Activation Mechanism for CRAC Current 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. 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 H.Y. Bugrim A. Cunningham K.W. Nuccitelli R. Keizer J.E. Hanley M.R. Blalock J.E. Marchase R.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 121-126Crossref PubMed Scopus (103) Google Scholar, 13Trepakova E.S. Csutora P. Marchase R.B. Cohen R.A. Bolotina V.M. J. Biol. Chem. 2000; 275: 26158-26163Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). For a complete review of the present knowledge related to CIF we will refer the readers to specialized reviews (1Parekh A.B. Putney Jr., J.W. Physiol. Rev. 2005; 85: 757-810Crossref PubMed Scopus (1807) Google Scholar, 14Bolotina V.M. Csutora P. Trends Biochem. Sci. 2005; 30: 378-387Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Here we will concentrate on some new questions, such as what is the physiological signal that triggers CIF production, how ubiquitous is production of CIF, and how interchangeable is its biological activity between different species. It is generally thought that activation of SOCE is triggered by a net Ca2+ loss from ER (for review, see Refs. 1Parekh A.B. Putney Jr., J.W. Physiol. Rev. 2005; 85: 757-810Crossref PubMed Scopus (1807) Google Scholar and 15Elliott A.C. Cell Calcium. 2001; 30: 73-93Crossref PubMed Scopus (104) Google Scholar). However, another attractive possibility exists that a simple reduction in free Ca2+ concentration within the stores (without physical Ca2+ loss) may be enough for triggering SOCE in a dose-dependent manner (16Hofer A.M. Fasolato C. Pozzan T. J. Cell Biol. 1998; 140: 325-334Crossref PubMed Scopus (205) Google Scholar). It is presently unknown what kind of changes in intraluminal Ca2+ may be sufficient for triggering CIF production; does it necessarily need to be a Ca2+ loss (as widely believed), or could it be a simple reduction in free Ca2+ concentration in the stores. If the later is true, CIF production and SOCE activation may be a natural response of the cell not only to agonist-induced discharge of Ca2+ from the stores but also to variations in free Ca2+ in the stores due to pathological changes in the expression levels of intraluminal Ca2+ binding proteins, such as calsequestrin and calreticulin (for review, see Refs. 17Meldolesi J. Pozzan T. Trends Biochem. Sci. 1998; 23: 10-14Abstract Full Text PDF PubMed Scopus (454) Google Scholar and 18Ma J. Pan Z. Cell Calcium. 2003; 33: 375-384Crossref PubMed Scopus (51) Google Scholar). Here we tested if simple Ca2+ buffering in the stores may or may not trigger CIF production and how it may relate to CIF produced upon physical Ca2+ loss. We also tested if CIF produced by different types of cells and species could be interchangeable and if exogenously applied CIF can mimic the effects of endogenous CIF in activating iPLA2 and SOC. It is now well established that iPLA2 (group VI encoded by PLA2G6) is not a single, but a growing family of enzymes (19Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1223) Google Scholar, 20Winstead M.V. Balsinde J. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 28-39Crossref PubMed Scopus (222) Google Scholar, 21Jenkins C.M. Mancuso D.J. Yan W. Sims H.F. Gibson B. Gross R.W. J. Biol. Chem. 2004; 279: 48968-48975Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar). Two major isoforms of iPLA2 have been identified and intensively studied (for reviews, see Ref. 20Winstead M.V. Balsinde J. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 28-39Crossref PubMed Scopus (222) Google Scholar and 22Jenkins C.M. Han X. Mancuso D.J. Gross R.W. J. Biol. Chem. 2002; 277: 32807-32814Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar): iPLA2β (group VIA) and iPLA2γ (group VIB). The signature feature of iPLA2β is that it contains CaM binding domain, making it possible for CaM to bind and inhibit its activity (23Wolf M.J. Gross R.W. J. Biol. Chem. 1996; 271: 20989-20992Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 24Jenkins C.M. Wolf M.J. Mancuso D.J. Gross R.W. J. Biol. Chem. 2001; 276: 7129-7135Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). This unique feature discriminates iPLA2β from other iPLA2s and provides a perfect design 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). Another prominent feature of some variants of iPLA2β is multiple ankyrin repeats in the N terminus, which may be important for their cellular localization and assembly into specific signaling domains. In vascular smooth muscle cells we found that iPLA2β remains functional at the inner leaflet of excised plasma membrane patches (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), which was consistent with its plasma membrane localization reported in some cell types (25Ramanadham S. Hsu F.F. Zhang S. Jin C. Bohrer A. Song H. Bao S. Ma Z. Turk J. Biochemistry. 