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• W2000665911 abstract "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 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. Store-operated Ca2+ entry (SOCE) 3The abbreviations used are: SOCEstore-operated calcium entryAngIIangiotensin IIERendoplasmic reticulumBAPTA1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acidFRBfragment of mTOR that binds FKBP12GFPgreen fluorescent proteinmRFPmonomeric red fluorescent proteinPI3Kphosphatidylinositol 3-kinasePI4Kphosphatidylinositol 4-kinasePLCphospholipase CPMplasma membraneICRACcalcium release-activated calcium currentInsinositolPtdInsphosphatidylinositolSTIMstromal interaction moleculeTgthapsigarginTIRFtotal internal reflection fluorescenceYFPyellow fluorescent proteinWmwortmannin5-PTasetype IV phosphoinositide 5-phosphatase. is a ubiquitous Ca2+ entry pathway that is regulated by the Ca2+ content of the endoplasmic reticulum (ER) (1Parekh A.B. Putney Jr., J.W. Phys. Rev. 2005; 85: 757-810Crossref PubMed Scopus (1800) Google Scholar). SOCE has been identified as the major route of Ca2+ entry during activation of cells of the immune system such as T cells and mast cells (2Hoth M. Penner R. Nature. 1992; 355: 353-356Crossref PubMed Scopus (1491) Google Scholar, 3Prakriya M. Lewis R.S. J. Gen. Physiol. 2002; 119: 487-507Crossref PubMed Scopus (267) Google Scholar), and it is also present and functionally important in other cells such as platelets (4Rosado J.A. Sage S.O. J. Biol. Chem. 2000; 275: 9110-9113Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) and developing myotubes (5Stiber J. Hawkins A. Zhang Z.S. Wang S. Burch J. Graham V. Ward C.C. Seth M. Finch E. Malouf N. Williams R.S. Eu J.P. Rosenberg P. Nat. Cell Biol. 2008; 10: 688-697Crossref PubMed Scopus (293) Google Scholar). The long awaited mechanism of how the ER luminal Ca2+ content is sensed and the information transferred to the plasma membrane (PM) has been clarified recently after identification of the ER Ca2+ sensor proteins STIM1 and -2 (6Liou J. Kim M.L. Heo W.D. Jones J.T. Myers J.W. Ferrell Jr., J.E. Meyer T. Curr. Biol. 2005; 15: 1235-1241Abstract Full Text Full Text PDF PubMed Scopus (1753) Google Scholar, 7Roos J. DiGregorio P.J. Yeromin A.V. Ohlsen K. Lioudyno M. Zhang S. Safrina O. Kozak J.A. Wagner S.L. Cahalan M.D. Veliçcelebi G. Stauderman K.A. J. Cell Biol. 2005; 169: 435-445Crossref PubMed Scopus (1519) Google Scholar) and the PM Ca2+ channels Orai1, -2, and -3 (8Feske S. Gwack Y. Prakriya M. Srikanth S. Puppel S.H. Tanasa B. Hogan P.G. Lewis R.S. Daly M. Rao A. Nature. 2006; 441: 179-185Crossref PubMed Scopus (1854) Google Scholar, 9Vig M. Peinelt C. Beck A. Koomoa D.L. Rabah D. Koblan-Huberson M. Kraft S. Turner H. Fleig A. Penner R. Kinet J.P. Science. 2006; 312: 1220-1223Crossref PubMed Scopus (1153) Google Scholar, 10Zhang S.L. Yeromin A.V. Zhang X.H. Yu Y. Safrina O. Penna A. Roos J. Stauderman K.A. Cahalan M.D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9357-9362Crossref PubMed Scopus (745) Google Scholar). According to current views, a decrease in the ER Ca2+ 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. 11Hewavitharana T. Deng X. Soboloff J. Gill D.L. Cell Calcium. 2007; 42: 173-182Crossref PubMed Scopus (150) Google Scholar, 12Putney Jr., J.W. Cell Calcium. 2007; 42: 103-110Crossref PubMed Scopus (189) Google Scholar, 13Lewis R.S. Nature. 2007; 446: 284-287Crossref PubMed Scopus (424) Google Scholar). 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 (14Huang G.N. Zeng W. Kim J.Y. Yuan J.P. Han L. Muallem S. Worley P.F. Nat. Cell Biol. 2006; 8: 1003-1010Crossref PubMed Scopus (568) Google Scholar), whereas the latter requires its C-terminal membrane-adjacent cytoplasmic tail to be fully activated by the STIM proteins (15Li Z. Lu J. Xu P. Xie X. Chen L. Xu T. J. Biol. Chem. 2007; 282: 29448-29456Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 16Muik M. Frischauf I. Derler I. Fahrner M. Bergsmann J. Eder P. Schindl R. Hesch C. Polzinger B. Fritsch R. Kahr H. Madl J. Gruber H. Groschner K. Romanin C. J. Biol. Chem. 2008; 283: 8014-8022Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). 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)P2 (17Heo W.D. Inoue T. Park W.S. Kim M.L. Park B.O. Wandless T.J. Meyer T. Science. 2006; 314: 1458-1461Crossref PubMed Scopus (540) Google Scholar). 