Dependence of STIM1/Orai1-mediated Calcium Entry on Plasma Membrane Phosphoinositides*

Recent studies identified two main components of store-operated calcium entry (SOCE): the endoplasmic reticulum-localized Ca2+ sensor protein, STIM1, and the plasma membrane (PM)-localized Ca2+ channel, Orai1/CRACM1. In the present study, we investigated the phosphoinositide dependence of Orai1 channel activation in the PM and of STIM1 movements from the tubular to PM-adjacent endoplasmic reticulum regions during Ca2+ store depletion. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) levels were changed either with agonist stimulation or by chemically induced recruitment of a phosphoinositide 5-phosphatase domain to the PM, whereas PtdIns4P levels were decreased by inhibition or down-regulation of phosphatidylinositol 4-kinases (PI4Ks). Agonist-induced phospholipase C activation and PI4K inhibition, but not isolated PtdIns(4,5)P2 depletion, substantially reduced endogenous or STIM1/Orai1-mediated SOCE without preventing STIM1 movements toward the PM upon Ca2+ store depletion. Patch clamp analysis of cells overexpressing STIM1 and Orai1 proteins confirmed that phospholipase C activation or PI4K inhibition greatly reduced ICRAC currents. These results suggest an inositide requirement of Orai1 activation but not STIM1 movements and indicate that PtdIns4P rather than PtdIns(4,5)P2 is a likely determinant of Orai1 channel activity.

Recent studies identified two main components of store-operated calcium entry (SOCE): the endoplasmic reticulum-localized Ca 2؉ sensor protein, STIM1, and the plasma membrane (PM)-localized Ca 2؉ channel, Orai1/CRACM1. In the present study, we investigated the phosphoinositide dependence of Orai1 channel activation in the PM and of STIM1 movements from the tubular to PM-adjacent endoplasmic reticulum regions during Ca 2؉ store depletion. Phosphatidylinositol 4,5bisphosphate (PtdIns(4,5)P 2 ) levels were changed either with agonist stimulation or by chemically induced recruitment of a phosphoinositide 5-phosphatase domain to the PM, whereas PtdIns4P levels were decreased by inhibition or down-regulation of phosphatidylinositol 4-kinases (PI4Ks). Agonist-induced phospholipase C activation and PI4K inhibition, but not isolated PtdIns(4,5)P 2 depletion, substantially reduced endogenous or STIM1/Orai1-mediated SOCE without preventing STIM1 movements toward the PM upon Ca 2؉ store depletion. Patch clamp analysis of cells overexpressing STIM1 and Orai1 proteins confirmed that phospholipase C activation or PI4K inhibition greatly reduced I CRAC currents. These results suggest an inositide requirement of Orai1 activation but not STIM1 movements and indicate that PtdIns4P rather than PtdIns(4,5)P 2 is a likely determinant of Orai1 channel activity.
Store-operated Ca 2ϩ entry (SOCE) 3 is a ubiquitous Ca 2ϩ entry pathway that is regulated by the Ca 2ϩ content of the endoplasmic reticulum (ER) (1). SOCE has been identified as the major route of Ca 2ϩ entry during activation of cells of the immune system such as T cells and mast cells (2,3), and it is also present and functionally important in other cells such as platelets (4) and developing myotubes (5). The long awaited mechanism of how the ER luminal Ca 2ϩ content is sensed and the information transferred to the plasma membrane (PM) has been clarified recently after identification of the ER Ca 2ϩ sensor proteins STIM1 and -2 (6, 7) and the PM Ca 2ϩ channels Orai1, -2, and -3 (8 -10). According to current views, a decrease in the ER Ca 2ϩ concentration is sensed by the luminal EF-hand of the single-transmembrane STIM proteins causing their multimerization. This oligomerization occurs in the tubular ER, where it promotes the interaction of the cytoplasmic C termini of STIM with PM components and association with the PM-localized Orai channels, causing both their clustering and activation in the PM (reviewed recently in Refs. [11][12][13]. Analysis of the interacting domains within the STIM1 and Orai1 proteins suggests that the cytoplasmic domain of STIM1 is necessary and sufficient to activate Orai1 (14), whereas the latter requires its C-terminal membrane-adjacent cytoplasmic tail to be fully activated by the STIM proteins (15,16). Both STIM1 and -2 contain a polybasic segment in their C termini, and such regions are often responsible for the PM localization of proteins (mostly of the small GTP-binding protein class) via interaction with anionic phospholipids such as phosphatidylserine or PtdIns(4,5)P 2 (17). However, the role of this domain in STIM1 function(s) remains controversial. Deletion of the polybasic tail is reported to prevent PM association but not clustering of STIM1 upon ER store depletion (18). In other studies, truncated STIM1 lacking the polybasic domain shows only slightly altered activation (15) or inactivation (19) kinetics without major defects in supporting Orai1-mediated Ca 2ϩ influx. The most recent studies identify the minimal Orai1 activation domain in STIM1 (20,21) and find that the polybasic domain is not essential for this function but makes electrostatic interaction with classical transient receptor potential channels (22).
