Ca 2+ ⁄ H + antiporter-like activity of human recombinant Bax inhibitor-1 reconstituted into liposomes Taeho Ahn 1 , Chul-Ho Yun 2 , Ho Zoon Chae 2 , Hyung-Ryong Kim 3 and Han-Jung Chae 4 1 Department of Biochemistry, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea 2 School of Biological Sciences and Technology, Chonnam National University, Gwangju, Korea 3 Department of Dental Pharmacology, School of Dentistry, Wonkwang University, Iksan, Korea 4 Department of Pharmacology and Institute of Cardiovascular Research, Medical School, Chonbuk National University, Jeonju, Korea Bax inhibitor-1 (BI-1; also known as ‘testis-enhanced gene transcript’) is an antiapoptotic protein capable of inhibiting Bax activation and translocation to mito- chondria [1]. Cells isolated from BI-1 ) ⁄ ) mice exhibited hypersensitivity to apoptosis induced by endoplasmic reticulum (ER) stress [2]. In BI-1 ) ⁄ ) mice, the ische- mia ⁄ reperfusion-induced unfolded protein response increased significantly, leading to increased cell death [3]. This ubiquitously expressed protein has 237 amino acids and a molecular mass of  26 kDa. Computer predictions and experimental observations suggest that BI-1 is a membrane-spanning protein with six to seven transmembrane domains and a cytoplasmic C-terminus localizing to the ER and nuclear envelope [4]. Sequence homology among several species indicates that the characteristic hydrophobicity and ER mem- brane localization have been evolutionarily conserved [5]. Functionally, BI-1 affects the leakage of calcium ions from the ER, as measured with Ca 2+ -sensitive, ER-targeted fluorescent proteins and Ca 2+ -sensitive dyes [2]. BI-1 also regulates the production of reactive oxygen species through functional inhibition of Bax [6,7]. In the BI-1 overexpression system, the increase in heme oxygenase-1 expression was also suggested as a Keywords antiporter; Bax inhibitor-1; Ca 2+ -release; proteoliposome; reconstitution Correspondence T. Ahn, Department of Biochemistry, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Korea Fax: +82 62 530 2809 Tel: +82 62 530 2823 E-mail: thahn@chonnam.ac.kr H J. Chae, Department of Pharmacology and Institute of Cardiovascular Research, Medical School, Chonbuk University, Jeonju, Chonbuk 561-181, Korea Fax: +82 63 275 2855 Tel: +82 63 270 3092 E-mail: hjchae@chonbuk.ac.kr (Received 11 November 2008, revised 30 December 2008, accepted 10 February 2009) doi:10.1111/j.1742-4658.2009.06956.x We investigated the functional activity of recombinant Bax inhibitor-1 reconstituted into liposomes. When proteoliposomes were suspended in acidic solutions, encapsulated Ca 2+ was released from the membranes, as previously suggested [Kim HR, Lee GH, Ha KC, Ahn T, Moon JY, Lee BJ, Cho SG, Kim S, Seo YR, Shin YJ et al. (2008) J Biol Chem 283, 15946–15955]. Concomitantly, proton ions were internalized when assayed using the time-dependent change in the fluorescence of the pH-sensitive dye oxonol V entrapped in the proteoliposomes. The influx of proton ions was confirmed by observing tritium accumulation in the membranes. However, the external acidity of the membranes per se did not induce proton ion influx without internalized Ca 2+ . These results suggest that reconstituted Bax inhibitor-1 has a Ca 2+ ⁄ H + antiporter-like activity. Abbreviations BI-1, Bax inhibitor-1; ER, endoplasmic reticulum; InsP 3 , inositol 1,4,5-trisphosphate; PICR, proton ion-induced Ca 2+ release. FEBS Journal 276 (2009) 2285–2291 ª 2009 The Authors Journal compilation ª 2009 FEBS 2285 regulatory mechanism for reactive oxygen species through the activation of Nrf2 transcription factor [8]. Recently, we also suggested that BI-1 acts as a pH-dependent Ca 2+ channel in the ER, which increases Ca 2+ leakage via a mechanism dependent on both the pH and the C-terminal cytosolic region of the protein [9]. However, the precise role of BI-1 remains unknown. Our results, following the reconstitution of recombinant BI-1 into membranes, support a role for BI-1 as a Ca 