Bax Inhibitor-1 Is a pH-dependent Regulator of Ca2+ Channel Activity in the Endoplasmic Reticulum

Abstract

In this study, Bax inhibitor-1 (BI-1) overexpression reduces the ER pool of Ca2+ released by thapsigargin. Cells overexpressing BI-1 also showed lower intracellular Ca2+ release induced by the Ca2+ ionophore ionomycin as well as agonists of ryanodine receptors and inositol trisphosphate receptors. In contrast, cells expressing carboxyl-terminal deleted BI-1 (CΔ-BI-1 cells) displayed normal intracellular Ca2+ mobilization. Basal Ca2+ release rates from the ER were higher in BI-1-overexpressing cells than in control or CΔ-BI-1 cells. We determined that the carboxyl-terminal cytosolic region of BI-1 contains a lysine-rich motif (EKDKKKEKK) resembling the pH-sensing domains of ion channels. Acidic conditions triggered more extensive Ca2+ release from ER microsomes from BI-1-overexpressing cells and BI-1-reconsituted liposomes. Acidic conditions also induced BI-1 protein oligomerization. Interestingly subjecting BI-1-overexpressing cells to acidic conditions induced more Bax recruitment to mitochondria, more cytochrome c release from mitochondria, and more cell death. These findings suggest that BI-1 increases Ca2+ leak rates from the ER through a mechanism that is dependent on pH and on the carboxyl-terminal cytosolic region of the BI-1 protein. The findings also reveal a cell death-promoting phenotype for BI-1 that is manifested under low pH conditions. In this study, Bax inhibitor-1 (BI-1) overexpression reduces the ER pool of Ca2+ released by thapsigargin. Cells overexpressing BI-1 also showed lower intracellular Ca2+ release induced by the Ca2+ ionophore ionomycin as well as agonists of ryanodine receptors and inositol trisphosphate receptors. In contrast, cells expressing carboxyl-terminal deleted BI-1 (CΔ-BI-1 cells) displayed normal intracellular Ca2+ mobilization. Basal Ca2+ release rates from the ER were higher in BI-1-overexpressing cells than in control or CΔ-BI-1 cells. We determined that the carboxyl-terminal cytosolic region of BI-1 contains a lysine-rich motif (EKDKKKEKK) resembling the pH-sensing domains of ion channels. Acidic conditions triggered more extensive Ca2+ release from ER microsomes from BI-1-overexpressing cells and BI-1-reconsituted liposomes. Acidic conditions also induced BI-1 protein oligomerization. Interestingly subjecting BI-1-overexpressing cells to acidic conditions induced more Bax recruitment to mitochondria, more cytochrome c release from mitochondria, and more cell death. These findings suggest that BI-1 increases Ca2+ leak rates from the ER through a mechanism that is dependent on pH and on the carboxyl-terminal cytosolic region of the BI-1 protein. The findings also reveal a cell death-promoting phenotype for BI-1 that is manifested under low pH conditions. The endoplasmic reticulum (ER) 3The abbreviations used are: ER, endoplasmic reticulum; IP3, inositol trisphosphate; BI-1, Bax inhibitor-1; ICM, intracellular-like medium; HA, hemagglutinin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TRITC, tetramethylrhodamine isothiocyanate; IP3R, IP3 receptor; Rhod-2, Rhodamine-2. contains the largest calcium reserve in the cell (1Berridge M.J. Cell Calcium. 2002; 32: 235-249Crossref PubMed Scopus (794) Google Scholar, 2Van Coppenolle F. Vanden Abeele F. Slomianny C. Flourakis M. Hesketh J. Dewailly E. Prevarskaya N. J. Cell Sci. 2004; 117: 4135-4142Crossref PubMed Scopus (103) Google Scholar). Agonist-induced ER calcium release occurs through Ca2+ channels such as inositol trisphosphate (IP3) and ryanodine receptors (3Clapham D.E. Cell. 2007; 131: 1047-1058Abstract Full Text Full Text PDF PubMed Scopus (3027) Google Scholar). Calcium uptake into the ER occurs when the calcium release channels are closed (i.e. negative feedback to the IP3 receptor) (4Bezprozvanny I. Watras J. Ehrlich B.E. Nature. 