PM phosphoinositides have been widely reported as regulators of the activity of several ion channels and transporters (23). However, only a few studies have addressed the inositide requirement of SOCE and none specifically that of the Orai1mediated Ca 2ϩ entry process. Sensitivity of SOCE to phosphatidylinositol 3-kinases (PI3K) inhibitors has been reported, but this required concentrations that suggested inhibition of targets other than PI3Ks, possibly myosin light chain kinase or the type-III PI4Ks (4, 24 -26

EXPERIMENTAL PROCEDURES
Materials-Rapamycin and thapsigargin were purchased from Calbiochem. Angiotensin II (human octapeptide) was from Peninsula Laboratories (Bachem, Torrance, CA), and ATP was obtained from Sigma. All other chemicals were of the highest analytical grade.
DNA Constructs-The YFP-and mRFP-STIM1 plasmids as well as the Orai1 constructs used in this study have been described previously (27). The plasmids designed for the rapamycin-induced PM recruitment of the type IV 5-phosphatase domain as well as those for PLC␦ 1 PH-GFP and OSH2-2xPH-GFP have also been described elsewhere (28,29). For siRNAmediated knockdown of the various PI4K isoforms, the duplexes and treatment protocols have been described previously (29).
Cytoplasmic Ca 2ϩ [Ca 2ϩ ] i and TIRF Measurements in Single Cells-COS-7 cells were cultured on glass coverslips (3 ϫ 10 5 cells/35-mm dish) and transfected with the indicated constructs (0.5 g of DNA/dish) using Lipofectamine 2000 for 24 h as described previously (27). TIRF analysis was performed at room temperature in an Olympus IX81 microscope-based through-the-lens dual-launch TIRF system equipped with a Hammamatsu EM-CCD camera and a PlanApo 60ϫ/1.45 objective. Excitation with 488 or 568 nm lasers were used for the YFP or Fluo4 and mRFP, respectively, and scans were performed at every 10 s. For data acquisition OpenLab Software (Improvision) was used, and the pictures were exported as TIFF files for processing with the MetaMorph software (Molecular Devices). Quantification of the membrane intensities was determined after defining the regions of individual cells and thresholding. Because of the large variations in the intensities of individual cells due to different footprint size and translocation responses, these responses were normalized and their maximal Tg-induced translocation taken as 100%. These recordings were then averaged and their S.E. calculated and plotted against time.
For calcium experiments cells were loaded with Fura2/AM (3 M) for 45 min, room temperature). Calcium measurements with Fura2 were performed in modified Krebs-Ringer solution (see Ref. 27 for composition) supplemented with 200 M sulfinpyrazone. Calcium studies were also performed in individual cells attached to coverslips at room temperature using an Olympus IX70 inverted microscope equipped with a Lamda-DG4 illuminator and a MicroMAX-1024BFT digital camera and the appropriate filter sets. MetaFluor (Molecular Devices) software was used for data acquisition. When cells were studied in suspension, they were removed from the culture plates with mild trypsinization and loaded with 3-5 M Fura2/AM at room temperature as described previously (30). Loaded cells were kept in HEPES-buffered M199, Hanks' salt solution containing 0.1% bovine serum albumin and 200 M sulfinpyrazone, and an aliquot of the cells was centrifuged immediately before [Ca 2ϩ ] i measurement in the modified Krebs-Ringer solution contain-ing sulfinpyrazone but not bovine serum albumin. [Ca 2ϩ ] i measurements in suspension were performed at 34°C in a PTI DeltaScan fluorescence spectrophotometer (Photon Technology International).
