Activation Mechanism for CRAC Current and Store-operated Ca2+ Entry

Abstract

Here we tested the role of calcium influx factor (CIF) and calcium-independent phospholipase A2 (iPLA2) in activation of Ca2+ release-activated Ca2+ (CRAC) channels and store-operated Ca2+ entry in rat basophilic leukemia (RBL-2H3) cells. We demonstrate that 1) endogenous CIF production may be triggered by Ca2+ release (net loss) as well as by simple buffering of free Ca2+ within the stores, 2) a specific 82-kDa variant of iPLA2β and its corresponding activity are present in membrane fraction of RBL cells, 3) exogenous CIF (extracted from other species) mimics the effects of endogenous CIF and activates iPLA2β when applied to cell homogenates but not intact cells, 4) activation of ICRAC can be triggered in resting RBL cells by dialysis with exogenous CIF, 5) molecular or functional inhibition of iPLA2β prevents activation of ICRAC, which could be rescued by cell dialysis with a human recombinant iPLA2β, 6) dependence of ICRAC on intracellular pH strictly follows pH dependence of iPLA2β activity, and 7) (S)-BEL, a chiral enantiomer of suicidal substrate specific for iPLA2β, could be effectively used for pharmacological inhibition of ICRAC and store-operated Ca2+ entry. These findings validate and significantly advance our understanding of the CIF-iPLA2-dependent mechanism of activation of ICRAC and store-operated Ca2+ entry. Here we tested the role of calcium influx factor (CIF) and calcium-independent phospholipase A2 (iPLA2) in activation of Ca2+ release-activated Ca2+ (CRAC) channels and store-operated Ca2+ entry in rat basophilic leukemia (RBL-2H3) cells. We demonstrate that 1) endogenous CIF production may be triggered by Ca2+ release (net loss) as well as by simple buffering of free Ca2+ within the stores, 2) a specific 82-kDa variant of iPLA2β and its corresponding activity are present in membrane fraction of RBL cells, 3) exogenous CIF (extracted from other species) mimics the effects of endogenous CIF and activates iPLA2β when applied to cell homogenates but not intact cells, 4) activation of ICRAC can be triggered in resting RBL cells by dialysis with exogenous CIF, 5) molecular or functional inhibition of iPLA2β prevents activation of ICRAC, which could be rescued by cell dialysis with a human recombinant iPLA2β, 6) dependence of ICRAC on intracellular pH strictly follows pH dependence of iPLA2β activity, and 7) (S)-BEL, a chiral enantiomer of suicidal substrate specific for iPLA2β, could be effectively used for pharmacological inhibition of ICRAC and store-operated Ca2+ entry. These findings validate and significantly advance our understanding of the CIF-iPLA2-dependent mechanism of activation of ICRAC and store-operated Ca2+ entry. Store-operated channels (SOC) 2The abbreviations used are: SOC, store-operated channel; SOCE, store-operated Ca2+ entry; iPLA2, calcium-independent phospholipase A2; rec-iPLA2β, recombinant iPLA2β; CIF, Ca2+ influx factor; CRAC, Ca2+ release-activated Ca2+-conducting channel; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; BEL, bromoenol lactone (formula name, 6E-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one); CaM, calmodulin; ER, endoplasmic reticulum; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; RBL, rat basophilic leukemia; (R)-BEL, (R) enantiomer of bromoenol lactone; (S)-BEL, (S) enantiomer of bromoenol lactone; TG, thapsigargin; TPEN, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine; pF, picofarad(s). and Ca2+ entry (SOCE) are triggered by depletion of intracellular Ca2+ stores in a wide variety of cell types (for review, see Ref. 1Parekh A.B. Putney Jr., J.W. Physiol. Rev. 2005; 85: 757-810Crossref PubMed Scopus (1807) Google Scholar). Recently we discovered Ca2+-independent phospholipase A2 (iPLA2) to be a crucial determinant of SOCE (2Smani T. Zakharov S. Csutora P. Leno E. Trepakova E.S. Bolotina V.M. Nat. Cell Biol. 2004; 6: 113-120Crossref PubMed Scopus (235) Google Scholar, 3Smani T. Zakharov S. Leno E. Csutora P. Trepakova E.S. Bolotina V.M. J. Biol. Chem. 2003; 278: 11909-11915Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and a physiological target for a mysterious Ca2+ influx factor (CIF) that is produced by the endoplasmic reticulum (ER) upon Ca2+ store