2+ ⁄ H + antiporter. Results and Discussion Proton ion-induced Ca 2+ release measurement using indo-1 fluorescence and 45 Ca 2+ We have previously suggested that BI-1 is a pH-depen- dent regulator of Ca 2+ channel activity [9]. To ascer- tain the validity of the proper refolding and reconstitution of recombinant BI-1 into lipid bilayers, a functional assay was performed using proteolipo- somes to monitor proton ion-induced Ca 2+ release (PICR) from the liposomes. Rapid dilution of BI-1- reconstituted vesicles in acidic solution induced the release of entrapped Ca 2+ (Fig. 1), confirming BI-1 activity as a Ca 2+ channel. In kinetic analyses (Fig. 1A), the relaxation time was  37 s. Using other pH values (e.g. pH 6.5 and 5.0), relaxation times were calculated to be in the region of 35)42 s (results not shown), in which any tendency for acidity was not observed. The amounts of Ca 2+ released were similar when assayed using a fluorescent dye and 45 Ca 2+ (Fig. 1B). These results also suggest that increasing acidity is linked to increased Ca 2+ release by the pro- teoliposomes, implying that concomitant refolding and reconstitution of BI-1 can be used as a functional assay of the recombinant protein. As a control experi- ment, Ca 2+ efflux from proteoliposomes was measured under alkaline conditions, such as pH 8.5 and 9.0, but any remarkable Ca 2+ release was not observed. PICR was also performed with liposomes in the absence of reconstituted BI-1. Acidic or alkaline conditions did not induce the release of encapsulated Ca 2+ in the absence of BI-1, when assayed using indo-1 and 45 Ca 2+ . This suggests that the liposome was stable in acidic or alkaline solutions and that the PICR described in Fig. 1 was entirely mediated by reconsti- tuted BI-1. External Ca 2+ effect on PICR of reconstituted BI-1 External (cytosolic) Ca 2+ is a well-known modulator of the channel activity of the inositol 1,4,5-trisphos- phate (InsP 3 ) receptor [10,11]. To compare the pattern for stimulus-induced Ca 2+ release between BI-1 and the InsP 3 receptor, we investigated the effect of external Ca 2+ on the PICR. External Ca 2+ consistently inhib- ited the PICR, regardless of the pH value outside the liposomes, when the Ca 2+ concentration was increased Fig. 1. Acidic or alkaline pH induced Ca 2+ release from BI-1 recon- stituted into lipid vesicles. (A) The change in the fluorescence inten- sity of indo-1 was recorded kinetically after the rapid dilution of proteoliposomes into an acidic solution (pH 5.5). (B) The amounts of Ca 2+ released by acidic or alkaline pH were expressed as a per- centage of the maximum releasable Ca 2+ using the fluorescence and the radioactivity of 45 Ca 2+ , and were compared with those caused by the addition of 1% Triton X-100 (TX-100), which was set to 100%. (C)pH 5.0 and (C)pH 9.0 represent the Ca 2+ release at pH 5.0 and 9.0 in the absence of reconstituted BI-1, respectively. Data shown are the mean ± SE of five independent experiments. Ca 2+ ⁄ H + antiporter activity of Bax inhibitor-1 T. Ahn et al. 2286 FEBS Journal 276 (2009) 2285–2291 ª 2009 The Authors Journal compilation ª 2009 FEBS to 5 lm (Fig. 2). It has been suggested that a biphasic mode of external Ca 2+ on the channel activity of the InsP 3 receptor is important in generating characteristic Ca 2+ signaling within cells [12–15]. Moreover, although the effect of external Ca 2+ differs according to the type of InsP 3 receptor, Ca 2+ efflux exhibits a biphasic pattern, with a maximal peak near 0.2)0.3 lm of cytoplasmic Ca 2+ [11,12]. Therefore, the mode of regulation by external Ca 2+ suggests that BI-1 shows Ca 2+ signaling different from that of the conventional InsP 3 receptor, even though the other regulation modes were not experimentally compared. Proton influx into proteoliposomes by exchange with Ca 2+ efflux To obtain more insight into the functional influence of proton ions on the PICR and to test the possibility that BI-1 incorporated into membranes might exhibit a Ca 2+ ⁄ H + antiporter-like