Electrophysiological Recordings-All voltage clamp recordings were performed at room temperature using an Axopatch 200 B patch clamp amplifier (Axon Instruments, Foster City, CA) and were low-pass filtered at 2 kHz. Ramp generation and data acquisition were done with a PC equipped with a Digidata 1322A A/D interface in conjunction with Clampex 10 (Axon Instruments). The standard HEPES-buffered saline solution contained (mM): 140 NaCl, 2.5 KCl, 1 MgCl 2 , 2 CaCl 2 , 15 glucose, and 10 HEPES (pH to 7.4 with NaOH). Fire-polished pipettes fabricated from borosilicate glass capillaries (World Precision Instruments, Sarasota, FL) with 3-5-megohm resistance were filled with the following (mM): 100 cesium methanesulfonate, 20 BAPTA (dissolved in 0.3 M CsOH), 10 HEPES, 10 NaCl, and 6 MgATP (pH to 7.2 with CsOH). In all experiments, the pipette also contained 25 M inositol 1,4,5-trisphosphate (InsP 3 , hexasodium salt; Sigma). Voltage ramps (Ϫ100 to ϩ100 mV) of 250 ms were recorded every 2 s immediately after gaining access to the cell from a holding potential of 0 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 and 10 megohms. Wortmannin (Wm) and angiotensin II (AngII) were applied in some experiments using a gravity-driven microperfusion system, RSC-200 (Bio-Logic SAS, Claix, France).

Rapid Dephosphorylation of PtdIns(4,5)P 2 to PtdIns4P Does Not Inhibit SOCE and Has Only Minor Effects on STIM1
Translocation from the Tubular to PM-adjacent ER-First we wanted to determine whether isolated changes in PtdIns(4,5)P 2 could alter STIM1 movements or the activation of Orai1-mediated Ca 2ϩ influx in intact cells. To this end, we used the recently described chemically (rapamycin) induced recruitment of the 5-phosphatase domain of the type IV phosphoinositide 5-phosphatase enzyme (5-PTase domain) to rapidly reduce PtdIns(4,5)P 2 levels in the PM (28). This method allows changing the level of PtdIns(4,5)P 2 without setting off the signaling cascade downstream of PLC activation. For these studies we used COS-7 cells, as they show very robust elimination of PtdIns(4,5)P 2 upon rapamycin treatment (28). Cells were transfected with the PM-targeted FRB construct and the mRFP-FKBP12-fused 5-phosphatase domain, which is cytosolic under basal conditions but becomes recruited to the PM after the addition of rapamycin causing rapid depletion of PtdIns(4,5)P 2 from the membrane. This process can be followed in TIRF experiments, where both the recruitment of the phosphatase and the release of the PtdIns(4,5)P 2 reporter PLC␦ 1 PH-GFP from the membrane can be monitored simultaneously (see Ref. 31).
In previous studies we showed that PtdIns(4,5)P 2 elimination from the PM by this method does not prevent Tg-induced STIM1 translocation to PM-adjacent regions (27). However, analysis of STIM1 translocation from many cells in TIRF exper-iments required normalization to the maximum translocation. Therefore, those experiments could not demonstrate whether STIM1 translocation was altered in the PtdIns(4,5)P 2 -depleted state. To overcome this problem, we first induced YFP-STIM1 translocation toward the PM by addition of ATP and Tg, and only when translocation has fully developed did we induce PtdIns(4,5)P 2 depletion by recruitment of the 5-phosphatase domain to the PM with rapamycin. Fig. 1A shows that under these conditions, we could see a some reduction in the YFP-STIM1 intensity at the TIRF plane after 5-phosphatase domain recruitment but only a slight change when the construct con-taining only the mRFP-FKBP12 protein without the 5-phosphatase was recruited to the membrane (Fig. 1B). In parallel experiments, the translocation response of the PLC␦1PH-GFP was also followed using the same sequence of stimulation. This showed that ATP/Tg only slightly reduced PtdIns(4,5)P 2 levels, which showed a large decrease only after rapamycin addition (see on Fig. 3). These experiments indicated that PM PtdIns(4,5)P 2 may contribute to the stabilization of STIM1-PM interaction.