2004; 43: 918-930Crossref PubMed Scopus (92) Google Scholar, 26Larsson Forsell P.K.A. Kennedy B.P. Claesson H.E. Eur. J. Biochem. 1999; 262: 575-585Crossref PubMed Scopus (119) Google Scholar). However, in numerous studies iPLA2β was found in cytosol (27Hazen S.L. Stuppy R.J. Gross R.W. J. Biol. Chem. 1990; 265: 10622-10630Abstract Full Text PDF PubMed Google Scholar, 28Yang H.C. Mosior M. Ni B. Dennis E.A. J. Neurochem. 1999; 73: 1278-1287Crossref PubMed Scopus (110) Google Scholar, 29Bao S. Miller D.J. Ma Z. Wohltmann M. Eng G. Ramanadham S. Moley K. Turk J. J. Biol. Chem. 2004; 279: 38194-38200Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), the inner membrane of mitochondria (30Williams S.D. Gottlieb R.A. Biochem. J. 2002; 362: 23-32Crossref PubMed Scopus (111) Google Scholar), and nucleus (25Ramanadham S. Hsu F.F. Zhang S. Jin C. Bohrer A. Song H. Bao S. Ma Z. Turk J. Biochemistry. 2004; 43: 918-930Crossref PubMed Scopus (92) Google Scholar). There is also some evidence for its possible translocation to perinuclear and plasma membranes (25Ramanadham S. Hsu F.F. Zhang S. Jin C. Bohrer A. Song H. Bao S. Ma Z. Turk J. Biochemistry. 2004; 43: 918-930Crossref PubMed Scopus (92) Google Scholar, 31Tay H.K. Melendez A.J. J. Biol. Chem. 2004; 279: 22505-22513Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 32Bao S. Jin C. Zhang S. Turk J. Ma Z. Ramanadham S. Diabetes. 2004; 53: 186-189Crossref PubMed Google Scholar). The existence of multiple splice variants of iPLA2β isoform (33Turk J. Ramanadham S. Can. J. Physiol. Pharmacol. 2004; 82: 824-832Crossref PubMed Scopus (31) Google Scholar) as well as their post-translational modifications (34Song H. Hecimovic S. Goate A. Hsu F.F. Bao S. Vidavsky I. Ramanadham S. Turk J. J. Am. Soc. Mass Spectrom. 2004; 15: 1780-1793Crossref PubMed Scopus (16) Google Scholar) may be some of the reasons for inconsistence in iPLA2β localization. Further studies are necessary to determine which splice variant of iPLA2β is required and sufficient for activation of plasma membrane channels and SOCE. Here we test the main steps in the CIF-mediated iPLA2-dependent SOCE pathway and unveil new important details in CIF production, iPLA2 specificity, and CIF-iPLA2-dependent mechanism of activation of ICRAC and capacitative Ca2+ entry in RBL-2H3 cells. Rat basophilic leukemia RBL-2H3 cells were obtained from ATCC and maintained in minimum essential medium supplemented with 2 mml-glutamine and 1% penicillin/streptomycin (10,000 IU/10,000 μg/ml). Cells were passed every 3 days at a ratio of 1:5 and used for up to 12 passages. For Ca2+ imaging and patch clamp experiments, RBL cells were grown on small coverslips (∼5 × 5 mm) placed into 6-well plates. For CIF preparations, Western blots, and iPLA2 activity measurements, RBL cells were grown in 10-cm tissue culture dishes. Bromoenol lactone (BEL), thapsigargin (TG), N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN), and most other drugs were purchased from Sigma. Fura-2 and fura-2 AM were from Invitrogen. Anti-iPLA2β, a polyclonal antiserum against a 19-amino acid peptide (NQIHSKDPRYGASPLHWAK) specific to the ankyrin region of iPLA2β (24Jenkins C.M. Wolf M.J. Mancuso D.J. Gross R.W. J. Biol. Chem. 2001; 276: 7129-7135Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), was a generous gift of Dr. R. W. Gross. Human recombinant iPLA2β (rec-iPLA2β) was expressed in and purified from Sf9 cells as previously described by Dr. Gross (21Jenkins C.M. Mancuso D.J. Yan W. Sims H.F. Gibson B. Gross R.W. J. Biol. Chem. 2004; 279: 48968-48975Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar, 35Wolf M.J. Gross R.W. J. Biol. Chem. 1996; 