depletion. We proposed a mechanism (2Smani T. Zakharov S. Csutora P. Leno E. Trepakova E.S. Bolotina V.M. Nat. Cell Biol. 2004; 6: 113-120Crossref PubMed Scopus (235) Google Scholar) that mediates not only activation but also termination of SOCE. We demonstrated that CIF displaces inhibitory calmodulin (CaM) from iPLA2 leading to its activation and production of lysophospholipids that in turn activate SOC and SOCE in vascular smooth muscle cells. Upon refilling of the stores and termination of CIF production, CaM binds to iPLA2 and inhibits it, and the activity of SOC and SOCE is halted. Consistent with a major role of iPLA2, a growing number of studies demonstrated that its irreversible inhibition impairs SOCE in different cell types (2Smani T. Zakharov S. Csutora P. Leno E. Trepakova E.S. Bolotina V.M. Nat. Cell Biol. 2004; 6: 113-120Crossref PubMed Scopus (235) Google Scholar, 3Smani T. Zakharov S. Leno E. Csutora P. Trepakova E.S. Bolotina V.M. J. Biol. Chem. 2003; 278: 11909-11915Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 4Nowatzke W. Ramanadham S. Ma Z. Hsu F.F. Bohrer A. Turk J. Endocrinology. 1998; 139: 4073-4085Crossref PubMed Scopus (55) Google Scholar, 5Vanden Abeele F. Lemonnier L. Thebault S. Lepage G. Parys J. Shuba Y. Skryma R. Prevarskaya N. J. Biol. Chem. 2004; 279: 30326-30337Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 6Abeele F.V. Shuba Y. Roudbaraki M. Lemonnier L. Vanoverberghe K. Mariot P. Skryma R. Prevarskaya N. Cell Calcium. 2003; 33: 357-373Crossref PubMed Scopus (100) Google Scholar, 7Zablocki K. Wasniewska M. Duszynski J. Acta Biochim. Pol. 2000; 47: 591-599Crossref PubMed Scopus (7) Google Scholar, 8Martinez J. Moreno J.J. Biochem. Pharmacol. 2005; 70: 733-739Crossref PubMed Scopus (21) Google Scholar, 9Shin K.J. Chung C. Hwang Y.A. Kim S.H. Han M.S. Ryu S.H. Suh P.G. Toxicol. Appl. Pharmacol. 2002; 178: 37-43Crossref PubMed Scopus (25) Google Scholar). However, no detailed study of CIF-iPLA2-dependent mechanism has been done in the cells in which SOCE is mediated by a highly Ca2+-selective SOC (historically called CRAC) (10Hoth M. Penner R. Nature. 1992; 355: 353-356Crossref PubMed Scopus (1495) Google Scholar). The whole-cell current through CRAC channels (ICRAC) remains a golden standard for the studies of SOC and mechanisms of its regulation (for review, see Ref. 1Parekh A.B. Putney Jr., J.W. Physiol. Rev. 2005; 85: 757-810Crossref PubMed Scopus (1807) Google Scholar). Our present studies were designed to test the validity and functionality of the CIF-iPLA2-dependent pathway in CRAC channel activation, to obtain new important information related to the signal that triggers CIF production, the specific isoform of iPLA2 that may be involved as well as the functional regulation of ICRAC through regulation of iPLA2 in rat basophilic leukemia (RBL-2H3) cells, one of the well established models for CRAC channel studies. CIF is known to be produced in the cells when their Ca2+ stores are depleted after either active Ca2+ release (usually as a result of agonist-induced Ca2+ discharge from the stores) or as a result of passive Ca2+ loss (when Ca2+ back-sequestration into the stores is prevented). Although the molecular identity of CIF remains a mystery, its presence and biological activity was detected in numerous cell types, from yeast to humans (11Thomas D. Hanley M.R. J. Biol. Chem. 1995; 270: 6429-6432Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 12Csutora P. Su Z. Kim A. R. Hanley M.R. S. A. PubMed Scopus Google Scholar, E.S. Csutora P. Bolotina V.M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). a of the present related to CIF we the to A.B. Putney Jr., J.W. Physiol. Rev. 2005; 85: 757-810Crossref PubMed Scopus (1807) Google Scholar, V.M. Csutora P. Biochem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). Here we on new as is the physiological signal that triggers CIF production, is production of CIF, and is its biological activity different is that activation of SOCE is triggered by a Ca2+ loss from (for review, see 1Parekh A.B. Putney Jr., J.W. Physiol. Rev. 2005; 85: 757-810Crossref PubMed Scopus (1807) Google and Cell Calcium. PubMed Scopus Google Scholar). However, that a simple in free Ca2+ within the stores Ca2+ loss) may be for SOCE in a C. T. J. Cell Biol. 1998; PubMed Scopus Google Scholar). is of in Ca2+ may be for CIF to be a Ca2+ loss or could be a simple in free Ca2+ in the the is CIF production and SOCE activation may be a of the cell not only to agonist-induced discharge of Ca2+ from the stores but also to in free Ca2+ in the stores to in the of Ca2+ as and (for review, see J. T. Biochem. 