function, we measured the changes in emission fluorescence for encapsulated oxo- nol V (a pH-sensitive and non-permeable fluorescent probe), which was entrapped in membranes in the presence of internalized Ca 2+ , as described previously [16]. Figure 3A shows the time course of the decrease in fluorescence of the probe upon mixing proteoliposomes with acidic solutions. Relaxation times were calculated to be in the range of  32)45 s, Fig. 2. Effect of external Ca 2+ on PICR of reconstituted BI-1. The effects of external Ca 2+ on the PICR were plotted as external Ca 2+ concentration versus the amount of 45 Ca 2+ released from proteo- liposomes. The external Ca 2+ concentration was buffered by the EGTA–Ca 2+ chelating system and the experiment was performed using the method described in Fig. 1. Inset: the radioactivity by 45 Ca 2+ efflux was normalized to 100% upon the absence of exter- nal Ca 2+ and expressed as a relative percentage. Fig. 3. Proton influx into proteoliposomes. (A) The time course for the decrease in fluorescence of oxonol V encapsulated into proteo- liposomes was measured. After rapid mixing of proteolipsomes containing internalized Ca 2+ with acidic solutions, the emission fluo- rescence at 630 nm (excitation at 610 nm) was measured as a function of time. Lines a, b, c and d indicate an exterior pH of 6.5, 6.0, 5.5 and 5.0, respectively. Line e represents the emission fluo- rescence in the absence of entrapped Ca 2+ . (B) The amount of pro- ton uptake into liposomes as a result of the decrease in pH was assayed by measuring the radioactivity of tritium, as described in Materials and methods (In the figure, the y-axis represents the cpm values of pellet). Black and hatched bars represent the radio- activities of liposomes consisting of pure phospholipids without reconstituted BI-1 protein and the cpm values of proteoliposomes without internalized Ca 2+ , respectively, after the same procedure described above was performed. White and gray bars represent the cpm values of proteoliposomes after mixing the vesicles with pH 7.4 and each indicated acidic solution, respectively. T. Ahn et al. Ca 2+ ⁄ H + antiporter activity of Bax inhibitor-1 FEBS Journal 276 (2009) 2285–2291 ª 2009 The Authors Journal compilation ª 2009 FEBS 2287 which were very similar to the rate of Ca 2+ release. However, no correlation between the relaxation time and the acidity was apparent. As a control experiment, the change in fluorescence was also analyzed under the conditions described above, without encapsulated Ca 2+ . In this case, we did not observe any significant decrease in the emission fluorescence of oxonol V (Fig. 3A, line e). This indicates the importance of entrapped Ca 2+ for the uptake of protons into proteo- liposomes. However, the experiment was not per- formed in the presence of various concentrations of internalized Ca 2+ . Therefore, it is not, at present, pos- sible to determine how many proton ions are moved into liposomes in response to entrapped Ca 2+ ions. Proton influx was also ascertained by measuring the radioactivity of proteoliposomes containing encapsu- lated Ca 2+ (Fig. 3B). When the proteoliposomes were suspended in the indicated acidic buffer solutions containing [ 3 H], the radioactivity inside the proteolipo- somes increased with decreasing external pH, indica- tive of a pH-dependent proton uptake into the proteoliposomes. However, at pH 5.0, the radioactivity of the proteoliposomes decreased, compared with the radioactivity measured at pH 5.5, which was not con- sistent with the results obtained using oxonol V. As expected, without reconstituted BI-1, the remarkable proton influx could not be observed, regardless of the external acidity. By contrast, significant tritium accu- mulations were shown with proteoliposomes in the absence of internal Ca 2+ , which is different from the change in oxonol V-mediated fluorescence described in Fig. 3A. Furthermore, the cpm values increased with increasing acidity. This may be related to the