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• W2124353324 abstract "Store-operated channels (SOCs) provide an important means for mediating longer-term Ca2+ signals and replenishment of Ca2+stores in a multitude of cell types. However, the coupling mechanism between endoplasmic reticulum stores to activate plasma membrane SOCs remains unknown. In DT40 chicken B lymphocytes, the permeant inositol trisphosphate receptor (InsP3R) modifier, 2-aminoethoxydiphenyl borate (2-APB), was a powerful activator of store-operated Ca2+ entry between 1–10 μm. 2-APB activated authentic SOCs because the entry was totally selective for Ca2+ (no detectable entry of Ba2+ or Sr2+ ions), and highly sensitive to La3+ ions (IC50 30–100 nm). To assess the role of InsP3Rs in this response, we used the DT40 triple InsP3R-knockout (ko) cell line, DT40InsP3R-ko, in which the absence of full-length InsP3Rs or InsP3R fragments was verified by Western analysis using antibodies cross-reacting with N-terminal epitopes of all three chicken InsP3R subtypes. The 2-APB-induced activation of SOCs was identical in the DT40InsP3R-ko, cells indicating InsP3Rs were not involved. With both wild type (wt) and ko DT40 cells, 2-APB had no effect on Ca2+ entry in store-replete cells, indicating that its action was restricted to SOCs in a store-coupled state. 2-APB induced a robust activation of Ca2+ release from stores in intact DT40wt cells but not in DT40InsP3R-ko cells, indicating an InsP3R-mediated effect. In contrast, 2-APB blocked InsP3Rs in permeabilized DT40wt cells, suggesting that the stimulatory action of 2-APB was restricted to functionally coupled InsP3Rs in intact cells. Uncoupling of ER/PM interactions in intact cells by calyculin A-induced cytoskeletal rearrangement prevented SOC activation by store-emptying and 2-APB; this treatment completely prevented 2-APB-induced InsP3R activation but did not alter InsP3R activation mediated by phospholipase C-coupled receptor stimulation. The results indicate that the robust bifunctional actions of 2-APB on both SOCs and InsP3Rs are dependent on the coupled state of these channels and suggest that 2-APB may target the coupling machinery involved in mediating store-operated Ca2+ entry. Store-operated channels (SOCs) provide an important means for mediating longer-term Ca2+ signals and replenishment of Ca2+stores in a multitude of cell types. However, the coupling mechanism between endoplasmic reticulum stores to activate plasma membrane SOCs remains unknown. In DT40 chicken B lymphocytes, the permeant inositol trisphosphate receptor (InsP3R) modifier, 2-aminoethoxydiphenyl borate (2-APB), was a powerful activator of store-operated Ca2+ entry between 1–10 μm. 2-APB activated authentic SOCs because the entry was totally selective for Ca2+ (no detectable entry of Ba2+ or Sr2+ ions), and highly sensitive to La3+ ions (IC50 30–100 nm). To assess the role of InsP3Rs in this response, we used the DT40 triple InsP3R-knockout (ko) cell line, DT40InsP3R-ko, in which the absence of full-length InsP3Rs or InsP3R fragments was verified by Western analysis using antibodies cross-reacting with N-terminal epitopes of all three chicken InsP3R subtypes. The 2-APB-induced activation of SOCs was identical in the DT40InsP3R-ko, cells indicating InsP3Rs were not involved. With both wild type (wt) and ko DT40 cells, 2-APB had no effect on Ca2+ entry in store-replete cells, indicating that its action was restricted to SOCs in a store-coupled state. 2-APB induced a robust activation of Ca2+ release from stores in intact DT40wt cells but not in DT40InsP3R-ko cells, indicating an InsP3R-mediated effect. In contrast, 2-APB blocked InsP3Rs in permeabilized DT40wt cells, suggesting that the stimulatory action of 2-APB was restricted to functionally coupled InsP3Rs in intact cells. Uncoupling of ER/PM interactions in intact cells by calyculin A-induced cytoskeletal rearrangement prevented SOC activation by store-emptying and 2-APB; this treatment completely prevented 2-APB-induced InsP3R activation but did not alter InsP3R activation mediated by phospholipase C-coupled receptor stimulation. The results indicate