1991; 351: 751-754Crossref PubMed Scopus (1441) Google Scholar) and is performed by sarcoplasmic reticulum/ER-associated calcium-activated ATPase pumps (5Pozzan T. Rizzuto R. Volpe P. Meldolesi J. Physiol. Rev. 1994; 74: 595-636Crossref PubMed Scopus (30) Google Scholar). In the resting state, the Ca2+ content of the ER reflects a balance between active uptake by sarcoplasmic reticulum/ER-associated calcium-activated ATPase and passive efflux or basal leakage through other Ca2+ channels. This leakage is revealed when sarcoplasmic reticulum/ER-associated calcium-activated ATPase pumps are inhibited by agents such as thapsigargin (6Hofer A.M. Curci S. Machen T.E. Schulz I. FASEB J. 1996; 10: 302-308Crossref PubMed Scopus (75) Google Scholar), causing Ca2+ to leak out of the ER into the cytosol. The Bax inhibitor-1 (BI-1) (also known as “testis enhanced gene transcript” (TEGT)) is an antiapoptotic protein capable of inhibiting Bax activation and translocation to mitochondria (7Xu Q. Reed J.C. Mol. Cell. 1998; 1: 337-346Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). This ubiquitously expressed protein contains several transmembrane domains and localizes to the ER. The homology of BI-1 sequences among species is striking, and the characteristic hydrophobicity and ER membrane localization are evolutionarily conserved (8Chae H.J. Ke N. Kim H.R. Chen S. Godzik A. Dickman M. Reed J.C. Gene (Amst.). 2003; 323: 101-113Crossref PubMed Scopus (160) Google Scholar). BI-1 affects calcium leakage from the ER as measured with Ca2+-sensitive, ER-targeted fluorescent proteins and Ca2+-sensitive dyes (9Chae H.J. Kim H.R. Xu C. Bailly-Maitre B. Krajewska M. Krajewski S. Banares S. Cui J. Digicaylioglu M. Ke N. Kitada S. Monosov E. Thomas M. Kress C.L. Babendure J.R. Tsien R.Y. Lipton S.A. Reed J.C. Mol. Cell. 2004; 15: 355-366Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). However, the mechanism by which BI-1 regulates ER Ca2+ fluxes remains unclear. Here we have provided additional evidence that BI-1 induces passive Ca2+ leakage from the ER and also show that BI-1 activity is regulated by pH in a manner dependent on the carboxyl-terminal cytosolic domain of this protein. Cell Lines and Cell Culture—Human fibrosarcoma cells (HT1080), stably transfected with pcDNA3, pcDNA3-BI-1, or pcDNA3-BI-1ΔC, have been described previously (9Chae H.J. Kim H.R. Xu C. Bailly-Maitre B. Krajewska M. Krajewski S. Banares S. Cui J. Digicaylioglu M. Ke N. Kitada S. Monosov E. Thomas M. Kress C.L. Babendure J.R. Tsien R.Y. Lipton S.A. Reed J.C. Mol. Cell. 2004; 15: 355-366Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Human cervical carcinoma cells (HeLa) expressing BI-1 under a tetracycline-inducible promoter have been described previously (10Wang Z. Cuddy M. Samuel T. Welsh K. Schimmer A. Hanaii F. Houghten R. Pinilla C. Reed J.C. J. Biol. Chem. 2004; 279: 48168-48176Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 1 mm l-glutamine, and antibiotics. When investigating the effect of intracellular acidification, the cells were cultured in the presence of the K+ ionophore nigericin (5 μm) in medium buffered to a pH ranging from 8.0 to 5.0. Nigericin-containing media were supplemented with 140 mm K+, which facilitates pH equilibration (11Maidorn R.P. Cragoe Jr., E.J. Tannock I.F. Br. J. Cancer. 