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J. Biol. Chem., Vol. 283, Issue 23, 15946-15955, June 6, 2008

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

From the ^bDepartment of Pharmacology and Institute of Cardiovascular Research, School of Medicine, Chonbuk National University, Jeonju, Chonbuk 561-181, Republic of Korea, ^aDepartment of Dental Pharmacology and Wonkwang Biomaterial Implant Research Institute, School of Dentistry, Wonkwang University, Iksan, Chonbuk 570-749, Republic of Korea, ⁱClinical Trial Center for Functional Foods, Chonbuk Hospital, Jeonju, Chonbuk 561-712, Republic of Korea, ^cDepartment of Biochemistry, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea, ^dCollege of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea, ^eDepartment of Animal Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea, ^fDivision of Molecular and Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea, ^gDepartment of Pharmacology, Medical Research Center (MRC), College of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea, ^hDivision of Rheumatology, Department of Internal Medicine, Catholic Research Institutes of Medical Science, School of Medicine, The Catholic University of Korea, Seoul 150-010, Republic of Korea, and ^jBurnham Institute for Medical Research, La Jolla, California 02037

Received for publication, January 4, 2008 , and in revised form, March 17, 2008.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 (C-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 C-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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The endoplasmic reticulum (ER)³ contains the largest calcium reserve in the cell (1, 2). Agonist-induced ER calcium release occurs through Ca²⁺ channels such as inositol trisphosphate (IP₃) and ryanodine receptors (3). Calcium uptake into the ER occurs when the calcium release channels are closed (i.e. negative feedback to the IP₃ receptor) (4) and is performed by sarcoplasmic reticulum/ER-associated calcium-activated ATPase pumps (5). 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 (6), 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 (7). 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 (8). BI-1 affects calcium leakage from the ER as measured with Ca²⁺-sensitive, ER-targeted fluorescent proteins and Ca²⁺-sensitive dyes (9). 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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Lines and Cell Culture—Human fibrosarcoma cells (HT1080), stably transfected with pcDNA3, pcDNA3-BI-1, or pcDNA3-BI-1C, have been described previously (9). Human cervical carcinoma cells (HeLa) expressing BI-1 under a tetracycline-inducible promoter have been described previously (10). 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 (11, 12).

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 = K_d(F_{380 max}/F_{380 min}) (R - R_min)/(R_max - R); a K_d value of 229 nM was assumed for the binding of calcium to Fura-2/AM. R_max and R_min were determined in each experimental group by the consecutive addition of 30 µM digitonin (R_max) and 50 mM EGTA (R_min). 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 (11, 12).

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 1x protease inhibitor complex (Roche Applied Science). Homogenates were centrifuged at 1600 x g for 10 min to remove the nuclear fraction. The supernatant was then centrifuged at 10,000 x g for 10 min to pellet the mitochondria. The resulting supernatant was then centrifuged at 100,000 x g for 60 min to yield the ER-enriched microsomal pellet. Microsomes were used immediately for experiments.

To monitor cytosolic-mitochondrial translocation of proteins, neomycin control vector (Neo)- or BI-1-overexpressing cells were exposed to normal or acidic pH (5.0, 5.4, 6.0, 6.4, and 7; acidic medium: 15 mM HEPES in Dulbecco's modified Eagle's medium without bicarbonate) for 24 h. The cells were washed with phosphate-buffered saline, resuspended in an isotonic mitochondrial buffer (210 mM sucrose, 70 mM mannitol, 1 mM EDTA, and 10 mM HEPES, pH 7.4), and broken by six passages through a 25-gauge needle fitted to a syringe. Unbroken cells and nuclei were removed by centrifugation at 700 x g for 10 min at 4 °C. The resulting supernatant was further centrifuged at 10,000 x g for 30 min at 4 °C to collect the heavy membrane fraction in which mitochondria are enriched. The resulting supernatant was used as a cytosolic fraction, and the heavy membrane pellet was resuspended in 50 µl of the same mitochondrial buffer and used as a mitochondrial fraction.