1998; Full Text PDF PubMed Scopus Google and J. Z. Cell Calcium. 2003; 33: PubMed Scopus Google Scholar). Here we tested simple Ca2+ buffering in the stores may or may not CIF production and may to CIF produced upon Ca2+ We also tested CIF produced by different types of cells and could be and applied CIF can the effects of endogenous CIF in iPLA2 and is well established that iPLA2 by is not a but a growing of Biochim. 2000; PubMed Scopus Google Scholar, J. Biochim. 2000; PubMed Scopus Google Scholar, W. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). major of iPLA2 been and (for see Ref. J. Biochim. 2000; PubMed Scopus Google and Han J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google iPLA2β and The of iPLA2β is that CaM for CaM to and its activity J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). iPLA2β from other and a for its specific role in the SOCE pathway (2Smani T. Zakharov S. Csutora P. Leno E. Trepakova E.S. Bolotina V.M. Nat. Cell Biol. 2004; 6: 113-120Crossref PubMed Scopus (235) Google Scholar). of of iPLA2β is in the which may be important for their and into specific vascular smooth muscle cells we that iPLA2β remains functional the of membrane (2Smani T. Zakharov S. Csutora P. Leno E. Trepakova E.S. Bolotina V.M. Nat. Cell Biol. 2004; 6: 113-120Crossref PubMed Scopus (235) Google which was with its membrane in cell types S. Hsu F.F. S. C. Bohrer A. S. Ma Z. Turk J. 2004; PubMed Scopus Google Scholar, J. Biochem. PubMed Scopus Google Scholar). However, in numerous studies iPLA2β was in J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. J. PubMed Scopus Google Scholar, S. Ma Z. M. G. Ramanadham S. K. Turk J. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google the membrane of Biochem. J. 2002; PubMed Scopus Google and S. Hsu F.F. S. C. Bohrer A. S. Ma Z. Turk J. 2004; PubMed Scopus Google Scholar). is also for its to and S. Hsu F.F. S. C. Bohrer A. S. Ma Z. Turk J. 2004; PubMed Scopus Google Scholar, J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, S. C. S. Turk J. Ma Z. Ramanadham S. 2004; PubMed Google Scholar). The of of iPLA2β isoform J. Ramanadham S. J. Physiol. Pharmacol. 2004; PubMed Scopus Google Scholar) as well as their S. A. Hsu F.F. S. Ramanadham S. Turk J. J. 2004; PubMed Scopus Google Scholar) may be of the for in iPLA2β studies are to which variant of iPLA2β is and for activation of membrane channels and SOCE. Here we test the in the SOCE pathway and new important in CIF production, iPLA2 and CIF-iPLA2-dependent mechanism of activation of ICRAC and Ca2+ entry in cells. basophilic leukemia cells were from and in with and were a of and used for to Ca2+ and RBL cells were on into CIF and iPLA2 activity RBL cells were in lactone and other were from and were from a a specific to the of iPLA2β J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google was a of R. W. recombinant iPLA2β was in and from cells as by W. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cells were with the human recombinant of the cells, iPLA2β was from the by in were for iPLA2 activity as The was of and were by high performance liquid a of to a as Han J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). is important to that and its are suicidal for inhibition is activity of and on of and used T. Zakharov S. Leno E. Csutora P. Trepakova E.S. Bolotina V.M. J. Biol. Chem. 2003; 278: 11909-11915Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). with intact cells the for inhibition of are the intact cells to be not or with for and can be the of the iPLA2 is significantly may the CIF were from human and from cells. CIF were as E.S. Csutora P. Bolotina V.M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). resting from the were and used for of the CIF that no or CIF activity high CIF activity (CIF) were from with depleted Ca2+ stores, which was by to with of for were in with and in of the The was with of for the was and