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 (18Liou J. Fivaz M. Inoue T. Meyer T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9301-9306Crossref PubMed Scopus (520) Google Scholar). In other studies, truncated STIM1 lacking the polybasic domain shows only slightly altered activation (15Li Z. Lu J. Xu P. Xie X. Chen L. Xu T. J. Biol. Chem. 2007; 282: 29448-29456Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar) or inactivation (19Spassova M.A. Soboloff J. He L.P. Xu W. Dziadek M.A. Gill D.L. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 4040-4045Crossref PubMed Scopus (277) Google Scholar) kinetics without major defects in supporting Orai1-mediated Ca2+ influx. The most recent studies identify the minimal Orai1 activation domain in STIM1 (20Yuan J.P. Zeng W. Dorwart M.R. Choi Y.J. Worley P.F. Muallem S. Nat. Cell Biol. 2009; 11: 337-343Crossref PubMed Scopus (559) Google Scholar, 21Park C.Y. Hoover P.J. Mullins F.M. Bachhawat P. Covington E.D. Raunser S. Walz T. Garcia K.C. Dolmetsch R.E. Lewis R.S. Cell. 2009; 136: 876-890Abstract Full Text Full Text PDF PubMed Scopus (763) Google Scholar) and find that the polybasic domain is not essential for this function but makes electrostatic interaction with classical transient receptor potential channels (22Zeng W. Yuan J.P. Kim M.S. Choi Y.J. Huang G.N. Worley P.F. Muallem S. Mol. Cell. 2008; 32: 439-448Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). store-operated calcium entry angiotensin II endoplasmic reticulum 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid fragment of mTOR that binds FKBP12 green fluorescent protein monomeric red fluorescent protein phosphatidylinositol 3-kinase phosphatidylinositol 4-kinase phospholipase C plasma membrane calcium release-activated calcium current inositol phosphatidylinositol stromal interaction molecule thapsigargin total internal reflection fluorescence yellow fluorescent protein wortmannin type IV phosphoinositide 5-phosphatase. PM phosphoinositides have been widely reported as regulators of the activity of several ion channels and transporters (23Gamper N. Shapiro M.S. Nat. Rev. Neurosci. 2007; 8: 921-934Crossref PubMed Scopus (198) Google Scholar). However, only a few studies have addressed the inositide requirement of SOCE and none specifically that of the Orai1-mediated Ca2+ 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 (4Rosado J.A. Sage S.O. J. Biol. Chem. 2000; 275: 9110-9113Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 24Takahashi R. Watanabe H. Zhang X.X. Kakizawa H. Hayashi H. Ohno R. Biochem. Biophys. Res. Commun. 1997; 235: 657-662Crossref PubMed Scopus (21) Google Scholar, 25Hashimoto Y. Ogihara A. Nakanishi S. Matsuda Y. Kurokawa K. Nonomura Y. J. Biol. Chem. 1992; 267: 17078-17081Abstract Full Text PDF PubMed Google Scholar, 26Broad L.M. Braun F.J. Lievremont J.P. Bird G.S. Kurosaki T. Putney Jr., J.W. J. Biol. Chem. 2001; 276: 15945-15952Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Here we have described studies addressing the role of PM phosphoinositides in STIM1 movements as well as in Orai1 channel gating. Our results show that phosphoinositides do not have a major role in the prominent reorganization of STIM1 after Ca2+ store depletion but suggest a function of PtdIns4P rather than PtdIns(4,5)P2 in supporting the Orai1-mediated Ca2+ entry process. 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. The YFP- and mRFP-STIM1 plasmids as well as the Orai1 constructs used in this study have been described previously (27Várnai P. Tóth B. Tóth D.J. Hunyady L. Balla T. J. Biol. Chem. 2007; 282: 29678-29690Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). The plasmids designed for the rapamycin-induced PM recruitment of the type IV 5-phosphatase domain as well as those for PLCδ1PH-GFP and OSH2-2xPH-GFP have also been described elsewhere (28Varnai P. Thyagarajan B. Rohacs T. Balla T. J. Cell Biol. 2006; 175: 377-382Crossref PubMed Scopus (278) Google Scholar, 29Balla A. Kim Y.J. Varnai P. Szentpetery Z. Knight Z. Shokat K.M. Balla T. Mol. Biol. Cell. 2008; 19: 711-721Crossref PubMed Scopus (146) Google Scholar). For siRNA-mediated knockdown of the various PI4K isoforms, the duplexes and treatment protocols have been described previously (29Balla A. Kim Y.J. Varnai P. Szentpetery Z. Knight Z. Shokat K.M. Balla T. Mol. Biol. Cell. 2008; 19: 711-721Crossref PubMed Scopus (146) Google Scholar). COS-7 cells were cultured on glass coverslips (3 × 105 cells/35-mm dish) and transfected with the indicated constructs (0.5 μg of DNA/dish) using Lipofectamine 2000 for 24 h as described previously (27Várnai P. Tóth B. Tóth D.J. Hunyady L. Balla T. J. Biol. Chem. 2007; 282: 29678-29690Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). 