We also determined the effect of PtdIns(4,5)P 2 removal on the cytosolic Ca 2ϩ signal after store depletion. For this, [Ca 2ϩ ] i was monitored with Fura2 in COS-7 cells expressing the 5-phosphatase recruitment system either alone or with mRFP-STIM1 and untagged Orai1 to boost SOCE. The endogenous P2Y purinergic receptors of COS-7 cells were stimulated with ATP together with Tg to rapidly release and deplete the ER Ca 2ϩ stores and activate SOCE. This was followed by rapamycin addition to recruit the 5-phosphatase and deplete PtdIns(4,5)P 2 . As shown in Fig. 2, the addition of rapamycin failed to affect either the endogenous SOCE or the one enhanced by overexpression of STIM1/Orai1. These results suggested that change in the PM PtdIns(4,5)P 2 was not a major factor in the regulation of SOCE in these cells, despite its minor effect on STIM1 translocation.
PI4K Inhibition Affects SOCE but Not STIM1 Movements-Previous data had shown that PI3K inhibitors inhibit SOCE at concentrations that could also inhibit PI4Ks (26). To investigate whether PtdIns4P might be a regulatory factor of SOCE activity, the PI 3K inhibitor LY294002 was added to the cells at concentrations that inhibit type III PI4Ks (32). Our experience with the use of Wm in microscopy studies suggests that this inhibitor is not reliable because illumination with 488 nm (or shorter wavelengths) on the microscope stage rapidly inactivates this compound 4 (also see Ref. 33). For this reason we used LY294002 in these experiments, first studying its effects on the movements of STIM1 in TIRF experiments. LY294002 was added after STIM1 translocation had already been induced by ATP/Tg treatment and PtdIns(4,5)P 2 had been eliminated by recruitment of the 5-phosphatase. As shown in Fig. 1, the addition of LY294002 (30 -300 M) to such pretreated COS-7 cells failed to affect the STIM1 signal in the TIRF plane, suggesting that PtdIns4P is not a factor in keeping STIM1 at the PM. (A delayed increase in the TIRF signal was observed in both channels after LY294002 addition in this set of studies, and we attributed it to changes in the attachment of the cells or a slight change in focus.) In contrast, LY294002 addition rapidly inhibited both endogenous SOCE and that enhanced by Orai1/ STIM1 expression in a dose-dependent manner (Fig. 2). Importantly, this effect was also observed without prior elimination of PtdIns(4,5)P 2 when LY294002 was applied to naive, untransfected COS-7 cells (data not shown).
To demonstrate the effects of LY294002 treatment on PtdIns4P, two approaches were used. First, [ 32 P]phosphate-labeled COS-7 cells were treated with increasing doses of LY294002 and the labeled phospholipids analyzed by TLC anal-4 P. Varnai and T. Balla, unpublished observation.  (27) together with a PM-targeted FRB construct and either the mRFP-FKBP12-5-phosphatase or mRFP-FKBP12 without the phosphatase domain (28). After 24 h, cells were examined in a TIRF fluorescence microscope, and the STIM1 (black) and 5-phosphatase (gray) translocation was monitored in the TIRF plane. Stimulation with ATP/Tg (50 M/200 nM) leads to clustering and translocation of STIM1 to the membrane-adjacent region. Recruitment of 5-PTase (A) but not the control, mRFP-FKBP12 protein (FKBP-only) (B), by the addition of rapamycin (Rapa, 100 nM) (gray traces) causes elimination of PtdIns(4,5)P 2 and also decreases by about 30% the amount of STIM1 at the TIRF plane. The addition of 100 M LY294002 had no further effect on STIM1 movements. Data were normalized such that the minimum and maximum average intensities in the TIRF plane belonging to distinct cells were treated as 0 and 100%, respectively. Means Ϯ S.E. values derived from 127 and 58 cells are shown as recorded in seven and four separate experiments for A and B, respectively.