was to the was and the was with with for The was on a and the was with The were and in of The was by through a obtain the were to by the in study were done with CIF RBL cells in were with and with either or for to CIF CIF was from the cells as for human the were in of of the used for and were from with in were for to in standard of the with and CIF were by of into P. Su Z. Kim A. R. Hanley M.R. S. A. PubMed Scopus Google Scholar). were to and with of free in a were of and used for the was into a on the of a in intracellular free Ca2+ were through a a and and a and the CIF was into the the the of the a was to the to test the for Ca2+ and to that the membrane the was and Ca2+ was not CIF was to Ca2+ influx the CIF activity was on the in intracellular Ca2+ as which was as the the after and its RBL cells were of cells were in of by the of of or with of and specific to iPLA2β was used as in our studies T. Zakharov S. Leno E. Csutora P. Trepakova E.S. Bolotina V.M. J. Biol. Chem. 2003; 278: 11909-11915Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). was done the of the after the RBL cells were to and on the were used for after RBL cells were on by in the or presence of in the and The cell was in for and the was for to membrane and The membrane fraction was in the The in was the The were with for and was in in a with of in of was used as a standard in were membrane in a were for with in phosphate-buffered to were with for The was and were for with were with in in and with for were to and the was the The activity of iPLA2 was as (2Smani T. Zakharov S. Csutora P. Leno E. Trepakova E.S. Bolotina V.M. Nat. Cell Biol. 2004; 6: 113-120Crossref PubMed Scopus (235) Google Scholar). after RBL cells were as The activity of iPLA2 was in either cell or in membrane and as in the The iPLA2 activity was a designed for the phospholipase A2 the activity of iPLA2 of phospholipase A2 the were to no Ca2+ is for but not for iPLA2 activity was by the with the for in a of the and The free were by the of for and the was a standard The of activity was in when specific iPLA2 activity was with for and was from the The presence of in the was crucial for the not by activation of Ca2+-independent The specific activity of iPLA2 was in of RBL cells were to and with for the cells were for and to the of the the was to the cells to Ca2+ influx different as Ca2+ were done see was used for studies of RBL cells. The in intracellular Ca2+ were as which was as the the after Ca2+ was and its Ca2+ are of the Ca2+ were from the number of cells cells tested in different from cell were in RBL cells the standard whole-cell as we T. Zakharov S. Leno E. Csutora P. Trepakova E.S. Bolotina V.M. J. Biol. Chem. 2003; 278: 11909-11915Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). was were and were used with of into the the was and to were applied of the current was in The of current was for and for cells are in the are after the current its current with the of into the cell or after CRAC inhibition with was BAPTA, of was to was with exogenous CIF and recombinant iPLA2β, were to intracellular as were are as or test was used to the of the The was of were CIF by RBL cells were used to test the in free Ca2+ in the may production of CIF and the or different effects to depletion (net loss) of Ca2+ from the a depletion of the stores, was used to reticulum Ca2+ Ca2+ and the passive of Ca2+ from the free Ca2+ in the its we used TPEN, a Ca2+ that is known to and free Ca2+ in the C. T. J. Cell Biol. 1998; PubMed Scopus Google Scholar). depletion of the stores with for triggered activation of a SOCE which not when iPLA2 was by its suicidal substrate for produced CIF was from RBL cells, and its biological activity was by the of to activate SOCE when into a for CIF studies P. Su Z. Kim A. R. Hanley M.R. S. A. PubMed Scopus Google Scholar). that CIF from but not cells produced activation of SOCE when into These that CIF is produced by RBL cells when their stores are cells were with for Ca2+ influx was which was to SOCE produced by to SOCE, was on the functional activity of iPLA2 and not in cells. We also that CIF from RBL cells were from produced by when into SOCE the for and SOCE in RBL cells, and the for Ca2+ influx in triggered by of CIF from and RBL cells. of we demonstrated that