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 60×/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 μm) for 45 min, room temperature). Calcium measurements with Fura2 were performed in modified Krebs-Ringer solution (see Ref. 27Várnai P. Tóth B. Tóth D.J. Hunyady L. Balla T. J. Biol. Chem. 2007; 282: 29678-29690Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar for composition) supplemented with 200 μm 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 μm Fura2/AM at room temperature as described previously (30Nakanishi S. Catt K.J. Balla T. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 5317-5321Crossref PubMed Scopus (310) Google Scholar). Loaded cells were kept in HEPES-buffered M199, Hanks' salt solution containing 0.1% bovine serum albumin and 200 μm sulfinpyrazone, and an aliquot of the cells was centrifuged immediately before [Ca2+]i measurement in the modified Krebs-Ringer solution containing sulfinpyrazone but not bovine serum albumin. [Ca2+]i measurements in suspension were performed at 34 °C in a PTI DeltaScan fluorescence spectrophotometer (Photon Technology International). 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 MgCl2, 2 CaCl2, 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 μm inositol 1,4,5-trisphosphate (InsP3, hexasodium salt; Sigma). Voltage ramps (−100 to +100 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 ICRAC 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). First we wanted to determine whether isolated changes in PtdIns(4,5)P2 could alter STIM1 movements or the activation of Orai1-mediated Ca2+ 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)P2 levels in the PM (28Varnai P. Thyagarajan B. Rohacs T. Balla T. J. Cell Biol. 2006; 175: 377-382Crossref PubMed Scopus (278) Google Scholar). This method allows changing the level of PtdIns(4,5)P2 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)P2 upon rapamycin treatment (28Varnai P. Thyagarajan B. Rohacs T. Balla T. J. Cell Biol. 2006; 175: 377-382Crossref PubMed Scopus (278) Google Scholar). 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)P2 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)P2 reporter PLCδ1PH-GFP from the membrane can be monitored simultaneously (see Ref. 31Balla T. J. Physiol. 2007; 582: 927-937Crossref PubMed Scopus (56) Google Scholar). In previous studies we showed that PtdIns(4,5)P2 elimination from the PM by this method does not prevent Tg-induced STIM1 translocation to PM-adjacent regions (27Várnai P. Tóth B. Tóth D.J. Hunyady L. Balla T. J. Biol. Chem. 2007; 282: 29678-29690Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). However, analysis of STIM1 translocation from many cells in TIRF experiments required normalization to the maximum translocation. Therefore, those experiments could not demonstrate whether STIM1 translocation was altered in the PtdIns(4,5)P2-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)P2 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 containing only the mRFP-FKBP12 protein without the 5-phosphatase was recruited to the membrane (Fig. 1B). In parallel experiments, the translocation response of the PLCδ1PH-GFP was also followed using the same sequence of stimulation. This showed that ATP/Tg only slightly reduced PtdIns(4,5)P2 levels, which showed a large decrease only after rapamycin addition (see on Fig. 3). These experiments indicated that PM PtdIns(4,5)P2 may contribute to the stabilization of STIM1-PM interaction.FIGURE 3Inhibition of PtdIns4P production by the PI3K inhibitors wortmannin and LY294002. A, COS-7 cells were labeled with [32P]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 (30Nakanishi S. Catt K.J. Balla T. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 5317-5321Crossref PubMed Scopus (310) Google Scholar). 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 Ca2+ signaling is also plotted (B, red circles and dashed line). C, detection of PtdIns4P and PtdIns(4,5)P2 in the PM by the OSH2-2xPH-GFP and PLCδ1PH-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)P2 binding of this domain. Phosphatase recruitment caused rapid decrease in PLCδ1PH-GFP localization (blue) indicating PtdIns(4,5)P2 depletion. (The 5-phosphatase trace from the PLCδ1PH-GFP experiment was omitted for clarity, but it was almost identical to that shown in the red trace). The addition of LY294002 (100 μm) induced an immediate and gradual loss of OSH2-2xPH-GFP from the membrane. The addition of 10 μm 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 ± S.E. derived from 25 cells are shown as recorded in 4–5 independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also determined the effect of PtdIns(4,5)P2 removal on the cytosolic Ca2+ signal after store depletion. For this, [Ca2+]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 Ca2+ stores and activate SOCE. This was followed by rapamycin addition to recruit the 5-phosphatase and deplete PtdIns(4,5)P2. 