1993; 67: 297-303Crossref PubMed Scopus (73) Google Scholar, 12Zanke B.W. Lee C. Arab S. Cancer Res. 1998; 58: 2801-2808PubMed Google Scholar). Measurement of Intracellular Ca2+—The fluorescent calcium indicator Fura-2/AM (1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzoFURAn-5-oxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N′,N′-tetraacetic acid pentaacetoxymethyl ester; Molecular Probes, Eugene, OR) was used to measure changes in intracellular (cytosolic) free Ca2+. Cells were plated onto glass-bottomed perfusion chambers that were mounted on the stage of an inverted microscope (Nikon Eclipse TE2000) and incubated with Fura-2/AM (6 μm) for 30 min at room temperature in Hanks' balanced salt solution. After loading, cells were washed three times in isotonic buffer without Ca2+ (KH buffer: 132 mm NaCl, 5 mm KCl, 10 mm dextrose, 10 mm HEPES, and 1.05 mm MgCl2). Cells were then promptly treated with various agents, including thapsigargin, ionomycin, ryanodine, histamine, and ATP. Changes in [Ca2+]i were determined as a ratio of 340/380-nm excitation (512-nm emission) using an integrated spectrofluorometer (Photon Technology International, Birmingham, NJ). Ca2+ concentrations were calculated using the equation [Ca2+]i = Kd(F380 max/F380 min) (R - Rmin)/(Rmax - R); a Kd value of 229 nm was assumed for the binding of calcium to Fura-2/AM. Rmax and Rmin were determined in each experimental group by the consecutive addition of 30 μm digitonin (Rmax) and 50 mm EGTA (Rmin). When investigating the effect of intracellular acidification, cells were incubated with the proton ionophore nigericin (5 μm) in a pH ranging from 7.4 to 5.4 (5 μm nigericin-containing modified KH buffer: 140 mm KCl, 5 mm NaCl, 10 mm dextrose, 10 mm HEPES, and 1.05 mm MgCl2) to facilitate pH equilibration between the intra- and extracellular environment (11Maidorn R.P. Cragoe Jr., E.J. Tannock I.F. Br. J. Cancer. 1993; 67: 297-303Crossref PubMed Scopus (73) Google Scholar, 12Zanke B.W. Lee C. Arab S. Cancer Res. 1998; 58: 2801-2808PubMed Google Scholar). Subcellular Fractionation—To obtain microsome preparations, all procedures were performed at 4 °C using cold reagents. Cells were washed and homogenized using 20–30 strokes of a motor-driven glass-Teflon homogenizer at 4 °C in an ice-cold, pH 7.4 solution containing 250 mm sucrose, 10 mm HEPES, 10 mm KCl, 1.5 mm MgCl2, 1.0 mm EGTA, 1.0 mm dithiothreitol, and 1× protease inhibitor complex (Roche Applied Science). Homogenates were centrifuged at 1600 × g for 10 min to remove the The was then centrifuged at × g for 10 min to the The was then centrifuged at × g for min to the were used for translocation of control or BI-1-overexpressing cells were to normal or acidic pH and acidic mm in Dulbecco's modified Eagle's medium without for The cells were washed with in an isotonic buffer mm sucrose, mm 1 mm and 10 mm HEPES, pH and by through a to a cells and were by at × g for 10 min at 4 The was centrifuged at × g for 30 min at 4 °C to the membrane in which mitochondria are The was used as a cytosolic and the membrane was in 50 of the buffer and used as a Measurement of ER of we used the low fluorescent Ca2+ medium for ER Ca2+ mm KCl, mm NaCl, mm 10 mm HEPES, pH mm 1.0 mm EGTA, mm 10 mm and 1.0 mm also 5 mm and The were with Ca2+ in for min at the min of ER Ca2+ loading, μm was to the solution. After loading, the ER were washed with The (Photon Technology was on a inverted was and at 1 at nm was using a and a and then was and on a The changes in the nm were used as an of changes in The Kd of is the described experimental changes of the ratio = changes in cells were in 50 mm HEPES, pH mm NaCl, mm 1 mm 10 mm 1 mm and mm were for protein content with a and were in an of buffer containing and then for 5 min or without when BI-1 protein. were to with and incubated with various or was protein or a and to and of of were at °C in of with 50 the was to the protein by for 4 were in binding buffer mm pH mm NaCl, 5 mm 1 mm and 5 and The cells were by through an Cell were to a with binding The was washed with binding buffer and buffer mm pH mm NaCl, and mm The proteins were in binding buffer containing 250 mm The protein concentrations were with acid to the of into bovine bovine and bovine in were at of and The was 5 mm in a of 1 The was under a of and the was by The were in buffer mm HEPES, pH mm NaCl, mm dithiothreitol, 1 mm and