CIF production is of iPLA2 and is not by inhibition of iPLA2 in SOCE pathway and from iPLA2β; and in and Cell that depletion or of free Ca2+ in the stores of intact RBL cells produced activation of iPLA2, which was with CIF production and biological effects we that iPLA2 could not be by in cell in which Ca2+ stores were and CIF could not be produced We also that iPLA2 functional in cell after cell could be by of exogenous CIF from the cells with depleted stores but not by from cells. CIF could from RBL cells or from other and cell types (for human also that iPLA2 in cell homogenates could be by a with Ca2+ buffering and to be to inhibitory CaM from iPLA2, the physiological of buffering of was used in to that only the activity of Ca2+-independent is was not to iPLA2 activation in RBL cell the activity was of or exogenous CIF of demonstrated that of exogenous CIF to intact cells was with CIF and iPLA2 the intracellular of the The was to which variant of iPLA2 may be present and for phospholipase activity in membrane of RBL cells. and of RBL cells were by and used for and iPLA2 activity that a in iPLA2β human recombinant iPLA2β J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) we used as a in and that is in the membrane but not in the fraction of RBL cells. membrane was by iPLA2 activity of the iPLA2 activity was in the membrane fraction of in the iPLA2β and the corresponding activity from membrane to fraction These demonstrate that 82-kDa iPLA2β is present and active in the membrane of RBL cells. is to a human recombinant iPLA2β; has a molecular and is by the specific to the of that CIF is produced in RBL cells and activates specific iPLA2β that is with we tested intracellular of exogenous CIF could activate CRAC channels in resting RBL cells, when endogenous production of CIF and ICRAC are not We 1) that resting RBL cells not we that ICRAC not a dialysis with current 6) with and 6) with in the However, when exogenous CIF from human was in the a ICRAC and to ICRAC upon passive depletion of the stores cell dialysis with and current and was by The only were and of the current upon CIF ICRAC of its of with of its in from resting no CIF activity, as in was to the ICRAC not and ICRAC in RBL cells could be not only by CIF produced upon store depletion but also by CIF from other cell and applied into of the resting cells with iPLA2β and for of test a iPLA2β is not only but also for CRAC channel activation, were we tested its molecular could ICRAC exogenous iPLA2β could ICRAC when the endogenous is and that of iPLA2β in the of ICRAC, with for CRAC channel CIF dialysis of RBL cells with to iPLA2β also to activate the current was 6) in cells with to iPLA2β with in cells. and we tested exogenous iPLA2β could the endogenous iPLA2β and activation of RBL cells were with to iPLA2β the of to of the see the in and with a human recombinant iPLA2β J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google which has a CaM and can be by the as endogenous iPLA2β in of the recombinant iPLA2β into the to be to activation of ICRAC and that was in the cells in which endogenous iPLA2β was of ICRAC with recombinant iPLA2β was also in RBL cells in which functional activity of endogenous iPLA2β was by be in ICRAC was 5) in the and 5) in the presence of in the when cells were with These that the presence and functional activity of iPLA2β is and for activation of ICRAC in RBL cells. iPLA2β of ICRAC to and J. Physiol. 1998; PubMed Scopus Google Scholar) demonstrated that ICRAC is to intracellular pH and that pH may in the with the with a recombinant iPLA2β we that its activity highly on intracellular These iPLA2 may be a pH that the dependence of ICRAC activation on intracellular pH in RBL cells. test we intracellular pH dependence of ICRAC and iPLA2β and a that the of ICRAC to when intracellular pH and pH was to the pH dependence of iPLA2β which to be active pH and but demonstrated a loss of when pH was from to These demonstrate a of of ICRAC and SOCE regulation of the activity of iPLA2β, not only a crucial determinant of SOC channel activation but also important target for of the ICRAC and SOCE (S)-BEL, a for the of iPLA2β and ICRAC in Ca2+ and Han J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) demonstrated that used suicidal substrate for