finding that the C-terminal region of BI-1, which contains a cluster of charged residues, EKDKKKEKK, is impor- tant in the protein function as a pH sensor [9], similar to observations made for the TREK-1 potassium chan- nel [17]. Therefore, this cytoplasmic motif may bind to proton ions and result in the increased cpm values (as shown in Fig. 3B), without proton uptake into proteo- liposomes in the absence of internal Ca 2+ . However, at present, there is no direct evidence for the associa- tion of BI-1 with proton ions. Taken together, these results suggest that encapsulated Ca 2+ facilitates proton movement into proteoliposomes. As a control experiment, liposomes without reconsti- tuted BI-1 protein displayed background levels of radioactivity, suggesting that proton uptake was not induced by differences in the concentration of proton ions between the interior and exterior of proteolipo- somes per se (black bars in Fig. 3B). Collectively, these results support the possible role of reconstituted BI-1 as a Ca 2+ ⁄ H + antiporter. Proteolytic cleavage of reconstituted BI-1 On the basis of the result described in Fig. 3, it was considered that the proton influx associated with Ca 2+ efflux resulted from the membrane topology of BI-1, because recombinant BI-1 proteins could be incorpo- rated into artificial lipid bilayers with different confor- mations during proteoliposome formulation. To test this possibility and to obtain insight into the confor- mation of reconstituted BI-1, proteolytic cleavage was performed with the prepared proteoliposomes using carboxypeptidase B, which was added externally. The protease preferentially catalyzes the hydrolysis of the basic amino acids lysine, arginine and ornithine from the C-terminal end of polypeptides. After cleavage, SDS ⁄ PAGE analysis revealed that nearly all the recon- stituted BI-1 was partially digested by the protease and intact BI-1 could not be observed (Fig. 4). This suggests that most recombinant BI-1 proteins have a protease-accessible structure in membranes during the reconstitution procedure although the precise confor- mation of BI-1 in a lipid bilayer is not currently known. In relation to the membrane topology of BI-1, previous observations also support that the C-terminus of BI-1 is outside the ER [18,19]. After proteolytic digestion, the PICR was examined with the resulting proteoliposomes. However, we could not detect any remarkable Ca 2+ release and proton influx (results not shown). Considering the proteolytic pattern on SDS ⁄ PAGE, these results imply that the C-terminal region of BI-1 recognizing external acidity may be exposed to the outer surface of proteoliposomes, where they can be cleaved by the protease. In terms of the BI-1-induced protective function, the interplay of Ca 2+ and H + needs to be carefully Fig. 4. Proteolytic cleavage of proteoliposomes. Reconstituted BI-1 was cleaved by carboxypeptide B and the products were analyzed by SDS ⁄ PAGE. Lanes 1 and 2 represent the reconstituted BI-1 and the digested sample, respectively. Ca 2+ ⁄ H + antiporter activity of Bax inhibitor-1 T. Ahn et al. 2288 FEBS Journal 276 (2009) 2285–2291 ª 2009 The Authors Journal compilation ª 2009 FEBS interpreted. At normal pH, BI-1 has been suggested to leak Ca 2+ , leading to a decrease in the Ca 2+ concen- tration in the ER [9,20]. In this study, a unique func- tion for the BI-1–Ca 2+ ⁄ H + antiporter is suggested. At normal pH, Ca 2+ homeostasis may be maintained even in the presence of other stimuli such as ER stress, because of Ca 2+ porter activity. In view of pH homeo- stasis, proton uptake may be accelerated by a Ca 2+ ⁄ H + antiporter-like function of BI-1, leading to a cell-protective function. Therefore, it may be antici- pated that the antiporter activity of BI-1 contributes to adaptation of the channel protein and ⁄ or ER Ca 2+ regulation. However, in exceptional surroundings, such as at severely acidic pH, BI-1-overexpressed cells may be highly sensitized to cell death, consistent with an in