that the robust bifunctional actions of 2-APB on both SOCs and InsP3Rs are dependent on the coupled state of these channels and suggest that 2-APB may target the coupling machinery involved in mediating store-operated Ca2+ entry. Ca2+ signals control a vast array of cellular functions ranging from short-term responses such as contraction and secretion to longer-term regulation of cell growth and proliferation (1Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1752) Google Scholar, 2Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4319) Google Scholar). The cytosolic Ca2+ signals generated in response to receptors are complex involving two closely coupled components: rapid, transient release of Ca2+ stored in the endoplasmic reticulum (ER) 1ERendoplasmic reticulumTRPtransient receptor potentialInsP3Rinositol 1,4,5-trisphosphate receptorfura-2/AMfura-2 acetoxymethylester2-APB2-aminoethoxydiphenyl borateSOCstore-operated channelkoknockoutwtwild typeICMintracellular-like medium 1ERendoplasmic reticulumTRPtransient receptor potentialInsP3Rinositol 1,4,5-trisphosphate receptorfura-2/AMfura-2 acetoxymethylester2-APB2-aminoethoxydiphenyl borateSOCstore-operated channelkoknockoutwtwild typeICMintracellular-like medium followed by slowly developing extracellular Ca2+ entry (1Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1752) Google Scholar, 3Putney J.W. Bird G.S. Cell. 1993; 75: 199-201Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 4Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2250) Google Scholar, 5Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1284) Google Scholar, 6Putney J.W. McKay R.R. Bioessays. 1999; 21: 38-46Crossref PubMed Scopus (357) Google Scholar, 7Putney J.W. Ribeiro C.M. Cell Mol. Life Sci. 2000; 57: 1272-1286Crossref PubMed Google Scholar). G protein-coupled receptors and tyrosine kinase receptors, through activation of phospholipase C, generate the second messenger, InsP3, which diffuses rapidly within the cytosol to interact with InsP3Rs on the ER; the InsP3Rs serve as Ca2+ channels to release luminally stored Ca2+ and generate the initial Ca2+ signal phase (1Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1752) Google Scholar, 4Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2250) Google Scholar). The resulting depletion of Ca2+ stored within the ER lumen serves as the primary trigger for a message that is returned to the plasma membrane resulting in the slow activation of “store-operated channels” (SOCs), which mediate capacitative Ca2+ entry (3Putney J.W. Bird G.S. Cell. 1993; 75: 199-201Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 5Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1284) Google Scholar, 6Putney J.W. McKay R.R. Bioessays. 1999; 21: 38-46Crossref PubMed Scopus (357) Google Scholar, 7Putney J.W. Ribeiro C.M. Cell Mol. Life Sci. 2000; 57: 1272-1286Crossref PubMed Google Scholar, 8Gill D.L. Waldron R.T. Rys-Sikora K.E. Ufret-Vincenty C.A. Graber M.N. Favre C.J. Alfonso A. Biosci. Rep. 1996; 16: 139-157Crossref PubMed Scopus (67) Google Scholar). This second Ca2+ entry phase of Ca2+ signals serves to mediate longer-term cytosolic Ca2+ elevations and provides a means of replenishing intracellular stores (3Putney J.W. Bird G.S. Cell. 1993; 75: 199-201Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 5Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1284) Google Scholar). The mechanism for coupling ER Ca2+ store depletion with Ca2+entry remains a crucial but unresolved question (5Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1284) Google Scholar, 6Putney J.W. McKay R.R. Bioessays. 