Phosphoinositides in Store-operated Calcium Entry
ysis. As shown in Fig. 3A, a 10-min LY294002 treatment reduced the level of labeled PtdIns4P in a dose-dependent manner without a similar decrease in labeled [ 32 P]PtdIns(4,5)P 2 , essentially mimicking the effects of 10 M Wm as described previously in bovine adrenal and HEK293 cells (29,30). A slight increase in PtdIns(4,5)P 2 already at a lower LY294002 concentration was also observed. This was attributed to the inhibition of PI3Ks and thence sparing PtdIns(4,5)P 2 usage via that pathway. The potency of LY294002 to inhibit PtdIns4P synthesis was almost identical to that for inhibition of Ca 2ϩ influx (Fig.  3B). Because 32 P could label PtdIns4P pools other than those found in the PM, we also wanted to show that LY294002 acted on the PM pool of PtdIns4P. For this we used the PtdIns4P reporter OSH2-2xPH-GFP (34) that has proven to be a reasonable probe for following changes in PM PtdIns4P levels (29,35). In these TIRF experiments, we used the same treatment regime as in previous experiments in order to assess the lipid changes evoked by the ATP/Tg treatment. These studies showed negligible changes in OSH2-2xPH-GFP localization by ATP/Tg, but recruitment of the 5-phosphatase to the PM caused a slight decrease in the membrane-bound fraction of the OSH2-2xPH-GFP (Fig. 3C). This can be attributed to a weak PtdIns(4,5)P 2 binding of this reporter, as it was shown to also bind PtdIns(4,5)P 2 in vitro (36). Note the rapid decrease in the localization of the PLC␦1PH-GFP probe reporting on PtdIns(4,5)P 2 changes. Curiously, we were unable to detect an increased PtdIns4P after 5-phosphatase recruitment with any of the PtdIns4P binding reporter constructs (FAPP1-PH, OSH1-PH, OSBP-PH, OSH2-PH) for reasons that are yet to be understood. 5 Nevertheless, a very significant fraction of OSH2-2xPH-GFP remained associated with the PM after Ptd-Ins(4,5)P 2 elimination, which was then rapidly released after the addition of 100 M LY294002 (Fig. 3C). These experiments showed that LY294002 eliminates most of the PtdIns4P from the PM within 5 min of incubation. Thus, although PtdIns(4,5)P 2 level in the PM had only a small impact on STIM1 oligomerization and PM interaction in store-depleted cells, LY294002 had a major impact on SOCE that correlated with PtdIns4P rather than PtdIns(4,5)P 2 depletion.
PI4K Inhibition and PLC Activation Inhibit I CRAC -To study the effects of PtdIns4P manipulations directly on I CRAC , electrophysiological measurements were performed in HEK293 cells stably expressing the Ca 2ϩ -mobilizing AT1a angiotensin receptors (HEK-AT1). Cells were transiently transfected with YFP-STIM1 alone or in combination with Orai1. Experiments were performed only with cells exhibiting comparable YFP (STIM1) fluorescence. The pipette solution contained 25 M InsP 3 and 20 mM BAPTA, and 2 mM extracellular Ca 2ϩ was present in the bath solution. These conditions allowed depletion of the ER Ca 2ϩ store to activate Ca 2ϩ influx. Voltage ramps from Ϫ100 to ϩ100 mV of 250 ms were applied every 2 s immediately after gaining access to the cell from a holding potential of 0 mV. Fig. 4A illustrates a typical pattern of response to application of the voltage ramp in cells transfected with both Orai1 and STIM1. The time course of whole cell current activated by depletion of the ER calcium store, estimated at Ϫ80 mV potential, is shown in Fig. 4B. In cells expressing both proteins, the current developed fully within 50 to 100 s, with a peak amplitude of 30.7 Ϯ 3.1 pA/pF; (n ϭ 31). Once the current was developed, in a fraction of the cells it stayed unchanged for at least 300 s (Fig. 4B, black trace), whereas in the majority of the cells a slow and linear decay of current was consistently observed (blue trace). The current-voltage relationship indicated strong inward rectification and reversal potential at about ϩ50 mV, both characteristic of I CRAC (Fig. 4C, blue trace). Neither untransfected cells (data not shown) nor STIM1 aloneexpressing cells (Fig. 4C, gray trace)   We also examined the effects of PI4K inhibition on the amplitude and patterns of I CRAC . For this, Wm was used (as no light exposure was present during the patch clamp recording) at concentrations (10 M) that inhibit both PI3Ks and PI4Ks (30). To avoid the effects of dialysis on cells during the whole cell recording, intact cells were treated with Wm for 15 min prior to establishing the current recording, as described above. Fig. 4D illustrates a typical pattern of current response during a 300-s recording. As in untreated cells, the current developed with similar kinetics, but the amplitude of the current was substantially lower (12.3 Ϯ 2.7 pA/pF; n ϭ 11). To limit the impact of expression efficiency in different experiments on the amplitude of current, the mean values of current responses recorded in controls and Wm-treated cells were compared from traces generated in the same experiments (Fig. 4E), and a statistical comparison was done at two time points (at 100 and 200 s of recording; Fig. 4F). These results indicated that the inhibition of type III PI4Ks strongly affected the amplitude of the I CRAC current.