vivo cell-based study [9]. More detailed studies are needed to establish the functional relevance of BI-1 antiporter activity to cell death. Conclusion Proton ions induce Ca 2+ efflux from BI-1-reconsti- tuted liposomes containing entrapped Ca 2+ , and con- comitantly proton influx. Taking into account that the movement of these ions was not observed without encapsulated Ca 2+ and acidity, our results suggest that the reconstituted recombinant BI-1 has a Ca 2+ ⁄ H + antiporter-like function. Materials and methods All phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL, USA). The fluorescent Ca 2+ indicator, indo-1, and the pH-sensitive dye, oxonol V, were purchased from Invitrogen (Carlsbad, CA, USA). Radioactive materi- als 45 Ca 2+ and 3 H were obtained from GE Healthcare Bio- Sciences (Piscataway, NJ, USA). Construction of Bax inhibitor-1 expression plasmid Human BI-1 cDNA was subcloned into the pET12a expres- sion vector to generate pET12a ⁄ BI-1 using PCR. PCR amplification was designed to include BamHI and HindIII restriction enzyme sites for forward and reverse primer oli- gonucleotides, respectively (forward: 5¢-GACGGATCCAT GAACATATTTGATCGAAAG-3¢, reverse: 5¢-GACAAGC TTTTATTTCTTCTCTTTCTTCTT-3¢). PCR was per- formed with Pfu polymerase (Stratagene, La Jolla, CA, USA). The mixture was preincubated for 5 min at 94 °C before the addition of the polymerase, followed by amplifi- cation for 30 cycles: 94 °C for 90 s, 60 °C for 90 s and 72 °C for 3 min. The resulting PCR product was purified, digested with BamHI and HindIII, and then ligated into a pET-12a vector treated with the same restriction enzymes. The nucleotide sequence of the entire region including the BI-1 gene was analyzed by dideoxy sequencing. Expression of recombinant BI-1 protein Cultures of Escherichia coli BL21(DE3) containing pET12a ⁄ BI-1 were grown at 37 °C in 500 mL of Luria–Ber- tani ⁄ ampicillin (50 lgÆmL )1 ) until an attenuance at 600 nm of 0.5 was attained. Induction of the recombinant protein was carried out by the addition of 0.5 mm isopropyl-b-d- thiogalactopyranoside and further incubation for 4 h. To obtain inclusion bodies of expressed BI-1, harvested bacte- rial pellets were resuspended in lysis buffer consisting of 25 mm Tris ⁄ HCl (pH 8.0), 100 mm NaCl, 1 mm phen- ylmethanesulfonyl fluoride and 5 lgÆmL )1 of benzamidine, leupeptin and pepstatin. The cells were lysed by passage through an Amicon French pressure cell (Millipore, Billeri- ca, MA, USA) and the lysates were centrifuged (12 000 g, 10 min, 4 °C) to recover the inclusion bodies. The pellets were resuspended with 5 mL of the lysis buffer using probe sonication and were centrifuged (12 000 g, 10 min, 4 °C). The resuspension and centrifugation steps were repeated five times. The final inclusion body-containing pellet was dissolved in buffer A, which consisted of 20 mm Hepes (pH 7.4), 8 m urea, 0.1 mm dithiothreitol, 1 mm CaCl 2 and 1.5% Chaps by incubating the sample at 25 °C for 10 min with gentle shaking. Refolding and reconstitution of BI-1 inclusion bodies into proteoliposomes Chloroform solutions of lipids were stored in sealed amp- ules under argon gas at )20 °C. Phosphatidylcholine, phos- phatidylethanolamine and phosphatidylserine (all from bovine brain) dissolved in chloroform were mixed to give a respective molar ratio of 5 : 3 : 2. The phospholipid con- centrations were determined using a phosphorus assay [21]. A phospholipid concentration of 5 mm was used to recon- stitute the BI-1 protein. The solvent was evaporated under a stream of argon gas and the residual chloroform was removed by centrifugal lyophilization. The dry lipids were hydrated with 1 mL of buffer A containing  20 lg BI-1. The mixtures were dialyzed for 12 h against an excess vol- ume of buffer B (buffer A without Chaps) containing 2 m urea. The dialysis step was repeated for 12 h against an excess volume of buffer C (buffer B without urea). The resulting proteoliposomes were pelleted by centrifugation at 100 000 g for 30 min at 4 °C