Measurement of ER Ca²⁺—For measurements of [Ca²⁺]_ER, we used the low affinity fluorescent Ca²⁺ dye mag-Fura-2/AM. Intracellular-like medium (ICM) for ER Ca²⁺ loading contained 120 mM KCl, 20 mM NaCl, 2.0 mM MgSO₄, 10 mM HEPES, pH 7.2, 3.0 mM ATP, 1.0 mM EGTA, 2.0 mM CaCl₂, 10 mM Mg-ATP, and 1.0 mM dithiothreitol. ICM also contained 5 mM creatine phosphate and 20 units/ml creatine phosphokinase. The isolated ER-enriched microsomal fractions were loaded with Ca²⁺ in ICM for 60 min at 37 °C. During the last 20 min of ER Ca²⁺ loading, 8 µM mag-Fura-2/AM was added to the ICM loading solution. After mag-Fura-2 loading, the ER fractions were briefly washed with ICM. The fluorescence system (Photon Technology International) was based on a Nikon Diaphot inverted microscope. Mag-Fura-2 was excited (340 and 380 nm) at 1 Hz; fluorescence emission at 495 nm was captured using a 200x objective and a photomultiplier tube and then was digitized, displayed, and stored on a computer. The resulting changes in the 340/380 nm ratios were used as an indication of changes in [Ca²⁺]. The K_d of mag-Fura-2 is 54 µM. Under the described experimental conditions, changes of the ratio R = F₃₄₀/F₃₈₀ reflect changes in [Ca²⁺]_ER.

Immunoblotting—For immunoblot analysis, cells were lysed in 1% Nonidet P-40, 50 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM EDTA, 1 mM pyrophosphate, 10 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 100 mM sodium fluoride. Lysates were normalized for total protein content with a BCA kit (Pierce), and 40–50-µg aliquots were suspended in an equal volume of 2x Laemmli buffer containing 2-mercaptoethanol and then boiled for 5 min before performing SDS-PAGE or loaded directly without boiling when analyzing BI-1 protein. Proteins were transferred to Immobilon-P membranes (Millipore, Billerica, MA), blocked with skim milk (5%), and incubated with various primary antibodies recognizing hemagglutinin (HA) or actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Antibody detection was accomplished via horseradish peroxidase-conjugated protein A or goat anti-rabbit immunoglobulin G (Bio-Rad), a chemiluminescent substrate (Amersham Biosciences), and exposure to x-ray film (Eastman Kodak Co.).

Expression and Purification of Recombinant His-BI-1 Protein—Bacterial cultures of pRSET-BI-1 were grown at 37 °C in 500 ml of LB with 50 µg/ml ampicillin until the A₆₀₀ reached 0.5. Isopropyl 1-thio-β-D-galactopyranoside (0.5 mM) was added to induce the recombinant protein followed by further incubation for 4 h. To purify recombinant His-BI-1 proteins, harvested bacterial pellets were resuspended in binding buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml benzamidine, leupeptin, and pepstatin). The cells were lysed by passing through an Amicon French pressure cell. Cell lysates were applied to a nickel-nitrilotriacetic acid-agarose column pre-equilibrated with binding buffer. The column was washed with binding buffer and washing buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 60 mM imidazole). The recombinant proteins were eluted in binding buffer containing 250 mM imidazole. The recombinant protein concentrations were quantified with bicinchoninic acid according to the manufacturer's instructions (Pierce).

Reconstitution of Purified His-BI-1 into Proteoliposomes— Phosphatidylcholine (from bovine brain), phosphatidylethanolamine (from bovine brain), and phosphatidylserine (from bovine brain) dissolved in chloroform were mixed at molar ratios of 50, 30, and 20%, respectively. The final lipid concentration was 5 mM in a total volume of 1 ml. The solvent was evaporated under a stream of argon gas, and the residual chloroform was removed by speed vacuuming. The dry lipids were hydrated in buffer A (20 mM HEPES, pH 7.5, 100 mM NaCl, 0.1 mM dithiothreitol, 1 mM CaCl₂, and 1% CHAPS) containing 20 µg/ml His-BI-1. The mixtures were dialyzed for 72 h against an excess volume of buffer B (buffer A without CHAPS). The resulting proteoliposomes were pelleted by centrifugation at 100,000 x g for 30 min at 4 °C, washed with buffer C (20 mM HEPES, pH 7.5, 100 mM NaCl, 0.1 mM dithiothreitol, and 1 M KCl), and then dialyzed against buffer D (buffer C without 1 mM KCl) for 24 h at 4 °C. The resulting proteoliposomes were passed through Chelex 100 to remove free Ca²⁺. The formation of proteoliposomes was monitored by measurement of light scattering during dialysis with a spectrofluorometer (excitation and emission wavelength of 450 nm). The amounts of reconstituted BI-1 protein were determined using the NanoOrange® Protein Quantitation kit (Molecular Probes). After reconstitution, the ratio of phospholipid concentration (w/v) to protein was 830 ± 48 in reaction samples.