Next we studied whether PLC activation has any effect on the I CRAC current. For this, AngII was applied using a gravity-driven microperifusion system after the development of the current, and the time of AngII application was varied between experiments. In the majority of cells (n ϭ 24), AngII was applied almost immediately once the current had fully developed; this treatment caused a rapid but incomplete inhibition of the current. A representative trace of the AngII-induced change in the current profile is shown in Fig. 5A. In these cells, the amplitude of peak current (31.4 Ϯ 4.6 Pa/pF, n ϭ 24) prior to the addition of AngII was almost identical to that observed in controls, suggesting similar levels of the STIM1/Orai1 protein expression. To compare the profiles of currents in control and AngII-treated cells more directly, the mean values of current were generated from traces recorded under the same experimental conditions, normalized for the peak amplitude of current (Fig. 5B). To analyze these results statistically, the mean Ϯ S.E. values for the change in current amplitude were calculated between time points from 100 to 200 s and from 100 to 300 s (Fig. 5C). There was only a small decrease in current amplitude between these time points without AngII addition (Fig. 5C, blue bars), but the current amplitude decreased by ϳ40% after AngII stimulation (red bars). Importantly, when AngII was applied at a later time (Ͼ200 s) after the development of the current, the inhibitory effect of AngII was reduced, and it was practically abolished when the agonist was added Ͼ300 s after establishing the whole cell recording (data not shown). These results indicated that in cells with activated I CRAC , stimulation of Ca 2ϩ -mobilizing AT1 receptors leads to inhibition of the current. The results also suggested that under FIGURE 3. Inhibition of PtdIns4P production by the PI3K inhibitors wortmannin and LY294002. A, COS-7 cells were labeled with [ 32 P]phosphate in phosphate-free medium for 3 h. LY294002 was added in increasing concentrations for 10 min before harvesting the cells with perchloric acid precipitation. After extraction, phospholipids were separated by TLC as detailed elsewhere (30). Radioactive spots were detected and quantified by a PhosphorImager. A representative TLC is shown in A; the dose-response curve of the inhibition was calculated from two experiments performed in duplicates (B). The inhibitory potency of LY294002 for Ca 2ϩ signaling is also plotted (B, red circles and dashed line). C, detection of PtdIns4P and PtdIns(4,5)P 2 in the PM by the OSH2-2xPH-GFP and PLC␦1PH-GFP domains, respectively, with TIRF analysis. COS-7 cells were transfected with a PM-targeted FRB construct and the mRFP-FKBP12-5-phosphatase in addition to the appropriate PH domain construct. Rapamycin-induced (Rapa) recruitment of 5-phosphatase (red) caused a small but consistent reduction in the membrane localization of OSH2-2xPH-GFP (green), probably reflecting to a small extent the PtdIns(4,5)P 2 binding of this domain. Phosphatase recruitment caused rapid decrease in PLC␦1PH-GFP localization (blue) indicating PtdIns(4,5)P 2 depletion. (The 5-phosphatase trace from the PLC␦1PH-GFP experiment was omitted for clarity, but it was almost identical to that shown in the red trace). The addition of LY294002 (100 M) induced an immediate and gradual loss of OSH2-2xPH-GFP from the membrane. The addition of 10 M ionomycin (Iono) at the end of the experiment was used to achieve complete removal of the OSH2-2xPH-GFP protein by massive PLC activation. TIRF data are normalized where the maximum and minimum (after ionomycin) fluorescent intensities recorded in the footprint of cells were considered to be 100 and 0%, respectively. Means Ϯ S.E. derived from 25 cells are shown as recorded in 4 -5 independent experiments. JULY 31, 2009 • VOLUME 284 • NUMBER 31 the whole cell recording conditions, which lead to dialysis of cells, the pathways responsible for AngII action are affected and that further experiments are warranted to identify these components.