271: 30879-30885Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Briefly, Sf9 cells were infected with baculovirus harboring the human recombinant iPLA2βHis6. After lysis of the cells, iPLA2β was purified from the cytosol using Co2+ affinity chromatography followed by ATP-agarose chromatography. Column fractions in each step were assayed for iPLA2 activity as described below. The final rec-iPLA2β concentration was typically 0.15–0.18 μg/μl. Chiral enantiomers of BEL ((S)- and (R)-BEL)) were separated by high performance liquid chromatography (HPLC) utilizing a Chirex column of 3,5-dinitrobenzoyl-(R)-phenylglycine attached to a silica matrix (Phenomenex) as previously described (22Jenkins C.M. Han X. Mancuso D.J. Gross R.W. J. Biol. Chem. 2002; 277: 32807-32814Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). It is important to notice that BEL and its enantiomers are suicidal substrates for iPLA2; inhibition is irreversible, requires basal activity of this enzyme, and strongly depends on temperature, duration of treatment, and concentration used (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). In experiments with intact cells the optimal conditions for BEL treatment (to ensure complete inhibition of iPLA2) are the following; intact cells need to be pretreated (in bath solution not containing bovine serum albumin or serum) with 10–25 μm BEL for 30 min at 37 °C, and then BEL can be washed away before the beginning of the experiments. In cases where iPLA2 is already active, significantly shorter (1–5 min) treatments may fully inhibit the enzyme. CIF extracts were purified from human platelets and from RBL-2H3 cells. Human Platelets—Crude CIF extracts were obtained as described before (13Trepakova E.S. Csutora P. Marchase R.B. Cohen R.A. Bolotina V.M. J. Biol. Chem. 2000; 275: 26158-26163Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Briefly, resting (unstimulated) platelets (obtained from the local Red Cross) were kept at room temperature and used immediately for preparation of the control CIF extracts that contained no or very little CIF activity (control extract). Extracts containing high CIF activity (CIF) were prepared from platelets with depleted Ca2+ stores, which was achieved by exposing them to cold (4 °C) overnight, with subsequent application of TG (2 μm for 20 min). To prepare crude extracts, platelets (50 ml per platelet pack, about 1011 cells) were washed in Hanks' balanced salt solution supplemented with 20 mm HEPES (20 ml) and resuspended in 0.85 ml of the same solution. The suspension was extracted with 0.2 ml of 1 m hydrochloric acid for 30 min at room temperature. After centrifugation, the supernatant was neutralized (10 m NaOH), and BaCl2 (10 mm) was added to precipitate compounds containing vicinal phosphates, including inositol 1,4,5-trisphosphate. After centrifugation the supernatant was lyophilized, and the residue was extracted with methanol (0.8 ml) with continuous mixing for 15 min. The methanol extract was loaded on a Sep-Pak Vac C18 cartridge (Waters), and the cartridge was washed with methanol (0.8 ml). The combined methanol elutes were dried at 30 °C under N2 gas and resuspended in 200 μl of 100 mm acetic acid. The reconstituted extract was clarified by centrifugal ultrafiltration through a Ultrafree-MC 30-kDa filter (Millipore). To obtain fine extracts, the crude extracts were further subjected to anion exchange HPLC followed by reversed-phase HPLC. Unless specified, all the experiments in this study were done with crude CIF extracts. RBL Cells—RBL cells grown in 10-cm culture dishes were washed with serum-free media and then treated with either 1 μm TG or 1 mm TPEN for 5 min at 37 °C to initiate CIF production. CIF was prepared from the cells exactly as described above for human platelets; however, the final dried methanol elutes were reconstituted in 50 μl of 100 mm acetic acid instead of the 200 μl used for platelets. Stage V and VI oocytes were harvested from albino Xenopus laevis frogs (Xenopus Express) under anesthesia with 3-aminobenzoic acid ethyl ester (MS-222, Sigma). After defolliculation in 2 mg/ml collagenase A (Roche Applied Science), oocytes were maintained for up to 5 days in standard ND-96 oocyte media of the following composition: 96 mm NaCl, 5 mm HEPES, 1.8 mm CaCl2, 2 mm KCl, 1 mm MgCl2 (pH 7.5) supplemented with 2.5 mm pyruvate, 50 μg/ml gentamycin, and 5% horse serum. CIF activities were assayed by microinjection of extracts into fura-2-loaded albino oocytes (12Csutora P. Su Z. Kim H.Y. Bugrim A. Cunningham K.W. Nuccitelli R. Keizer J.E. Hanley M.R. Blalock J.E. Marchase R.