Bax Inhibitor-1 Antibody—A BI-1 peptide (CAMNEKDKKKEKK, in single letter code) corresponding to the carboxyl terminus of BI-1 was synthesized with an additional amino-terminal cysteine. In rats, a polyclonal antibody was raised against this peptide coupled to keyhole limpet hemocyanin, and the antibody was purified by affinity chromatography on the immobilized peptide as described previously (13).

pH Decrease-induced Ca²⁺ Release Measurement Using Indo-1 Fluorescence—Ca²⁺ efflux from proteoliposomes was observed by measuring the changes in the fluorescence of external indo-1, a fluorescent Ca²⁺ indicator, after rapid dilution of the proteoliposomes with acidic solutions (50 mM sodium phosphate, pH 6.5, and 50 mM sodium citrate, pH 6.0, 5.5, and 5.0) in a ratio of 1:20 (v/v). Fluorometric measurements were performed at 30 °C using a Shimadzu RF-5301 PC spectrofluorometer. The fluorescence intensity was measured at an emission wavelength of 393 nm with an excitation wavelength of 355 nm. The fluorescence intensity of 10 µM indo-1 was calibrated to free Ca²⁺ concentration using a Ca²⁺-EGTA buffering system. To quantify the amount of Ca²⁺ released from proteoliposomes by hydrogen ions, the fluorescence intensity of indo-1 after rapidly mixing the proteoliposomes with buffer was compared with the fluorescence intensity after addition of Triton X-100 (1% (v/v) final concentration).

To examine the functional role of the carboxyl terminus of BI-1 in Ca²⁺ efflux, the anti-BI-1 antibody was mixed with reaction samples at a ratio of 1 µg of reconstituted BI-1 to 10, 20, or 40 µg of antibody. After preincubating the sample at 30 °C for 15 min, Ca²⁺ release was measured using the same method described above. As a control, all experiments were repeated using liposomes without reconstituted BI-1 protein under the same conditions.

Hydrogen Ion-mediated Release of Ca²⁺ from Proteoliposomes Using ⁴⁵Ca²⁺—The proteoliposomes were prepared in the presence of ⁴⁵Ca²⁺ to include 20,000 cpm ⁴⁵Ca²⁺ in buffer D. To remove residual Ca²⁺ bound to the vesicle surface, the sample was applied to a Sephadex G25 column equilibrated with buffer D, and the liposomes were pelleted by centrifugation (100,000 x g for 30 min at 4 °C). Each pellet was then redissolved and dialyzed against an excess volume of buffer D for 12 h at 4 °C. The proteoliposomes were rapidly mixed with pH buffer solutions and incubated for 20 min at 30 °C. The samples were diluted with buffer E (buffer D plus 1.5 M KCl), and the liposomes were pelleted by centrifugation (100,000 x g for 30 min at 30 °C). The pellet was then dissolved in 1% (v/v) Triton X-100, and the radioactivity of each fraction (pellet and supernatant) was determined by scintillation counting.

Analysis of BI-1 Oligomerization—Endoplasmic reticulum were isolated from Neo-, BI-1-, and C-BI-1-overexpressing cells. ER pellets were resuspended in buffer B containing 250 mM sucrose, 10 mM HEPES-NaOH, pH 7.5, 2 mM KH₂PO₄, 5 mM sodium succinate, and 25 µM EGTA and then treated with or without acidic pH (5.0, 6.0, 7.0, or 7.4) for 30 min at 30 °C. The ER pellets were harvested by centrifugation and then analyzed by immunoblotting using an anti-HA antibody.