major of iPLA2 J. Biol. Chem. Full Text PDF PubMed Google is of (S)-BEL, which has to iPLA2β, and (R)-BEL, which is specific to Here we to test the effects of on the activity of iPLA2β, and the activation of ICRAC and SOCE in RBL cells to could be used as pharmacological for the studies of the role of iPLA2β and store-operated channels in Ca2+ entry. we and that but not inhibits activation of iPLA2 with iPLA2β but not by depletion of Ca2+ stores in RBL cells. we tested the effects of and on ICRAC and SOCE. and that but not inhibits ICRAC in RBL cells. Ca2+ influx was also effectively by but not in that inhibits Ca2+ influx with or no inhibition of iPLA2 activity and SOCE by and not was also in vascular smooth muscle cells not These demonstrate that (S) enantiomer of could be used as a highly specific pharmacological to ICRAC, and SOCE. study tested and the and mechanism of CRAC channel activation in cells. We new important information on the for endogenous CIF production, the of CIF, and its biological activity different dependence of ICRAC activation on the presence and functional activity of a specific 82-kDa variant of iPLA2β, and new pharmacological for SOCE studies. We demonstrated that CIF is produced and can be from RBL cells after depletion of their stores, and its biological activity is to the activity of CIF from other cell types V.M. Csutora P. Biochem. 2005; Full Text Full Text PDF PubMed Scopus Google which that CIF is highly and may be the from cells and into exogenous CIF to the endogenous produced the activation of iPLA2β and exogenous CIF was active only upon intracellular 1) iPLA2β when to cell homogenates but not to intact cells, and 2) intracellular CIF dialysis was to activate exogenous CIF was to activate ICRAC in resting cells in which Ca2+ stores were not and ICRAC not upon dialysis with CIF produced activation of ICRAC is upon cell dialysis with BAPTA, as one from CIF iPLA2, which is only one from activation of the to the stores and CIF production of CIF to activate ICRAC in RBL cells in which iPLA2β was that iPLA2β is a physiological target for CIF and of CRAC channel The studies in RBL cells new important information on a specific variant of iPLA2β that is and for activation of CRAC channels as well as its and intracellular and The of of iPLA2β J. Ramanadham S. J. Physiol. Pharmacol. 2004; PubMed Scopus Google Scholar) may important role in of iPLA2β and be one of the for the variety of on its and role in Our that a variant of iPLA2β in may within specific or in to for the SOCE Our studies that is to the membrane and could be and its functional activity in membrane E.S. Csutora P. Bolotina V.M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google CIF to the intracellular of membrane was to activate in smooth muscle cells, and CaM to iPLA2 in could Here we demonstrated its with 82-kDa iPLA2β could be by and by other that of iPLA2β with membrane one of of regulation of SOCE in cells. Our also that are to iPLA2β in the membrane fraction after cell and may iPLA2β was in the in studies. of in study the of and the of its target in the store-operated we demonstrated that by mimics the effects of molecular of iPLA2β on not CIF production, which is from iPLA2, and not activation of SOCE by lysophospholipids (2Smani T. Zakharov S. Csutora P. Leno E. Trepakova E.S. Bolotina V.M. Nat. Cell Biol. 2004; 6: 113-120Crossref PubMed Scopus (235) Google of we demonstrated that only of the chiral of inhibits activation of iPLA2, ICRAC, and SOCE in RBL cells. may be used as a highly specific pharmacological for studies of Ca2+ in cells and in which molecular with iPLA2β and SOC channels may Our studies on the of the signal in that may CIF not only the to into a of CIF production but also the major of is a signal for SOCE. studies we that simple buffering of free Ca2+ within the stores triggers CIF production and activation of SOCE in RBL cells, which was from that triggered by a Ca2+ loss from a in free Ca2+ a for CIF production and SOCE our studies in RBL cells significantly our understanding of the crucial role of CIF and the specific variant of iPLA2β in activation and physiological regulation of CRAC channels and SOCE. We R. W. and C. M. for the specific the of iPLA2β and in of recombinant iPLA2β and of (S) and