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 121-126Crossref PubMed Scopus (103) Google Scholar). Oocytes were transferred to Ca2+-free oocyte Ringer (OR-2) buffer solution (82 mm NaCl, 5 mm HEPES, 5 mm MgCl2, 2 mm KCl (pH 7.5)) and microinjected with 14 nl of fura-2 free acid (1 mm in 125 mm KCl) using a Nanoject-II injector (Drummond). Injected oocytes were allowed at least 2 h of recovery and kept at 4 °C until used for the bioassay. Next, an individual oocyte was transferred into a homemade imaging chamber containing Ca2+-free OR-2 solution, mounted on the stage of a Nikon Eclipse TS-100 inverted microscope. Changes in intracellular free Ca2+ were measured through a Nikon 20× Super Fluor objective (NA = 0.75) using a rapid excitation filter changer alternating between 340 and 380 nm (Sutter Instruments) and a CCD camera (Cooke PixelFly) and analyzed using the InCyt IM2 software (Intracellular Imaging). A micropipette containing CIF extract was inserted into the oocyte around the equatorial plane before the beginning of the recording. After obtaining a stable recording base line, CaCl2 (5 mm) was added to the extracellular bath solution to first test the oocytes for nonspecific Ca2+ leak and to verify that the membrane seal around the pipette was tight and Ca2+ was not leaking in. About 1 min later CIF extract (28 nl) was injected to trigger Ca2+ influx across the oocyte membrane. CIF activity was determined based on the changes in intracellular Ca2+ (expressed as ΔRatio), which was calculated as the difference between the peak F340/F380 ratio (6 min after injection), and its basal level right before injection. RBL cells were transfected using Nucleofector II (Amaxa Biosystems). Batches of 1.5 × 106 cells were resuspended in 100 μl of Nucleofector solution R at room temperature followed by the addition of 2 μg of antisense or sense DNA together with 2 μg of green fluorescent protein. A 20-base-long antisense (5′-fluorescein-CTCCTTCACCCGGAATGGGT-3′) and sense (5′-fluorescein-ACCCATTCCGGGTGAAGGAG-3′) specific to iPLA2β was used as in our previous studies (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). Transfection was done at the T-20 setting of the Nucleofector II device. Immediately after transfection, the RBL cells were transferred to minimum essential medium and plated on the coverslips. Cells showing green protein fluorescence (excitation at 480 nm, emission at 515 nm) were used for experiments 38 ± 4 h after transfection. RBL cells were homogenized on ice by sonication in the absence or presence of 1% Triton X-100 in the homogenization buffer containing 300 mm sucrose and 10 mm Tris-HCl (pH 7.0). The cell homogenate was centrifuged in an Eppendorf centrifuge at 14,000 × rpm for 10 min, and the supernatant was further centrifuged at 100,000 × g for 1 h to separate membrane and cytosol fractions. The membrane fraction was resuspended in the same homogenization buffer. The total protein amount in each sample was determined using the Bio-Rad protein dye reagent (Bradford method). The protein samples were incubated with Laemmli sample buffer at 95 °C for 2 min, and then one-dimensional protein gel electrophoresis was performed in 7.5% SDS-PAGE gels in a Mini-Protein system (Bio-Rad) with 30 μg of total protein loaded in each lane. Rec-iPLA2β of 84 kDa was used as a standard (0.5–1.0 ng) in all Western blots. Separated proteins were electrophoretically transferred overnight onto nitrocellulose membrane in a Mini Trans-blot system (Bio-Rad). Blots were incubated for 1 h with 5% (w/v) skim milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBST) to block residual protein binding sites. Blocked membranes were then incubated with primary anti-iPLA2β (1:2000 dilution) for 2 h at room temperature. The primary antibody was removed, and blots were washed 3 times for 10 min with milk/PBST. Then blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Cell