Analysis of Mitochondrial Ca²⁺—Cells were allowed to adhere to glass coverslips and incubated with 5% CO₂ at 37 °C. All Ca²⁺ imaging experiments were performed using an inverted epifluorescence Nikon microscope and a digital imaging system consisting of a TILL polychrome IV monochromator illumination system, a Sensicam 12-bit charge-coupled device camera, and TILL VisION acquisition and analysis software as described previously (11). Mitochondrial Ca²⁺ uptake was confirmed by dual loading cells with MitoTracker Green FM and Rhod-2-TRITC. Cells were excited with light at 488 ± 15 (10 ms) and 550 ± 25 nm (50 ms), and the emitted fluorescence from both dyes was collected through a fluorescein isothiocyanate/TRITC dual emission dichroic beam splitter and band pass filter (520 ± 15 and 600 ± 25 nm; Chroma). All experiments were performed at 37 °C with 5% CO₂.

Statistical Analyses—Results are presented as mean ± S.E. of n cells, and paired and unpaired Student's t tests were applied to test and control conditions where appropriate. Microcal Origin software (Northampton, MA) was used for statistical calculations.

View larger version (25K): [in this window] [in a new window]   FIGURE 1. Internal stores of thapsigargin-releasable Ca2+ are reduced in BI-1 stable transfectants. A, expression of BI-1-HA tagged protein in HT1080-Neo, -BI-1, and -C-BI-1 cells was compared by immunoblotting using an anti-HA antibody (upper panel). Lysates were normalized for total protein content (20 µg/lane). Blots were reprobed with anti-β-actin antibody to confirm equivalent loading (lower panel). B, HT1080-Neo, -BI-1, and -C-BI-1 cells were loaded with Fura-2/AM, cultured in Ca2+-free medium, and incubated with 1 µM (left) or 5 µM (right) thapsigargin (Tg) at 1 min. Individual cells were imaged (n = 16), and average fluorescence intensity was recorded over time. C, the peak of Ca2+ release obtained in B was quantified and expressed as a percentage of the increase above base line prior to thapsigargin treatment (mean ± S.E. of four independent experiments). *, p < 0.05 by unpaired t test versus the percentage of increased cytoplasmic Ca2+ in the indicated concentration of thapsigargin in Neo cells.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

View larger version (30K): [in this window] [in a new window]   FIGURE 2. Internal stores of thapsigargin-releasable Ca2+ are modulated by BI-1 in cells with conditional expression. A, HeLa-Tet-On-BI-1 cells were incubated with 1 µM doxycycline for 6, 12, 18, 24, or 36 h, and the expression of BI-1-HA tagged protein (top panel) was analyzed by immunoblotting using lysates normalized for total protein content (20 µg/lane). The blot was reprobed with anti-β-actin antibody (bottom panel). B, HeLa-Tet-On-BI-1-transfected cells were incubated with or without 1 µM doxycycline for 12 h, and then cells were loaded with Fura-2/AM, cultured in Ca2+-free medium, and incubated with 1 µM thapsigargin (Tg) (arrow). Individual cells were imaged (n = 16). A representative tracing is provided showing Ca2+ release over time. C, the peak of Ca2+ release obtained in B was quantified, and data are expressed as a percentage of the increase over base line prior to thapsigargin treatment (mean ± S.E. of four independent experiments). *, p < 0.05 by paired t test versus Neo cells.

View larger version (35K): [in this window] [in a new window]   FIGURE 3. Total and ryanodine receptor- and IP3R-releasable intracellular pools of Ca2+ are lower in BI-1-overexpressing cells. HT1080-Neo, -BI-1, and -C-BI-1 cells were cultured in Ca2+-free medium and stimulated with 0.1 µM ionomycin following pretreatment with 5 µg/ml digitonin (A) or with 300 µM 4-chloro-m-cresol (4-CMC) (B), 1 mM ATP (C), or 100 µM histamine (D). Individual cells were imaged (n = 20), fluorescence intensity was recorded, and the peak of the Ca2+ release was quantified as a percentage of the increase above base line prior to stimulation (*, p < 0.05 by unpaired t test versus Neo cells).

