated Orai recordings, Cs + does not permeate well through STIM and store-depletion activated Orai channels. (G) Current-voltage relationships from the peak Cs + and Na + currents seen in the Orai + STIM expressing HEK9 cells after active ER Ca + store depletion. All traces are representatives of at least three similar recordings. Supplemental Figure : Effects of Orai and Orai overexpression on inhibition of STIM rearrangement. TIRFM measurements were carried out on cells co-expressing eyfp-stim with either CFP-Orai (A), or CFP-Orai (C). Shown are average fluorescence intensity plots from individual experiments. As indicated, cells were treated with thapsigargin (Tg; μm) followed 5 minutes later by application of 5 μm in the continuous presence of mm extracellular Ca +. (E) The average baseline-subtracted TIRFM fluorescence intensity 5 minutes following addition was divided by

12 that just prior to addition for experiments carried out as described in panels A and C. Also shown are data obtained from cells expressing eyfp-stim alone (see Figure B) and from cells expressing eyfp-stim and CFP-Orai (from Figure ). STIM alone: n = 6 cells, STIM+Orai: n = 8 cells, STIM+Orai: n = 7 cells, STIM+Orai: n = 5 cells; coverslips each. TIRFM measurements were also carried out in experiments in which cells expressing eyfp-stim with either CFP-Orai (B), or CFP-Orai (D) were pretreated with 5 μm for 5 minutes prior to store depletion with thapsigargin. (F) The fold increase in TIRFM fluorescence intensity in response to store depletion with thapsigargin was calculated for cells pretreated for 5 minutes with 5 μm (gray bars; STIM alone: n = cells, STIM+Orai: n = 6 cells, STIM+Orai: n = 7 cells, STIM+Orai: n = 5 cells) and for untreated control cells (black bars; STIM alone: n = 6 cells, STIM+Orai: n = 8 cells, STIM+Orai: n = 7 cells, STIM+Orai: n = 5 cells). Supplemental Figure : Inhibition of store depletion-dependent STIM movements and I CRAC by ML-9 is significantly reduced when STIM is co-expressed with Orai. (A) TIRFM recordings in which cells expressed either STIM alone (dashed line), or co-expressed STIM with Orai (solid lines), with (black) or without (gray) the pretreatment of μm ML-9. ( μm) was used to deplete stores and rearrange eyfp-stim as described earlier. (B) I CRAC recordings taken from HEK9 cells coexpressing STIM and Orai, in which cells were either bathed in μm Ca + alone (black trace; Control), or under the presence of μm ML-9 (gray trace; +ML-9). Stores were actively depleted with IP and BAPTA in the pipette and currents were elicited by voltage ramps (- mv to + mv) from a mv holding potential every seconds. (C) Current-voltage relationship taken from the experiments shown in (B) (- mv to + mv).. Putney, J. W. (986) Cell Calcium 7, - References. Smyth, J. T., DeHaven, W. I., Jones, B. F., Mercer, J. C., Trebak, M., Vazquez, G., and Putney, J. W. (6) Biochim. Biophys. Acta 76, 7-6. Hoth, M. and Penner, R. (99) Nature 55, Parekh, A. B. and Putney, J. W. (5) Physiol Rev. 85, Roos, J., DiGregorio, P. J., Yeromin, A. V., Ohlsen, K., Lioudyno, M., Zhang, S., Safrina, O., Kozak, J. A., Wagner, S. L., Cahalan, M. D., Velicelebi, G., and Stauderman, K. A. (5) J. Cell Biol. 69, Liou, J., Kim, M. L., Heo, W. D., Jones, J. T., Myers, J. W., Ferrell, J. E., Jr., and Meyer, T. (5) Curr. Biol. 5, 5-7. Feske, S., Gwack, Y., Prakriya, M., Srikanth, S., Puppel, S. H., Tanasa, B., Hogan, P. G., Lewis, R. S., Daly, M., and Rao, A. (6) Nature, Vig, M., Peinelt, C., Beck, A., Koomoa, D. L., Rabah, D., Koblan-Huberson, M., Kraft, S., Turner, H., Fleig, A., Penner, R., and Kinet, J. P. (6) Science, - 9. Zhang, S. L., Yeromin, A. V., Zhang, X. H., Yu, Y., Safrina, O., Penna, A., Roos, J., Stauderman, K. A., and Cahalan, M. D. (6) Proc. Natl. Acad. Sci U. S. A,

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15 F/F8 F/F8 A. WT HEK C. +STIM.5..5 Ca + 5 Ca + 5 B. F/F8 F/F8 D [] ( M) FIGURE 5.. [] ( M) 5

16 A +Ca+ B F/F C F/F (%) D F/F E F/F 5 FIGURE