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)P2 was not a major factor in the regulation of SOCE in these cells, despite its minor effect on STIM1 translocation. Previous data had shown that PI3K inhibitors inhibit SOCE at concentrations that could also inhibit PI4Ks (26Broad L.M. Braun F.J. Lievremont J.P. Bird G.S. Kurosaki T. Putney Jr., J.W. J. Biol. Chem. 2001; 276: 15945-15952Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). 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 (32Downing G.J. Kim S. Nakanishi S. Catt K.J. Balla T. Biochemistry. 1996; 35: 3587-3594Crossref PubMed Scopus (104) Google Scholar). 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 4P. Varnai and T. Balla, unpublished observation. (also see Ref. 33Warashina A. Arch. Biochem. Biophys. 1999; 367: 303-310Crossref PubMed Scopus (11) Google Scholar). 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)P2 had been eliminated by recruitment of the 5-phosphatase. As shown in Fig. 1, the addition of LY294002 (30–300 μm) 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)P2 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, [32P]phosphate-labeled COS-7 cells were treated with increasing doses of LY294002 and the labeled phospholipids analyzed by TLC analysis. 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 [32P]PtdIns(4,5)P2, essentially mimicking the effects of 10 μm Wm as described previously in bovine adrenal and HEK293 cells (29Balla A. Kim Y.J. Varnai P. Szentpetery Z. Knight Z. Shokat K.M. Balla T. Mol. Biol. Cell. 2008; 19: 711-721Crossref PubMed Scopus (146) Google Scholar, 30Nakanishi S. Catt K.J. Balla T. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 5317-5321Crossref PubMed Scopus (310) Google Scholar). A slight increase in PtdIns(4,5)P2 already at a lower LY294002 concentration was also observed. This was attributed to the inhibition of PI3Ks and thence sparing PtdIns(4,5)P2 usage via that pathway. The potency of LY294002 to inhibit PtdIns4P synthesis was almost identical to that for inhibition of Ca2+ influx (Fig. 3B). Because 32P 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 (34Roy A. Levine T.P. J. Biol. Chem. 2004; 279: 44683-44689Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar) that has proven to be a reasonable probe for following changes in PM PtdIns4P levels (29Balla A. Kim Y.J. Varnai P. Szentpetery Z. Knight Z. Shokat K.M. Balla T. Mol. Biol. Cell. 2008; 19: 711-721Crossref PubMed Scopus (146) Google Scholar, 35Yeung T. Terebiznik M. Yu L. Silvius J. Abidi W.M. Philips M. Levine T. Kapus A. Grinstein S. Science. 2006; 313: 347-351Crossref PubMed Scopus (256) Google Scholar). 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)P2 binding of this reporter, as it was shown to also bind PtdIns(4,5)P2 in vitro (36Yu J.W. Mendrola J.M. Audhya A. Singh S. Keleti D. DeWald D.B. Murray D. Emr S.D. Lemmon M.A. Mol. Cell. 2004; 13: 677-688Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Note the rapid decrease in the localization of the PLCδ1PH-GFP probe reporting on PtdIns(4,5)P2 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. 5M. Korzeniowski, Z. Szentpetery, and T. Balla, unpublished observations. Nevertheless, a very significant fraction of OSH2–2xPH-GFP remained associated with the PM after PtdIns(4,5)P2 elimination, which was then rapidly released after the addition of 100 μm 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)P2 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)P2 depletion. To study the effects of PtdIns4P manipulations directly on ICRAC, electrophysiological measurements were performed in HEK293 cells stably expressing the Ca2+-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 μm InsP3 and 20 mm BAPTA, and 2 mm extracellular Ca2+ was present in the bath solution. These conditions allowed depletion of the ER Ca2+ store to activate Ca2+ influx. Voltage ramps from −100 to +100 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 −80 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 ± 3.1 pA/pF; (n = 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 obs" @default.
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• W2000665911 title "Dependence of STIM1/Orai1-mediated Calcium Entry on Plasma Membrane Phosphoinositides" @default.
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