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

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 or the (4Rosado J.A. Sage S.O. J. Biol. Chem. 2000; 275: 9110-9113Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, R. H. Zhang H. H. R. PubMed Scopus Google Scholar, Y. A. S. Y. K. Y. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, J.P. T. Putney Jr., J.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we have studies the role of PM phosphoinositides in STIM1 movements as as in Orai1 channel results that phosphoinositides not have a major role in the 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. and thapsigargin were from II from and from other were of the The and as as the Orai1 used in this have been P. B. L. T. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google Scholar). The for the PM recruitment of the type IV 5-phosphatase domain as as for and have also been P. B. T. T. J. Cell Biol. 2006; PubMed Scopus Google Scholar, A. Kim Y.J. P. Z. Z. T. Mol. Biol. Cell. 2008; PubMed Scopus Google Scholar). of the PI4K the and have been A. Kim Y.J. P. Z. Z. T. Mol. Biol. Cell. 2008; PubMed Scopus Google Scholar). cells were and with the of for as P. B. L. T. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google Scholar). analysis in an system with a and a with or were used for the or and and were and the were as for with the of the membrane after the regions of cells and of the in the of cells to and were and their as These were and their and calcium cells were with for with were in P. B. L. T. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google for with studies were also in cells to an with a and a and the used for cells were in were from the with and with as S. T. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). cells were in and and an of the cells in the but not in were in a fluorescence clamp were an clamp and were and were with a with a in with The and to with from with were with the in and to with In the also inositol to of were after to the from a potential of and the were were by an current ICRAC and this from currents. and and angiotensin II were in a we to isolated in PtdIns(4,5)P2 STIM1 movements or the activation of Orai1-mediated Ca2+ in this we used the recently chemically induced recruitment of the 5-phosphatase domain of the type IV phosphoinositide 5-phosphatase to PtdIns(4,5)P2 levels in the PM P. B. T. T. J. Cell Biol. 2006; PubMed Scopus Google Scholar). This the of PtdIns(4,5)P2 without the of studies we used as of PtdIns(4,5)P2 upon P. B. T. T. J. Cell Biol. 2006; PubMed Scopus Google Scholar). were with the and the 5-phosphatase is but to the PM after the of causing depletion of PtdIns(4,5)P2 from the This be in where both the recruitment of the and the of the PtdIns(4,5)P2 from the membrane be T. J. Physiol. 2007; PubMed Scopus Google Scholar). In studies we that PtdIns(4,5)P2 from the PM by this not prevent STIM1 to PM-adjacent regions P. B. L. T. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google Scholar). However, analysis of STIM1 from cells in required to the not STIM1 altered in the this we induced toward the PM by of and and only has fully we PtdIns(4,5)P2 depletion by recruitment of the 5-phosphatase domain to the PM with shows that we a in the the after 5-phosphatase domain recruitment but only a the only the protein without the 5-phosphatase to the membrane In the of the also the of This that only slightly reduced PtdIns(4,5)P2 a decrease only after These that PM PtdIns(4,5)P2 to the of of PtdIns4P by the inhibitors wortmannin and cells were with in for in concentrations for the cells with acid phospholipids were by as S. T. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). were and by a is in the of the inhibition from two in The of for Ca2+ is also red and of PtdIns4P and PtdIns(4,5)P2 in the PM by the and with cells were with a and the in to the domain recruitment of 5-phosphatase a small but in the membrane localization of to a small the PtdIns(4,5)P2 of this recruitment decrease in localization PtdIns(4,5)P2 depletion. 5-phosphatase from the for but it to that in the red The of induced an and of from the The of the of the used to of the protein by are where the and fluorescent in the of cells were to be and from cells are as in also the of PtdIns(4,5)P2 the Ca2+ after store depletion. with in cells the 5-phosphatase recruitment system either or with and Orai1 to The endogenous of cells were with with to and the ER Ca2+ and activate This by to the 5-phosphatase and in the of to either the endogenous SOCE or the by of These results suggested that in the PM PtdIns(4,5)P2 not a major in the of SOCE in its STIM1 that inhibitors SOCE concentrations that also J.P. T. Putney Jr., J.