Phosphoinositides in Store-operated Calcium Entry
The effects of AngII on the Tg-induced [Ca 2ϩ ] i rise were also studied in HEK-AT1 cells in single cell [Ca 2ϩ ] i measurements. These studies showed an AngII-induced decrease in [Ca 2ϩ ] i both in naive cells (Fig. 5D) and in cells overexpressing STIM1/ Orai1 after store depletion (Fig. 5E). Because AngII stimulation decreases both PtdIns(4,5)P 2 and PtdIns4P levels, these results were compatible with inhibition of SOCE or I CRAC after robust PLC activation and PtdIns4P reduction, although such decreases in [Ca 2ϩ ] i could be caused by other mechanism(s).
PI4KIII␣ Down-regulation Inhibits SOCE-The sensitivity of SOCE to PI4K inhibition or PLC-mediated PtdIns4P depletion raised the question of whether any of the four mammalian PI4Ks was responsible for the production of PtdIns4P in support of Orai1 channel activity. To address this question, we performed [Ca 2ϩ ] i measurements with cells in which each of the four PI4Ks had been individually knocked down by RNA interference-mediated gene silencing. Although in such studies done previously we found that even significantly reduced levels of the PI4Ks had only modest effects on most signaling functions of the cells and complete knockdown could not be accomplished because of cell death (29), we hoped that at least a partial effect of PI4K knockdown could be detected. First, we examined the effects of knockdown of PI4KII␣ and -III␣ in COS-7 cells and PI4KIII␣ in HEK-AT1 cells on the Tg-induced [Ca 2ϩ ] i rise. As shown in Fig. 6B, knockdown of PI4KIII␣, but not PI4KII␣, partially inhibited the plateau phase of the Tginduced [Ca 2ϩ ] i increase in COS-7 cells. PI4KIII␣ or -II␣ knockdown did not inhibit Ca 2ϩ release by Tg as shown in cells analyzed in Ca 2ϩ -free medium, but PI4KIII␣ knockdown inhibited the [Ca 2ϩ ] i rise upon Ca 2ϩ readdition (Fig. 6B). In separate experiments the effects of PI4KIII␤ knockdown were also analyzed in COS-7 cells. However, these effects were more complex and appeared to be dominated by a prolonged disruption of Golgi function and, hence, will require further investigation. Importantly, acute inhibition by PI4KIII␤ with the more selective inhibitor, PIK93, was without effect on Ca 2ϩ entry (data not shown).
The effect of PI4KIII␣ down-regulation was also examined in single HEK-AT1 cells attached to coverslips. These experiments again showed a modest but significant decrease in the Tg-induced [Ca 2ϩ ] i signal (Fig. 6C). Taken together these studies indicated that PI4KIII␣ but not PI4KII␣ enzyme is important in the support of Orai1 function in the PM.

DISCUSSION
The present studies were designed to address the question of whether PM phosphoinositides contribute to the regulation of the STIM1/Orai1-mediated Ca 2ϩ entry process. The importance of this question is 2-fold. First, only a few ion channels and transporters are known that do not require phosphoinositides for their proper function in the PM (23). Second, the ERlocalized STIM1 protein makes a contact with the PM upon ER Ca 2ϩ store depletion, and it contains a polybasic domain at its C terminus that is perfectly suited to serve as a phosphoinositidebinding module, especially in the multimerized form in which STIM1 exists in the ER Ca 2ϩ -depleted state (18).
To address this question, we employed several methods to alter PM phosphoinositides. We used PLC activation via stimulation of expressed AT1 receptors by AngII, applied inhibitors or knockdown of PI4Ks, and acutely decreased PM PtdIns(4,5)P 2 levels with the recruitable 5-phosphatase system described recently (28,37). The effects of these manipulations were studied on STIM1 movements from the tubular to the PM-adjacent ER compartment on [Ca 2ϩ ] i changes after ER store depletion and on I CRAC recordings. The results of these experiments indicate that STIM1 movements do not depend on PM phosphoinositides, as neither PtdIns(4,5)P 2 depletion nor PI4K inhibition had a major impact on the ability of STIM1 to move and associate with the PM. The slight effect of the removal of PtdIns(4,5)P 2 on STIM1-PM association suggests some contribution of the lipid in the stabilization of these interactions. These data are consistent with findings that STIM1, lacking the polybasic domain, is fully functional in activating Orai1-mediated SOCE, although its activation and inactivation properties might be impaired (15,19,22).