1999; 21: 38-46Crossref PubMed Scopus (357) Google Scholar, 7Putney J.W. Ribeiro C.M. Cell Mol. Life Sci. 2000; 57: 1272-1286Crossref PubMed Google Scholar, 8Gill D.L. Waldron R.T. Rys-Sikora K.E. Ufret-Vincenty C.A. Graber M.N. Favre C.J. Alfonso A. Biosci. Rep. 1996; 16: 139-157Crossref PubMed Scopus (67) Google Scholar). endoplasmic reticulum transient receptor potential inositol 1,4,5-trisphosphate receptor fura-2 acetoxymethylester 2-aminoethoxydiphenyl borate store-operated channel knockout wild type intracellular-like medium endoplasmic reticulum transient receptor potential inositol 1,4,5-trisphosphate receptor fura-2 acetoxymethylester 2-aminoethoxydiphenyl borate store-operated channel knockout wild type intracellular-like medium Coupling to activate SOCs has been hypothesized to involve direct coupling between the ER and plasma membrane (9Irvine R.F. FEBS Lett. 1990; 263: 5-9Crossref PubMed Scopus (576) Google Scholar, 10Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1045) Google Scholar), and evidence indicates that physical docking of the ER with the plasma membrane may be involved in SOC activation (11Patterson R.L. van Rossum D.B. Gill D.L. Cell. 1999; 98: 487-499Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 12Putney J.W. Cell. 1999; 99: 5-8Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 13Yao Y. Ferrer-Montiel A.V. Montal M. Tsien R.Y. Cell. 1999; 98: 475-485Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 14Rosado J.A. Jenner S. Sage S.O. J. Biol. Chem. 2000; 275: 7527-7533Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Recent evidence has indicated that the InsP3R may be a component in mediating the coupling process between stores and Ca2+ entry channels. In particular, a role for the InsP3R has been implicated in the activation of the TRP family of receptor-activated channels, which share a number of functional parameters with SOCs including reports that they are store-operated (15Birnbaumer L. Zhu X. Jiang M. Boulay G. Peyton M. Vannier B. Brown D. Platano D. Sadeghi H. Stefani E. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15195-15202Crossref PubMed Scopus (352) Google Scholar, 16Philipp S. Cavalié A. Freichel M. Wissenbach U. Zimmer S. Trost C. Marquart A. Murakami M. Flockerzi V. EMBO J. 1996; 15: 6166-6171Crossref PubMed Scopus (255) Google Scholar, 17Kiselyov K.I., Xu, X. Mohayeva G. Kuo T. Pessah I.N. Mignery G.A. Zhu X. Birnbaumer L. Muallem S. Nature. 1998; 396: 478-482Crossref PubMed Scopus (557) Google Scholar, 18Philipp S. Hambrecht J. Braslavski L. Schroth G. Freichel M. Murakami M. Cavalie A. Flockerzi V. EMBO J. 1998; 17: 4274-4282Crossref PubMed Scopus (269) Google Scholar, 19Vannier B. Peyton M. Boulay G. Brown D. Qin N. Jiang M. Zhu X. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2060-2064Crossref PubMed Scopus (234) Google Scholar, 20Philipp S. Trost C. Warnat J. Rautmann J. Himmerkus N. Schroth G. Kretz O. Nastainczyk W. Cavalie A. Hoth M. Flockerzi V. J. Biol. Chem. 2000; 275: 23965-23972Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 21Liu X. Wang W. Singh B.B. Lockwich T. Jadlowiec J. O' Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Evidence from reconstitution studies has indicated a direct functional communication between TRP channels and InsP3Rs (17Kiselyov K.I., Xu, X. Mohayeva G. Kuo T. Pessah I.N. Mignery G.A. Zhu X. Birnbaumer L. Muallem S. Nature. 1998; 396: 478-482Crossref PubMed Scopus (557) Google Scholar, 22Putney J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14669-14671Crossref PubMed Scopus (136) Google Scholar, 23Boulay G. Brown D.M. Qin N. Jiang M. Dietrich A. Zhu M.X. Chen Z. Birnbaumer M. Mikoshiba K. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14955-14960Crossref PubMed Scopus (345) Google Scholar, 24Birnbaumer L. Boulay G. Brown D. Jiang M. Dietrich A. Mikoshiba K. Zhu X. Qin N. Recent Prog. Horm. Res. 2000; 55: 127-161PubMed Google Scholar), and a number of reports have revealed a physical interaction between TRP channels and the InsP3R (24Birnbaumer L. Boulay G. Brown D. Jiang M. Dietrich A. Mikoshiba K. Zhu X. Qin N. Recent Prog. Horm. Res. 2000; 55: 127-161PubMed Google Scholar, 25Kiselyov K.I. Mignery G.A. Zhu M.X. Muallem S. Mol. Cell. 1999; 4: 423-429Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 26Rosado J.A. Sage S.O. Biochem. J. 2000; 350: 631-635Crossref PubMed Scopus (160) Google Scholar, 27Mery L. Magnino F. Schmidt K. Krause K.H. Dufour J.F. FEBS Lett. 2001; 487: 377-383Crossref PubMed Scopus (65) Google Scholar, 28Zhang Z. Tang J. Tikunova S. Johnson J.D. Chen Z. Qin N. Dietrich A. Stefani E. Birnbaumer L. Zhu M.X. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3168-3173Crossref PubMed Scopus (203) Google Scholar, 29Tang J. Lin Y. Zhang Z. Tikunova S. Birnbaumer L. Zhu M.X. J. Biol. Chem. 2001; 276: 21303-21310Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Recently, we have probed the role played by InsP3Rs in Ca2+ entry mechanisms utilizing cells from the DT40 chicken B lymphocyte line (DT40InsP3R-ko) in which the InsP3R genes have been disrupted (30Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (373) Google Scholar). It was observed that these cells have store-operated Ca2+ entry that is functionally the same as that occurring in the wild type DT40 cells (30Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (373) Google Scholar, 31Ma H.-T. Venkatachalam K., Li, H.S. Montell C. Kurosaki T. Patterson R.L. Gill D.L. J. Biol. Chem. 2001; 276: 18888-18896Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 32Broad L.M. Braun F.J. Lievremont J.P. Bird G.S. Kurosaki T. Putney J.W., Jr. J. Biol. Chem. 2001; 276: 15945-15952Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Moreover, we observed that TRPC3 channels transiently expressed in DT40InsP3R-ko cells or DT40wt cells were in both cases functionally activated in response to phospholipase C-coupled receptors (33Venkatachalam K., Ma, H.T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). These results suggested that the InsP3R was not necessary for SOC or TRP channel activation. Recent work has also focused on the permeant InsP3 receptor antagonist, 2-aminoethoxydiphenyl borate (2-APB) (34Maruyama T. Kanaji T. Nakade S. Kanno T. Mikoshiba K. J. Biochem.(Tokyo). 1997; 122: 498-505Crossref PubMed Scopus (764) Google Scholar), to probe the role of the InsP3R in coupling to activate SOC and TRP channels. At concentrations around 50 μm, the 2-APB molecule inhibits store-operated Ca2+ entry, blocking the activation of SOCs in response to store depletion with Ca2+pump blockers or ionomycin (35Ma H.-T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (528) Google Scholar, 36van Rossum D.B. Patterson R.L., Ma, H.-T. Gill D.L. J. Biol. Chem. 2000; 275: 28562-28568Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). 2-APB also blocks activation of mammalian TRPC3 channels (35Ma H.-T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (528) Google Scholar) and TRP channels mediating theDrosophila light response (37Chorna-Ornan I. Joel-Almagor T. Ben Ami H.C. Frechter S. Gillo B. Selinger Z. Gill D.L. Minke B. J. Neurosci. 2001; 21: 2622-2629Crossref PubMed Google Scholar). However, in the case of both of these TRP channels, the action of 2-APB does not appear to be directly upon the TRP channels themselves but rather upon an upstream target that is involved perhaps in controlling the activation of the channels (35Ma H.-T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (528) Google Scholar, 37Chorna-Ornan I. Joel-Almagor T. Ben Ami H.C. Frechter S. Gillo B. Selinger Z. Gill D.L. Minke B. J. Neurosci. 2001; 21: 2622-2629Crossref PubMed Google Scholar). We recently determined that the blocking action of 2-APB on SOCs was still observed in the DT40InsP3R-ko cells (31Ma H.-T. Venkatachalam K., Li, H.S. Montell C. Kurosaki T. Patterson R.L. Gill D.L. J. Biol. Chem. 2001; 276: 18888-18896Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), suggesting that this action of 2-APB was not mediated by the InsP3R. Considering these recent results and the fact that they appeared to contradict a large body of evidence implicating a role for InsP3Rs in SOC activation, it was crucial to obtain more information on the function of SOCs and the actions of 2-APB in the DT40 cell lines. It was also essential to determine more definitively whether the DT40InsP3R-ko cells were truly devoid of InsP3R translation products. Thus, it was suggested (17Kiselyov K.I., Xu, X. Mohayeva G. Kuo T. Pessah I.N. Mignery G.A. Zhu X. Birnbaumer L. Muallem S. Nature. 1998; 396: 478-482Crossref PubMed Scopus (557) Google Scholar) that truncated InsP3R genes replacing the original genes in these cells could give rise to expression of C-terminally truncated InsP3Rs (17Kiselyov K.I., Xu, X. Mohayeva G. Kuo T. Pessah I.N. Mignery G.A. Zhu X. Birnbaumer L. Muallem S. Nature. 