View larger version (48K): [in this window] [in a new window]   FIGURE 4. BI-1 increases the Ca2+ permeability of ER membranes. Thapsigargin (100 nM) was added at 1 min, and ionomycin (10 µM) was added either concomitantly with thapsigargin or at later time points (1–5 min at 1-min intervals). A, typical experiments showing initial peak calcium ([Ca2+]c) elevations in Neo- and BI-1-expressing cells; four traces with ionomycin addition at different times are superimposed to facilitate comparison. B, ionomycin-releasable Ca2+ (expressed as a percentage of the initial peak [Ca2+]c) is plotted as a function of time after thapsigargin addition in untransfected, Neo-, BI-1-, and C-BI-1-transfected cells. Results shown in B represent the mean ± S.E. of four independent experiments (*, p < 0.05 compared with the amount of [Ca2+]c at 0 min). Tg, thapsigargin; Iono, ionomycin; UT, untransfected.

View larger version (46K): [in this window] [in a new window]   FIGURE 5. pH differentially regulates microsomal Ca2+ when BI-1 is overexpressed. A, mag-Fura-2/AM-loaded microsomal fractions were isolated from untransfected, Neo-, BI-1-, and C-BI-1-transfected cells and treated with acidic condition (pH 5.4) at the time indicated. B, the decrease in microsomal Ca2+ induced by acidic pH in A was expressed as a percentage of the maximum releasable Ca2+. C, Fura-2/AM-loaded cells were treated with nigericin (5 µM) in Ca2+-free extracellular medium (pH 6.4), and the increase of [Ca2+]c was measured. Results shown in B and C represent the mean ± S.E. of four independent experiments (*, p < 0.05 compared with Neo cells). UT, untransfected; cyto, cytosolic.

View larger version (38K): [in this window] [in a new window]   FIGURE 6. Acidic pH induces Ca2+ release from BI-1-reconstituted lipid vesicles. A, changes in fluorescence were recorded kinetically after the dilution of proteoliposomes into acidic solution (pH 6.5, 6.0, 5.5, and 5.0). B, the amount of Ca2+ released in acidic pH is expressed as a percentage of the maximum releasable Ca2+. The acidic pH-induced changes in fluorescence were compared with those changes caused by 1% Triton X-100 (this intensity was set as 100%). Gray bars indicate the amount of Ca2+ released in the presence of anti-BI-1 antibody at a ratio of 1 µg of reconstituted BI-1 to 10, 20, or 40 µg of antibody after mixing the proteoliposomes with pH 5.0 buffer for 15 min at 30 °C. Hatched bars represent the amount of Ca2+ released in the absence of reconstituted BI-1 protein. C, the proteoliposomes were incubated with each of the indicated acid solutions, and the radioactivities of released 45Ca2+ were measured as described under "Materials and Methods." Gray and hatched bars indicate the amount of Ca2+ efflux in the presence of anti-BI-1 antibody and in the absence of reconstituted BI-1 protein, respectively. Data shown are the mean ± S.E. of three independent experiments. F.I., fluorescence intensity; TX-100, Triton X-100.

View larger version (46K): [in this window] [in a new window]   FIGURE 7. BI-1 forms oligomers, and BI-1 oligomerization is enhanced by acidic conditions. A, Neo-, BI-1-, or C-BI-1-expressing cells were fractionated to collect membrane-bound organelle fractions. Proteins were resolved by gel electrophoresis under non-reducing conditions followed by immunoblot analysis with anti-HA antibody. B, microsomal fractions were isolated from BI-1-expressing cells, and then the fractions were exposed to different pH conditions (pH 5.0, 6.0, 7.0, or 7.4) followed by gel electrophoresis under non-reducing conditions for immunoblot analysis with anti-HA antibody. Results shown in B represent the mean ± S.E. of seven independent experiments.