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). PtdIns4P be a of SOCE the to the cells concentrations that type Kim S. S. T. PubMed Scopus Google Scholar). with the of in studies suggests that this is not with the this and T. A. PubMed Scopus Google Scholar). this we used in its the movements of STIM1 in after STIM1 been induced by and PtdIns(4,5)P2 been by recruitment of the 5-phosphatase. in the of to such cells to the STIM1 in the that PtdIns4P is not a in STIM1 the in the in both channels after in this of studies, and we it to in the of the cells or a in In both endogenous SOCE and that by in a this also without of PtdIns(4,5)P2 to cells not the of two were cells were with of and the phospholipids by in a reduced the of PtdIns4P in a without a decrease in the of as in and cells A. Kim Y.J. P. Z. Z. T. Mol. Biol. Cell. 2008; PubMed Scopus Google Scholar, S. T. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). in PtdIns(4,5)P2 a concentration also This to the inhibition of and PtdIns(4,5)P2 via that The of to PtdIns4P to that for inhibition of Ca2+ PtdIns4P other than in the PM, we also to that the PM of this we used the PtdIns4P A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that has to be a for in PM PtdIns4P levels A. Kim Y.J. P. Z. Z. T. Mol. Biol. Cell. 2008; PubMed Scopus Google Scholar, T. M. Yu L. J. M. T. A. S. Science. 2006; PubMed Scopus Google Scholar). In we used the as in in to the by the These studies in localization by but recruitment of the 5-phosphatase to the PM a decrease in the of the This be to a PtdIns(4,5)P2 of this as it to also PtdIns(4,5)P2 in J.W. A. S. D. D. M.A. Mol. Cell. Full Text Full Text PDF PubMed Scopus (277) Google Scholar). the decrease in the localization of the PtdIns(4,5)P2 we were to an PtdIns4P after 5-phosphatase recruitment with of the PtdIns4P for that are to be Z. and T. a of with the PM after PtdIns(4,5)P2 after the of These that most of the PtdIns4P from the PM within of PtdIns(4,5)P2 in the PM only a small STIM1 oligomerization and PM interaction in a major SOCE that with PtdIns4P rather than PtdIns(4,5)P2 depletion. the of PtdIns4P were in cells the angiotensin were with or in with were only with cells The and and Ca2+ present in the These depletion of the ER Ca2+ store to activate Ca2+ influx. from to of were after to the from a potential of a of to of the in cells with both Orai1 and The of current activated by depletion of the ER calcium is in In cells both the current fully within to with a of the current in a of the cells it for whereas in the of the cells a and of current The and potential both of ICRAC cells not STIM1 cells current the endogenous ICRAC current is this These results are with the that cells a ICRAC current with STIM1 and also the of PI4K inhibition the and of used light present during the clamp concentrations that both and S. T. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). the of cells during the cells were with for to the current as a of current during a in the current with but the of the current substantially the of in the of the of current in and cells were from in the and a two and of These results that the inhibition of type the of the ICRAC we activation has the ICRAC a system after the of the and the of In the of cells the current fully this a but inhibition of the of the in the current is in In the of current to the of to that in levels of the protein the of in and cells the of current were from the for the of current results the for the in current were from to and from to only a small decrease in current without but the current decreased by after stimulation a after the of the the of and it the agonist after the not These results that in cells with activated stimulation of to inhibition of the The results also suggested that the to of the responsible for are and that are to identify The of the were also in cells in These studies an decrease in both in cells and in cells overexpressing after store depletion stimulation both PtdIns(4,5)P2 and PtdIns4P results were with inhibition of SOCE or ICRAC after activation and PtdIns4P such in be by other The of SOCE to PI4K inhibition or PtdIns4P depletion the of of the responsible for the of PtdIns4P in of Orai1 channel activity. this we with cells in of the been by in such studies we that reduced levels of the only most of the cells and not be of A. Kim Y.J. P. Z. Z. T. Mol. Biol. Cell. 2008; PubMed Scopus Google Scholar), we that a of PI4K be we the of of and in cells and in cells the in of but not the of the in or not Ca2+ by as in cells in but the upon Ca2+ In the of were also in However, were and to be by a of function inhibition by with the without Ca2+ entry not The of down-regulation also in cells to These a but decrease in the studies that but not is important in the of Orai1 function in the The present studies were to the of PM phosphoinositides to the of the STIM1/Orai1-mediated Ca2+ entry process. The of this is only a few ion channels and transporters are that not phosphoinositides for their function in the PM (23Gamper N. Shapiro M.S. Nat. Rev. Neurosci. 