1998; 396: 478-482Crossref PubMed Scopus (557) Google Scholar), which might be sufficient for the activation of Ca2+ entry channels (22Putney J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14669-14671Crossref PubMed Scopus (136) Google Scholar, 23Boulay G. Brown D.M. Qin N. Jiang M. Dietrich A. Zhu M.X. Chen Z. Birnbaumer M. Mikoshiba K. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14955-14960Crossref PubMed Scopus (345) Google Scholar, 24Birnbaumer L. Boulay G. Brown D. Jiang M. Dietrich A. Mikoshiba K. Zhu X. Qin N. Recent Prog. Horm. Res. 2000; 55: 127-161PubMed Google Scholar, 25Kiselyov K.I. Mignery G.A. Zhu M.X. Muallem S. Mol. Cell. 1999; 4: 423-429Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 28Zhang Z. Tang J. Tikunova S. Johnson J.D. Chen Z. Qin N. Dietrich A. Stefani E. Birnbaumer L. Zhu M.X. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3168-3173Crossref PubMed Scopus (203) Google Scholar, 29Tang J. Lin Y. Zhang Z. Tikunova S. Birnbaumer L. Zhu M.X. J. Biol. Chem. 2001; 276: 21303-21310Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Here we provide the results of exhaustive Western analyses establishing that the DT40InsP3R-ko cells are devoid of detectable InsP3Rs or fragments thereof. We also reveal that robust bifunctional actions of 2-APB on both SOCs and InsP3Rs in DT40wt cells are dependent on the coupled state of these channels and suggest that 2-APB may target the coupling machinery involved in mediating store-operated Ca2+ entry. Cells of both the wild type DT40 chicken B cell line (DT40wt) and triple InsP3 receptor knockout cell line (DT40InsP3R-ko) were cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum, penicillin, streptomycin, and glutamine, as described previously (30Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (373) Google Scholar). Cells grown on coverslips for 1 h were transferred to Hepes-buffered Krebs medium (107 mm NaCl, 6 mm KCl, 1.2 mmMgSO4, 1 mm CaCl2, 1.2 mm KH2PO4, 11.5 mmglucose, 0.1% bovine serum albumin, 20 mm Hepes-KOH, pH 7.4) and loaded with fura-2/AM (2 μm) for 25 min at 20 °C. Cells were washed, and dye was allowed to de-esterify for a minimum of 15 min at 20 °C. Approximately 95% of the dye was confined to the cytoplasm as determined by the signal remaining after saponin permeabilization (38Short A.D. Klein M.G. Schneider M.F. Gill D.L. J. Biol. Chem. 1993; 268: 25887-25893Abstract Full Text PDF PubMed Google Scholar, 39Short A.D. Bian J. Ghosh T.K. Waldron R.T. Rybak S.L. Gill D.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4986-4990Crossref PubMed Scopus (247) Google Scholar). Fluorescence emission at 505 nm was monitored with excitation at 340 and 380 nm. Ca2+measurements are shown as 340/380 nm ratios obtained from groups of 10–12 cells. Resting Ca2+ levels in the two DT40 cell lines were similar at ∼100–130 nm. All measurements shown are representative of a minimum of three, and in most cases, a larger number of independent experiments. Cells of both of the DT40 lines as well as the A7r5 smooth muscle and Chinese hamster ovary cell lines (CHO; used as InsP3R positive controls) were homogenized and treated exactly as described previously (40Parys J.B., De Smedt H. Missiaen L. Bootman M.D. Sienaert I. Casteels R. Cell Calcium. 1995; 17: 239-249Crossref PubMed Scopus (117) Google Scholar), with the exception that for both DT40wt and DT40InsP3R-ko cells the analyses used cell homogenates and not isolated membranes. All samples were rapidly frozen in liquid N2 and stored at −80 °C, protein determinations were performed by the Lowry procedure. For InsP3R detection, samples were boiled for 2 min in sample buffer, separated on 3–12% Laemmli-type linear gradient gels, transferred to Immobilon-P, and probed with the following antibody preparations directed against InsP3R N-terminal domains: (a) the polyclonal anti-cytI3b-1 and anti-cytI3b-2 antisera directed against Ca2+-binding site 3b (amino acids 378–450) of the type-1 InsP3R (41Sipma H., De Smet P. Sienaert I. Vanlingen S. Missiaen L. Parys J.B. De Smedt H. J. Biol. Chem. 1999; 274: 