View larger version (35K): [in this window] [in a new window]   FIGURE 8. Acidic pH induces greater mitochondrial Ca2+ accumulation in BI-1-overexpressing cells. HT1080-Neo and HT1080-BI-1 cells were pre-loaded with 1 µM Rhod-2 for 1 h and then treated with nigericin (5 µM) in 140 mM K+, Ca2+-free buffer (pH 5.0, 6.0, and 7.0). A, Rhod-2 fluorescence intensity was measured over time. Data shown represent 16 independent samples. B, the peak of Rhod-2 fluorescence was recorded for several cells (n = 16 cells) (*, p < 0.05) versus Neo control cells at the same pH). Mitochondrial Rhod-2 Ca2+ fluoresence is shown (right).

View larger version (49K): [in this window] [in a new window]   FIGURE 9. BI-1 sensitizes cells to cell death induced by acidic conditions. A, cells were cultured in 140 mM K+-adjusted medium buffered to the indicated pH in the presence of 5 µM nigericin, and cell viability was measured 48 h later using the trypan blue method. B, mitochondria-enriched heavy membranes and cytosol fractions were obtained from cells at 48 h following treatment in the same medium as in A. The fractions were analyzed by SDS-PAGE/immunoblotting using antibodies recognizing Bax, cytochrome c, Hsp60 (mitochondrial marker), and tubulin (cytosolic marker). Data from blots of three replicate immunoblotting experiments were quantified by scanning densitometry, were normalized relative to Hsp60 for membrane fractions and tubulin for cytosol fractions, and are presented as bar graphs below the immunoblots. Cyt c, cytochrome c.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
BI-1 is a cytoprotective, integral membrane protein that is conserved in both animal and plant species and that resides in ER membranes. BI-1 function is associated with the regulation of intracellular Ca²⁺ homeostasis in both plant and mammalian systems (9, 19, 23, 24). Specifically ER free Ca²⁺ concentrations are reduced in BI-1-overexpressing cells, and cells with deficient BI-1 have elevated thapsigargin-releasable Ca²⁺ levels (9), suggesting that BI-1 protein controls ER Ca²⁺ levels. We have confirmed and extended these previous observations, showing that microsomes from BI-1-overexpressing cells contain reduced thapsigargin-releasable Ca²⁺ and showing that cells with either stable or conditional overexpression of BI-1 demonstrate less release of ER Ca²⁺ into the cytosol in response to thapsigargin. Furthermore the pools of ER Ca²⁺ regulated by ryanodine receptors and IP₃Rs, as shown by experiments using agonists of these receptors (4-chloro-m-cresol, histamine, and ATP), were reduced in BI-1-overexpressing cells as were total Ca²⁺ stores as shown by ionomycin and digitonin treatment.

We found that BI-1 renders ER membranes more porous to Ca²⁺, but the mechanism of this function is unknown. BI-1 could be an ion channel that conducts Ca²⁺, thus accounting for the passive leak of Ca²⁺ out of the lumen of the ER in cells overexpressing BI-1. Alternatively BI-1 may regulate the activity of other types of ER Ca²⁺ channels such as ryanodine receptors and IP₃Rs. Chemical antagonists of these channels failed to modify BI-1-mediated changes in Ca²⁺ regulation.⁴ Because these pharmacological approaches have scientific limitations (25), it is necessary to extend the studies using a more biological approach, e.g. an IP₃R knock-out system, in the future.

Here we show that the effect of BI-1 on ER Ca²⁺ permeability is pH-dependent and that the carboxyl-terminal cytosolic domain of BI-1 is required for regulating ER Ca²⁺. Interestingly this region contains a charged cluster of residues, EKDKKKEKK, within the proximal part of the cytosolic BI-1 carboxyl terminus. We hypothesized that this motif could function as a pH sensor similar to observations previously made for the TREK-1 potassium channel (17). For TREK-1, cytosolic acidosis increases the probability of channel opening; this is similar to the effects we observed for BI-1-overexpressing microsomes and cells with respect to release of ER Ca²⁺ regulation under acidic conditions. Furthermore we showed that BI-1 protein itself shows acidic pH-induced Ca²⁺ release in BI-1-reconsituted artificial liposomes (Fig. 6, A, B, C, and D). Thus, BI-1 itself, without interacting with another protein, may be a pH-sensitive Ca²⁺ regulator. Because the putative pH-sensing motif of BI-1 is similar among several BI-1 orthologs, this pH sensitivity may be evolutionarily conserved.