containing The were for an of buffer without The were by at × g for 30 min at 4 washed with buffer mm HEPES, pH mm NaCl, mm dithiothreitol, and 1 and then buffer without 1 mm for at 4 The were through to remove free Ca2+. The of was by of with a spectrofluorometer and of The of BI-1 protein were determined using the After the ratio of to protein was in Bax BI-1 in to the of BI-1 was with an additional In a was this to and the was by on the as described previously J. Kim C. M.J. PubMed Scopus Google Scholar). pH Ca2+ Measurement efflux from was by the changes in the of a fluorescent Ca2+ of the with acidic mm pH and 50 mm pH and in a ratio of were performed at 30 °C using a The was measured at an of nm with an excitation of The of 10 μm was to free Ca2+ using a the of Ca2+ released from by the of the with buffer was with the addition of the of the of BI-1 in Ca2+ the was with at a ratio of 1 of BI-1 to or of After the at 30 °C for Ca2+ release was measured using the described a all were using without BI-1 protein under the conditions. of Ca2+ from were in the presence of to in buffer remove Ca2+ to the the was to a with buffer and the were by × g for 30 min at 4 was then and an of buffer for at 4 The were with pH buffer and incubated for min at 30 The were with buffer 1.5 and the were by × g for 30 min at 30 The was then in and the of each and was determined by of BI-1 reticulum were from and cells. ER were in buffer containing 250 mm sucrose, 10 mm pH mm 5 mm and μm EGTA and then treated with or without acidic pH or for 30 min at 30 The ER were by and then by using an of were to to and incubated with at Ca2+ were performed using an inverted microscope and a of a a and and as described previously (11Maidorn R.P. Cragoe Jr., E.J. Tannock I.F. Br. J. Cancer. 1993; 67: 297-303Crossref PubMed Scopus (73) Google Scholar). Ca2+ uptake was by cells with and Cells were with at and nm and the from dyes was through a and and were performed at °C with are as of and and were to and control conditions was used for BI-1 ER of an inhibitor of the sarcoplasmic reticulum/ER-associated calcium-activated basal release of Ca2+ from ER by Ca2+ uptake into the ER. thapsigargin Ca2+ to leak from the ER into the an with cytosolic Ca2+-sensitive dyes such as Fura-2/AM in cells cultured in of BI-1, we used cells stably transfected with BI-1 protein cells) or BI-1 protein the carboxyl-terminal cytosolic domain (CΔ-BI-1 cells) or with of the BI-1 and CΔ-BI-1 proteins of thapsigargin to control cells cultured in medium to a in cytosolic free Ca2+ and We used a of Fura-2/AM (6 μm) that was for the integrated spectrofluorometer However, μm Fura-2/AM Ca2+ release into the was in BI-1 cells and In contrast, Ca2+ release was in CΔ-BI-1 cells with control cells. The of BI-1 overexpression to intracellular of Ca2+ was also using cells expressing BI-1 under a promoter BI-1 protein which release of Ca2+ from intracellular that BI-1 overexpression regulates ER of Ca2+ are by BI-1 in cells with cells were incubated with 1 μm for or and the of protein was by using for protein content The was with cells were incubated with or without 1 μm for and then cells were with cultured in and incubated with 1 μm thapsigargin cells were = is provided Ca2+ release the of Ca2+ release in was and are expressed as a of the to thapsigargin of by The of Ca2+ into the thapsigargin that intracellular of Ca2+ are lower in BI-1-overexpressing cells. this we cultured cells in medium and then treated with a 5 of the digitonin at 5 leakage of and the Ca2+ ionophore the cell to such as Ca2+ S. 2003; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar) and of calcium with In [Ca2+]i induced by ionomycin was the of control cells by release was in CΔ-BI-1 cells We also ER Ca2+ in BI-1, and CΔ-BI-1 cells using the ryanodine Ca2+ release into in BI-1-overexpressing cells with and agonists of receptors that of IP3 an of ER Ca2+ channels Cell Calcium. 