However, in contrast to STIM1 movements, Orai1-mediated store-operated Ca 2ϩ influx was sensitive to PI4K inhibition (but not to PtdIns(4,5)P 2 depletion) and was also inhibited by robust PLC activation, which evokes a simultaneous decrease in PtdIns(4,5)P 2 and PtdIns4P levels in these cells (29). SOCE was also inhibited by PI4K inhibitors under conditions in which PtdIns(4,5)P 2 levels showed no reductions, and similar conclusions could be drawn from the electrophysiological experiments detecting I CRAC . These data strongly suggest that PtdIns4P rather than PtdIns(4,5)P 2 is required for the optimal function of the Orai1-mediated Ca 2ϩ entry process.
Inhibition of agonist-induced Ca 2ϩ influx by PI4K inhibitors has been described in several studies (25,38,39), but this effect could be explained by the gradual depletion of the agonist-sensitive phosphoinositide pools and thence the calcium-mobilizing messenger, InsP 3 (30,40). Fewer studies have noted an effect of PI4K inhibition on SOCE activated by Ca 2ϩ store depletion (4,26). Broad et al. (26) has reported the most thorough analysis of the effects of a high concentration of Wm and LY294002 on SOCE and endogenous I CRAC in rat basophilic leukemia cells. These authors showed that Wm treatment strongly inhibits SOCE and I CRAC , noting that this is not caused by the lack of InsP 3 or diacylglycerol and that it correlates better with changes in the level of PtdIns4P than of PtdIns(4,5)P 2 (26). The present results have confirmed and extended these observations, showing that the PtdIns4P requirement is not at the level of STIM1 movements but at the activity of the Orai1 channels. Rosado et al. (4) also analyzed the effects of LY294002 on SOCE in platelets and concluded that the inhibitory effect is not due to depolarization. This is an important question that should always be asked with [Ca 2ϩ ] i measurements, as SOCE is very sensitive to changes in the driving force of Ca 2ϩ entry to the cells and hence to a drop in the membrane potential (41). Because a number of potassium channels require phosphoinositides for their proper function, depletion of phosphoinositides could, in theory, inhibit some of these channels leading to depolarization. Although this mechanism may contribute to the effects of phosphoinositide depletion on SOCE in intact cells, the current patch clamp studies and those reported by Broad et al. (26) clearly showed that I CRAC is dependent on phosphoinositide levels regardless of the membrane potential.
Our studies also suggest that the PI4K relevant for generating PtdIns4P is the type III␣ enzyme. Although the knockdown of PI4KIII␣ did not show strong inhibition, this was the only PI4K that had a consistent effect on SOCE, and knockdown of the PI4KII␣ enzyme or pharmacological blockade of PI4KIII␤ was without effect on Tg-induced [Ca 2ϩ ] i elevations. These results are in agreement with our previous studies in which the PI4KIII␣ enzyme was found to generate the PM phosphoinositide-signaling pool (29). This finding has been somewhat puz-

Phosphoinositides in Store-operated Calcium Entry
zling, as the PI4KIII␣ enzyme is found in the ER and in the ER/Golgi interface in mammalian cells without a noticeable amount in the PM either in quiescent or in stimulated cells (42). Because the enzyme has a long N-terminal sequence, the possibility exists that the ER-localized enzyme acts in trans on the PM at the ER/PM junctional sites, exactly where STIM1/Orai1 interaction occurs. Nevertheless, the enrichment of the enzyme in this compartment could not be demonstrated thus far, whether by immunostaining of the endogenous protein in fixed cells (Ϯstore depletion) or by testing the appearance of a GFPtagged PI4KIII␣ protein in STIM1 puncta in TIRF studies. 5 Therefore, this potentially interesting aspect of PI4K signaling awaits further clarification.
In summary, our experiments here suggest the presence of a phosphoinositide component in the control of SOCE involving the STIM1/Orai1 proteins. The site of phosphoinositide action is not at the level of STIM1 protein movement but rather at the level of Orai1 activation. Surprisingly, the regulatory lipid is not the most abundant PtdIns(4,5)P 2 but rather its precursor, PtdIns4P, most likely synthesized by the PI4KIII␣ enzyme. Further studies are needed to determine the molecular means by which PtdIns4P regulates the activity of the Orai1 channel or the mechanism by which the ER-localized PI4KIII␣ enzyme supplies the PM with its lipid product, PtdIns4P.