Signaling) diluted 1:2000 in milk/PBST, washed 3 times in PBST, and treated with enhanced chemiluminescence reagents (Super ECL, Pierce) for 1 min. Blots were then exposed to photographic films, and the optical density was determined using the Un-scan-it analysis software (Silk Scientific). The activity of iPLA2 was determined as previously described (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). Briefly, after each experimental treatment, RBL cells were homogenized as described above. The activity of iPLA2 was determined in either total cell homogenate or in membrane and cytosol fractions, as specified in the figures. The iPLA2 activity was measured using a modified commercial assay kit originally designed for the cytosolic phospholipase A2 (Cayman). To detect the activity of iPLA2 instead of cytosolic phospholipase A2 (cPLA2), the assay buffers were modified to contain no Ca2+ (Ca2+ is needed for cPLA2 but not for iPLA2 activity). Phospholipase activity was assayed by incubating the samples with the substrate, 1-hexadecyl-2-arachidonoylthio-2-deoxy-sn-glycero-3-phosphorylcholine for 1 h at room temperature in a modified Ca2+-free assay buffer of the following composition 300 mm NaCl, 60% glycerol, 10 mm HEPES, 8 mm Triton X-100, 4 mm EGTA, and 2 mg/ml bovine serum albumin (pH 7.4). The generated free thiols were visualized by the addition of 5,5′-dithiobis(2-dinitrobenzoic acid) for 5 min, and the absorbance was determined at 405 nm using a standard microplate reader. The background iPLA2-independent component of basal lipase activity was determined in control samples when all specific iPLA2 activity was inhibited with (S)-BEL (10 μm for 5 min) and was subtracted from all the readings. The presence of 4 mm EGTA in the assay buffer (which was crucial for suppressing the contaminant Ca2+-dependent cPLA2 activity) did not by itself cause any significant activation of Ca2+-independent BEL-sensitive iPLA2. The specific activity of iPLA2 was expressed in absorbance/mg of protein units. RBL cells were transferred to minimum essential culture medium without serum and loaded with fura-2 AM (2 μm) for 30 min at 37 °C. Then the cells were washed for 10 min and transferred to the bath solution of the following composition: 140 mm NaCl, 10 mm HEPES, 1 mm MgCl2, 0.1 mm EGTA (pH 7.4). During the experiment, CaCl2 (2 mm) was added to the cells to observe Ca2+ influx following different treatments as described under "Results." Ca2+ measurements were done at 20–22 °C. A dual-excitation fluorescence imaging system (Intracellular Imaging, see description above) was used for studies of individual RBL cells. The changes in intracellular Ca2+ were expressed as ΔRatio, which was calculated as the difference between the peak F340/F380 ratio after extracellular Ca2+ was added and its level right before Ca2+ addition. Summary data are shown without subtraction of the basal Ca2+ influx. Data were summarized from the large number of individual cells (20–40 cells tested each in 3–6 different experiments from at least 3 cell preparations). Whole-cell currents were recorded in RBL cells using the standard whole-cell (dialysis) patch clamp technique as we previously described (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). Axopatch 200B amplifier was used; data were digitized at 5 kHz and filtered at 1 kHz. Pipettes were used with tip resistance of 2–4 megaohms. After breaking into the cell, the holding potential was 0 mV, and ramp depolarizations (from –100 to +100 mV, 200 ms) were applied every 3 s. Amplitude of the current was expressed in pA/pF. The time course of current development was analyzed at –80 mV for each individual cell, and summary data for 5+ cells are shown in the figures. Average I/V relationships are shown during ramp depolarization after the current reached its maximum. Passive leakage current with zero reversal potential (at the moment of breaking into the cell or after CRAC inhibition with 10 μm diethylstilbestrol) was subtracted. Intracellular (pipette) solution contained 145 mm cesium glutamate, 10 mm BAPTA, 10 mm HEPES, 3 mm MgCl2 (pH 7.2). In some experiments concentration of BAPTA was reduced to 0.1–1 mm. Extracellular solution was 130 mm NaCl, 20 mm CaCl2, 5 mm HEPES, 3 mm CsCl, 1 mm MgC