10: PubMed Scopus Google Scholar), showed release of the pool of Ca2+ from BI-1-overexpressing cells and ryanodine and of ER Ca2+ to in BI-1-overexpressing the of ryanodine and was in the BI-1 cells BI-1 Ca2+ of the the of BI-1 on Ca2+ of the ER we the of Ca2+ was thapsigargin to Ca2+ in the ER by cytosolic [Ca2+]i in cells. the of Ca2+ released in to thapsigargin and ionomycin was in cells with and cells with and that used ER-targeted Ca2+-sensitive fluorescent proteins to ER free Ca2+ concentrations (9Chae H.J. Kim H.R. Xu C. Bailly-Maitre B. Krajewska M. Krajewski S. Banares S. Cui J. Digicaylioglu M. Ke N. Kitada S. Monosov E. Thomas M. Kress C.L. Babendure J.R. Tsien R.Y. Lipton S.A. Reed J.C. Mol. Cell. 2004; 15: 355-366Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). by ionomycin at various times thapsigargin we that the of Ca2+ release was in cells and at 1 min thapsigargin of the Ca2+ was released in was released in control cells. to the from revealed that the of Ca2+ release in cells was than that in control that BI-1 increases the Ca2+ of the ER of ER Ca2+ that the carboxyl-terminal domain of BI-1 is for ER Ca2+. in the carboxyl-terminal domain of BI-1 (EKDKKKEKK) is to the in the that of this ion in a manner A. A. B. J. Full Text Full Text PDF PubMed Scopus Google Scholar). We that BI-1 as a of ER Ca2+ this microsomes were from cells stably transfected with control or BI-1 or The microsomes from BI-1 and CΔ-BI-1 cells were with the Ca2+-sensitive fluorescent to Ca2+ concentrations and under acidic conditions. When microsomes were to pH a in was in microsomes from BI-1-overexpressing was in ER Ca2+ concentrations in microsomes from control or cells to pH 5.4 and of the induces Ca2+ efflux from the ER in and cells were treated with buffer with nigericin (5 μm) and mm to pH and Ca2+ into the was measured in cells. extracellular into the intracellular environment through the to (11Maidorn R.P. Cragoe Jr., E.J. Tannock I.F. Br. J. Cancer. 1993; 67: 297-303Crossref PubMed Scopus (73) Google Scholar, 12Zanke B.W. Lee C. Arab S. Cancer Res. 1998; 58: 2801-2808PubMed Google Scholar, S. A. 2007; PubMed Scopus Google Scholar). BI-1-overexpressing cells showed release of intracellular Ca2+ in to acidic pH with control and CΔ-BI-1 cells the Ca2+ of BI-1, we release of Ca2+ into the in cells and using cells cultured in medium this we also a release of Ca2+ from the ER in to acidic conditions. These suggest that a Ca2+ under acidic pH is a characteristic of We measured Ca2+ efflux from in The of a fluorescent Ca2+ of the of the with BI-1 and acidic that Ca2+ was released Changes in were dependent on the of the induced more Ca2+ and a of in the was by the to pH When with the of the with of Ca2+ was released by at pH of a BI-1 at a ratio of 1 of BI-1 to 10 of BI-1 inhibited of the Ca2+ release at pH of more BI-1 at a ratio of to the Ca2+ the release of Ca2+ through BI-1, we the using The of Ca2+ released by acidic solution and the of by BI-1 were to the using the fluorescent this the of BI-1 as a Ca2+ in the in with of cultured cells and BI-1 and BI-1 by Acidic previously that the BI-1 protein when by that we to the hydrophobicity of the protein Mol. Biol. 1998; 15: PubMed Scopus Google Scholar). However, and of BI-1 are this we used of cells to BI-1 which a of and that were to various pH conditions. we a of at and with with the of or of BI-1 The enhanced CΔ-BI-1 protein also than BI-1 protein. Interestingly acidic conditions the of BI-1 In the was to show the of in and acidic conditions. a in pH as a to of BI-1 and the release of Ca2+ from the ER. BI-1 Ca2+ in overexpression of BI-1 increases Ca2+ release from the ER under acidic we that Ca2+ in mitochondria to a in cells overexpressing BI-1 than in control cells under acidic this we and cells with a Ca2+-sensitive and and to acidic conditions. intracellular and extracellular we used buffer with nigericin C. J. Physiol. 