Acidic pH conditions and chemical cross-linkers promote BI-1 oligomerization (Fig. 7, A and B), suggesting that oligomerization of BI-1 regulates an ER Ca²⁺ channel or creates a BI-1-mediated Ca²⁺ channel, thus explaining why acidic pH more potently induces Ca²⁺ release from ER when BI-1 is overexpressed. Further studies are necessary to establish the mechanistic relevance of BI-1 oligomerization to pH-dependent ER Ca²⁺ permeabilization. ER Ca²⁺ levels and the mechanisms controlling its release into the cytosol regulate many cellular processes, including cell death, a variety of signal transduction events, regulation of ER protein folding (27), and gene expression (28). The interplay of H⁺ and Ca²⁺ is complex with transient receptor potential channels (29) and acid-sensing ionic channels among the possible mediators (30–32). Reports vary in terms of whether cytosolic pH rises or declines during apoptosis, but the preponderance of evidence favors acidification (33). In contrast, growth and survival factors typically induce cytosol alkalinization (34). Acidification of the cytosol is also a common occurrence in ischemia (35). Thus, BI-1 activity may be potentiated by acidic conditions induced during cellular stress and cytosolic acidification. In HT1080 cells overexpressing BI-1, exposure to acidic conditions resulted in enhanced rather than reduced cell death along with accentuated Bax activation and cytochrome c release from mitochondria as well as excessive Ca²⁺ accumulation in mitochondria. These observations thus demonstrate for the first time a cell death-promoting phenotype for BI-1 that is manifested during acidic stress. It remains to be demonstrated whether endogenous levels of BI-1 are sufficiently high to promote cell death during acid stress in vivo. More modest levels of BI-1 might even be protective during acid stress. For example, during acidification BI-1 may promote ER Ca²⁺ release to stimulate mitochondrial respiration, which would help restore cellular ATP levels and support plasma membrane ion transport mechanisms that restore physiological pH.

In the normal human body, pH compensation mechanisms such as HCO³⁺/CO₂ have been well established. Notably in severe pathologic conditions, systemic pH alterations do not occur, although local pH alterations including severe acidic environment may be observed. The dynamic accumulation pattern of Ca²⁺ at low pH may be related to the pathological sites of rheumatoid arthritis (36), hypoxia (37), the surrounding sites of metastatic cancers (38, 39), and atherosclerosis (40). The acidic sites around cancer have been reported to be as low as 5.2 (38). The pH of atherosclerotic lesions will depend on the rate of release of H⁺ from the cells including macrophages (26, 41).

The physiological and pathophysiological roles of BI-1 should be studied in detail in the near future. Additional studies will reveal the pathophysiological contexts in which pH-dependent regulation of BI-1 contributes to ER Ca²⁺ regulation and cell death.

	FOOTNOTES

 
^* This work was supported, in whole or in part, by National Institutes of Health Grant AG15393 (to J. C. R.). This work was also supported by Korea Research Foundation Grants KRF-2005-070-C00095, E00021 [GenBank] , and 2005-015-E00210 and Korea Science and Engineering Foundation Grants R01-2006-000-10422-0 and R01-2007-000-20275-0. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ To whom correspondence may be addressed: Burnham Inst. for Medical Research, 10901 N. Torrey Pines Rd., La Jolla, CA 02037. Tel.: 858-795-5301; Fax: 858-646-3194; E-mail: jreed{at}burnham.org. ² To whom correspondence may be addressed: Dept. of Pharmacology and Inst. of Cardiovascular Research, Medical School, Chonbuk University, Jeonju, Chonbuk 561-181, Republic of Korea. Tel.: 82-63-270-3092; Fax: 82-63-275-2855; E-mail: hjchae{at}chonbuk.ac.kr.

³ The 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.

⁴ H.-R. Kim, G.-H. Lee, K.-C. Ha, T. Ahn, J.-Y. Moon, B.-J. Lee, S.-G. Cho, S. Kim, Y.-R. Seo, Y.-J. Shin, S.-W. Chae, J. C. Reed, and H.-J. Chae, unpublished data.

	ACKNOWLEDGMENTS

 
We thank Prof. Mi-Jae Lim for critical review of this manuscript.

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