2007; 8: 921-934Crossref PubMed Scopus (198) Google Scholar). the STIM1 protein makes a with the PM upon ER Ca2+ store depletion, and it a polybasic domain its C that is to as a in the in STIM1 in the ER (18Liou J. Fivaz M. Inoue T. Meyer T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9301-9306Crossref PubMed Scopus (520) Google Scholar). this we several to PM used activation via stimulation of by inhibitors or of and decreased PM PtdIns(4,5)P2 levels with the 5-phosphatase system recently P. B. T. T. J. Cell Biol. 2006; PubMed Scopus Google Scholar, Inoue T. Meyer T. B. Science. 2006; 314: PubMed Scopus Google Scholar). The of were STIM1 movements from the tubular to the PM-adjacent ER after ER store depletion and ICRAC The results of indicate that STIM1 movements not PM as PtdIns(4,5)P2 depletion PI4K inhibition a major the of STIM1 to and with the The of the of PtdIns(4,5)P2 association suggests of the in the of These are with that STIM1, lacking the polybasic is fully in Orai1-mediated its activation and inactivation be (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, 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, 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). However, in to STIM1 Orai1-mediated store-operated Ca2+ to PI4K inhibition not to PtdIns(4,5)P2 and also by a decrease in PtdIns(4,5)P2 and PtdIns4P levels in cells A. Kim Y.J. P. Z. Z. T. Mol. Biol. Cell. 2008; PubMed Scopus Google Scholar). SOCE also by PI4K inhibitors in PtdIns(4,5)P2 levels and be from the These suggest that PtdIns4P rather than PtdIns(4,5)P2 is required for the function of the Orai1-mediated Ca2+ entry process. of Ca2+ by PI4K inhibitors has been in several studies Y. A. S. Y. K. Y. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, S. T. J. Biol. Chem. Full Text PDF PubMed Google Scholar, H. R. Zhang H. H. R. PubMed Scopus Google Scholar), but this be by the depletion of the phosphoinositide and the S. T. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). studies have an of PI4K inhibition SOCE activated by Ca2+ store depletion (4Rosado J.A. Sage S.O. J. Biol. Chem. 2000; 275: 9110-9113Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, J.P. T. Putney Jr., J.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). J.P. T. Putney Jr., J.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) has reported the most analysis of the of a concentration of and SOCE and endogenous ICRAC in These that SOCE and that this is not by the of or and that it with in the of PtdIns4P than of PtdIns(4,5)P2 J.P. T. Putney Jr., J.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The present results have confirmed and that the PtdIns4P requirement is not the of STIM1 movements but the activity of the Orai1 (4Rosado J.A. Sage S.O. J. Biol. Chem. 2000; 275: 9110-9113Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) also the of SOCE in platelets and that the is not to This is an important that be with as SOCE is to in the of Ca2+ entry to the cells and to a in the membrane potential B. A. G. PubMed Scopus Google Scholar). a of channels phosphoinositides for their depletion of phosphoinositides in of channels to this mechanism to the of phosphoinositide depletion SOCE in the current clamp studies and reported by J.P. T. Putney Jr., J.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that ICRAC is phosphoinositide levels of the membrane studies also suggest that the PI4K for PtdIns4P is the type the of not inhibition, this the only PI4K that a and of the or of without These results are in with studies in the to the PM A. Kim Y.J. P. Z. Z. T. Mol. Biol. Cell. 2008; PubMed Scopus Google Scholar). This has been as the is in the ER and in the in cells without a in the PM either in or in cells A. T. Cell Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). the has a long the that the in the PM the where interaction the of the in this not be by of the endogenous protein in cells or by the of a protein in STIM1 in this of PI4K In suggest the of a phosphoinositide in the of SOCE the The of phosphoinositide is not the of STIM1 protein but rather the of Orai1 the is not the most PtdIns(4,5)P2 but rather its most likely by the studies are to the by PtdIns4P the activity of the Orai1 channel or the mechanism by the the PM with its are to Y. for the monomeric red fluorescent protein and to W. for the type IV the and of the of with the of and T.