1996; PubMed Google Scholar, J. Physiol. Google Scholar). higher in BI-1-overexpressing cells to acidic conditions with pH of and normal pH was in Ca2+ between and BI-1 cells we calculated for cells These show a pH Ca2+ in BI-1 BI-1 Cell Bax to and c from Acidic the of BI-1 overexpression on the cell under acidic we of and cells cultured with K+ medium and nigericin buffered to pH ranging from to 8.0 cell with more cell in BI-1 than in control cells. we Bax and cytochrome c under the acidic conditions the of Bax and cytochrome c in cytosolic and membrane Acidic pH induced Bax with mitochondria to a in BI-1-overexpressing cells than in control cells with of this protein from the to mitochondria J.C. Cell PubMed Scopus Google Scholar). Bax in the to a in BI-1-overexpressing cells to acidic conditions. cytochrome c were in mitochondria and in to a in BI-1-overexpressing cells with control cells with release of cytochrome c from mitochondria in BI-1-overexpressing cells. BI-1 is a membrane protein that is conserved in and species and that in ER BI-1 is with the of intracellular Ca2+ in and (9Chae H.J. Kim H.R. Xu C. Bailly-Maitre B. Krajewska M. Krajewski S. Banares S. Cui J. Digicaylioglu M. Ke N. Kitada S. Monosov E. Thomas M. Kress C.L. Babendure J.R. Tsien R.Y. Lipton S.A. Reed J.C. Mol. Cell. 2004; 15: 355-366Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, Mol. Biol. 1998; 15: PubMed Scopus Google Scholar, N. 2007; PubMed Scopus Google Scholar, M. S. M. Physiol. 2007; PubMed Scopus Google Scholar). ER free Ca2+ concentrations are in BI-1-overexpressing and cells with BI-1 have Ca2+ (9Chae H.J. Kim H.R. Xu C. Bailly-Maitre B. Krajewska M. Krajewski S. Banares S. Cui J. Digicaylioglu M. Ke N. Kitada S. Monosov E. Thomas M. Kress C.L. Babendure J.R. Tsien R.Y. Lipton S.A. Reed J.C. Mol. Cell. 2004; 15: 355-366Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), that BI-1 protein ER Ca2+ We have and that microsomes from BI-1-overexpressing cells Ca2+ and that cells with or overexpression of BI-1 release of ER Ca2+ into the in to thapsigargin. the of ER Ca2+ regulated by ryanodine receptors and as by using agonists of receptors histamine, and were in BI-1-overexpressing cells as were Ca2+ as by ionomycin and digitonin We that BI-1 ER more to the mechanism of this is BI-1 an ion that for the passive leak of Ca2+ out of the of the ER in cells overexpressing BI-1 the activity of other of ER Ca2+ channels such as ryanodine receptors and of channels to changes in Ca2+ T. S. J. C. and have M.J. FASEB J. 2002; PubMed Scopus Google Scholar), is to the using a more an in the Here we show that the effect of BI-1 on ER Ca2+ is and that the carboxyl-terminal cytosolic domain of BI-1 is for ER Ca2+. Interestingly this region contains a of the of the cytosolic BI-1 We that this motif as a pH to previously for the A. A. B. J. Full Text Full Text PDF PubMed Scopus Google Scholar). cytosolic increases the of this is to the we for BI-1-overexpressing microsomes and cells with to release of ER Ca2+ under acidic conditions. we showed that BI-1 protein acidic Ca2+ release in BI-1-reconsituted and BI-1 without with a Ca2+ the pH-sensing motif of BI-1 is among several BI-1 this pH evolutionarily Acidic pH conditions and BI-1 and that of BI-1 regulates an ER Ca2+ or a Ca2+ acidic pH more induces Ca2+ release from ER when BI-1 is are to the of BI-1 to ER Ca2+ ER Ca2+ and the release into the including cell a of of ER protein K. P. C. Thomas M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and gene S. Nature. PubMed Scopus Google Scholar). The of and Ca2+ is complex with channels E. T. Mol. Cell. Biol. 2007; PubMed Scopus Google Scholar) and channels among the B. 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