12157-12162Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar); (b) the polyclonal Rbt226 and Rbt227 antisera directed against amino acids 330–344 of the type-2 InsP3R (42Vanlingen S. Sipma H., De Smet P. Callewaert G. Missiaen L., De Smedt H. Parys J.B. Biochem. J. 2000; 346: 275-280Crossref PubMed Google Scholar); (c) the monoclonal antibody MMAtype3 against amino acids 22–230 of the type-3 InsP3R (I31220, Transduction Laboratories) (43De Smedt H. Missiaen L. Parys J.B. Henning R.H. Sienaert I. Vanlingen S. Gijsens A. Himpens B. Casteels R. Biochem. J. 1997; 322: 575-583Crossref PubMed Scopus (115) Google Scholar). Immunodepletion of the polyclonal anti-cytI3b-1 antiserum was performed by a 2-h preincubation of the antiserum with an excess of antigenic peptide. A new antibody was developed recognizing a conserved epitope localized in the N-terminal portion of the three InsP3R isoforms. The synthetic peptide KSNKYLTVNKRLPAL corresponding to amino acids 127–141 of the human type-1 InsP3R, which is conserved between isoforms and between diverse vertebrate species, was coupled to keyhole limpet hemocyanin through an additional C-terminal cysteine. Two rabbits (Rbt475 and Rbt476) were repeatedly immunized with the coupled peptide. Both antisera reacted with high affinity against each of the InsP3R isoforms from various sources. These antisera were further affinity-purified against the peptide. Finally, for some control experiments the N-terminal InsP3R antiserum AbC (44Cardy T.J. Traynor D. Taylor C.W. Biochem. J. 1997; 328: 785-793Crossref PubMed Scopus (99) Google Scholar) was also used. Analysis of the immunoreactive bands was performed after incubation with secondary antibodies coupled to alkaline phosphatase detected by using VistraTM ECF (Amersham Life Sciences, Inc.) and fluorimaged as described before (45Vanlingen S. Parys J.B. Missiaen L., De Smedt H. Wuytack F. Casteels R. Cell Calcium. 1997; 22: 475-486Crossref PubMed Scopus (45) Google Scholar). The procedures for cell permeabilization cells were as described earlier (46Rys-Sikora K.E. Ghosh T.K. Gill D.L. J. Biol. Chem. 1994; 269: 31607-31613Abstract Full Text PDF PubMed Google Scholar, 47Rys-Sikora K.E. Gill D.L. J. Biol. Chem. 1998; 273: 32627-32635Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Briefly, suspensions of DT40 cells (1 × 106 cells/ml) were stirred gently and incubated with 0.0025% saponin in an intracellular-like medium (ICM) (comprising 140 mmKCl, 10 mm NaCl, 2.5 mm MgCl2, and 10 mm Hepes-KOH, pH 7.0) at 37 °C until 95% permeabilization occurred (normally after 6–7 min). After permeabilization, cells were washed twice in saponin-free ICM at 4 °C and kept cold before use in experiments. To avoid problems of lipid dilution of added hydrophobic compounds, the final cell concentration in all experiments was kept at exactly 5 × 105 cells/ml. Ca2+ flux measurements were conducted as described previously (47Rys-Sikora K.E. Gill D.L. J. Biol. Chem. 1998; 273: 32627-32635Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 48Ghosh T.K. Bian J. Gill D.L. Science. 1990; 248: 1653-1656Crossref PubMed Scopus (326) Google Scholar, 49Waldron R.T. Short A.D. Gill D.L. J. Biol. Chem. 1995; 270: 11955-11961Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The accumulation of45Ca2+ into intracellular organelles was measured using permeabilized DT40 cells (5 × 105cells/ml) maintained by gentle stirring at 37 °C in ICM containing 50 μm CaCl2 (with 150 Ci/mol45Ca2+), EGTA to buffer free Ca2+ to exactly 0.1 μm, 3% polyethylene glycol, and 5 μm ruthenium red (to prevent mitochondrial Ca2+ accumulation) in a total volume of 2 ml. Effectors mentioned in the figures (2-APB) were added at the times shown. Oxalate with GTP when present was added immediately prior to the start of uptake. At the required times, 200-μl aliquots were removed from the stirred uptake medium, diluted immediately into 4 ml of ice-cold ICM containing 1 mm LaCl3, and then rapidly vacuum-filtered on glass fiber filters (Whatman GF/B), washed, and counted. The figures show ATP-dependent Ca2+accumulation with that component of Ca2+ retained by the cells and filters in the absence of ATP, subtracted (∼0.1% of total Ca2+). The experiments shown are typical of at least three separate experiments. ATP, GTP, EGTA, polyethylene glycol, saponin, ruthenium red, Hepes, and oxalate were purchased from Sigma. 2-APB was from Tocris (Ballwin, MO). Thapsigargin was from LC Services (Woburn, MA). Fura-2/AM was from Molecular Probes (Eugene, OR). The DT40 cell lines were generously provided by Dr. Tomohiro Kurosaki (Kyoto, Japan). For Ca2+ flux experiments, free Ca2+ concentrations were controlled using EGTA computing all complexes between EGTA, ATP, Ca2+, Mg2+, monovalent cations, and protons, as described previously (50Ghosh T.K. Mullaney J.M. Tarazi F.I. Gill D.L. Nature. 1989; 340: 236-239Crossref PubMed Scopus (134) Google Scholar). Anti-chicken IgM (supernatant, M-4 clone) was from Southern Biotechnology Associates (Birmingham, AL). The AbC antiserum was a generous gift from Dr. C. W. Taylor (University of Cambridge, UK). Considering the potential significance of 2-APB in targeting the activation of SOCs and TRP channels, we examined the details of the kinetics of action of 2-APB. Surprisingly, 2-APB was observed to induce a robust biphasic modification of SOC function in DT40 chicken B-lymphocytes. Using DT40wt cells in which Ca2+ stores had been previously depleted with thapsigargin, the addition of Ca2+ resulted in the onset of SOC-mediated Ca2+ entry, as shown in Fig.1. After SOC activation, the addition of low levels of 2-APB (1–10 μm) induced a rapid and profound stimulation of Ca2+ entry (Fig. 1,A–C) with a maximal effect at 10 μm. In contrast, at higher levels (25–75 μm), the action of 2-APB was strongly inhibitory on SOCs (Fig. 1, D–F). Thus, although there was an initial transient increase in Ca2+entry, this subsided to reveal a powerful blocking action. At the higher levels of 2-APB, the stimulatory effect was extremely brief such that at 75 μm there was an almost imperceptible activation followed rapidly by almost complete inhibition of Ca2+ entry. Indeed, we had previously observed the inhibitory action of 2-APB on SOCs (31Ma H.-T. Venkatachalam K., Li, H.S. Montell C. Kurosaki T. Patterson R.L. Gill D.L. J. Biol. Chem. 2001; 276: 18888-18896Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 35Ma H.-T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (528) Google Scholar) but had not examined the effects of acute addition of submaximally effective 2-APB levels on preactivated SOC activity. The biphasic effects of 2-APB on SOCs were not unique to the DT40 cells because we observed similar effects of 2-APB in HEK293 cells, DC3-F fibroblasts, and DDT1MF-2 smooth muscle cells (data not shown). In these cells the stimulatory effect with low levels of 2-APB was smaller, but the overall biphasic effect was similar. One particular reason why the nature and coupling of SOCs has remained elusive is that modifiers of the activation process have not been identified. Whereas there have been reports on a variety of agents that can inhibit SOC activity (51Putney J.W. Capacitative Calcium Entry. Springer, New York1997Crossref Google Scholar), almost invariably such agents have been shown to be highly nonselective, modifying a range of other channels for Ca2+ and/or other ions. In contrast, the action of 2-APB on SOCs reflects a much more selective function, 2-APB having little or no effect on a range of Ca2+ and related channels including ryanodine receptors (34Maruyama T. Kanaji T. Nakade S. Kanno T. Mikoshiba K. J. Biochem.(Tokyo). 1997; 122: 498-505Crossref PubMed Scopus (764) Google Scholar), voltage-sensitive Ca2+entry channels (34Maruyama T. Kanaji T. Nakade S. Kanno T. Mikoshiba K. J. Biochem.(Tokyo). 1997; 122: 498-505Crossref PubMed Scopus (764) Google Scholar), arachidonic acid-activated Ca2+ entry channels (52Luo D. Broad L.M. Bird G.S. Putney J.W., Jr. J. Biol. Chem. 2001; 276: 201" @default.
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• W2124353324 title "Modification of Store-operated Channel Coupling and Inositol Trisphosphate Receptor Function by 2-Aminoethoxydiphenyl Borate in DT40 Lymphocytes" @default.
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