and washed with buffer D (20 mm Hepes, pH 7.4, 100 mm NaCl, 0.1 mm dithiothrei- tol, 0.5 mm EGTA, 1 m KCl) and then were dialyzed against buffer E (buffer D without 1 m KCl and 0.5 mm EGTA) for 12 h at 4 °C. The resulting proteoliposomes T. Ahn et al. Ca 2+ ⁄ H + antiporter activity of Bax inhibitor-1 FEBS Journal 276 (2009) 2285–2291 ª 2009 The Authors Journal compilation ª 2009 FEBS 2289 were passed through Chelex 100 (Bio-Rad, Hercules, CA, USA) to remove free Ca 2+ . The formation of proteolipo- somes was monitored spectrofluorometrically by measure- ment of light scattering during dialysis at a wavelength of 450 nm. The amounts of reconstituted BI-1 protein were determined using the NanoOrangeÒ protein quantitation kit (Invitrogen). Hydrogen ion-mediated Ca 2+ release from proteoliposomes using indo-1 fluorescence and 45 Ca 2+ Ca 2+ release from the proteoliposomes was measured as described previously [4]. Briefly, Ca 2+ efflux was observed by measuring the fluorescence changes of external indo-1 (5 lm) after rapid dilution of the proteoliposomes with acidic solutions (50 mm sodium phosphate pH 6.5 or 50 mm sodium citrate pH 6.0 and 5.5) at a ratio of 1 : 20 (v ⁄ v). The fluorescence intensity was measured at emission and excitation wavelengths of 393 and 355 nm, respectively. To quantify the proton ion-mediated release of Ca 2+ from proteoliposomes, the acidity-induced fluorescence intensity of indo-1 was compared with the fluorescence intensity after addition of Triton X-100 to a final concentration of 1% (v ⁄ v). Proteoliposomes were also prepared in the pres- ence of 45 Ca 2+ to include  20 000 cpm of 45 Ca 2+ under the same conditions. After the dilution of proteoliposomes with acidic solutions to induce Ca 2+ efflux, the sample was centrifuged (100 000 g, 30 min, 30 °C). The amount of pro- ton-mediated Ca 2+ release was determined by the radioac- tivity of the pellet and supernatant using scintillation counting. Measurement of proton ion influx into proteoliposome To analyze proton influx into proteoliposomes coupling Ca 2+ efflux, the pH-sensitive fluorescent probe oxonol V was encapsulated inside proteoliposomes (pH 7.4) during membrane formulation in the presence of Ca 2+ , as previ- ously described [16]. After rapid mixing of the proteolipo- somes with each of the indicated buffer solutions (pH 9.0, 8.5, 6.5, 6.0, 5.5, and 5.0 as described above), the decrease in fluorescence was measured at an emission wavelength of 630 nm (excitation at 610 nm). Proton uptake was also investigated by measuring the tritium radioactivities. Prote- oliposomes with an interior pH of 7.4, in the presence or absence of internalized Ca 2+ , were incubated with each indicated buffer solution containing [ 3 H] ( 20 000 cpm) for 20 min at 30 ° C. The radioactivities of the pellet and supernatant fractions were measured after centrifugation of reaction samples using a Beckman TLA 100.2 rotor (Beck- man Coulter, Palo Alto, CA, USA) at 70 000 rpm for 30 min. Removal of Ca 2+ contamination Removal of Ca 2+ contamination was conducted as described previously [22]. Ca 2+ contamination during all experiments was checked using the fluorescence of the Ca 2+ indicator indo-1 before measurements. Proteolytic cleavage of reconstituted BI-1 with carboxypeptidase B After reconstitution of BI-1, proteoliposomes were ultracen- trifuged as described above. The pellet was suspended in 20 mm Hepes (pH 7.4) containing carboxypeptidase B at a BI-1 ⁄ carboxypeptidase B ratio of 50 : 1 (w ⁄ w). The sample was incubated at 25 °C for 20 min and the reaction was ter- minated by the addition of 1 m m EDTA (final concentra- tion). The products were analyzed by 12.5% SDS ⁄ PAGE and followed by Coomassie Brilliant Blue staining of the resolved proteins. Acknowledgements This work was supported by Korea Research Foun- dation Grant funded by the Korean Government (KRF-2008-314-C00231) and by Korea Science and Engineering Foundation Grant (R01-2006-000-10422-0). References 1 Xu Q & Reed JC (1998) Bax inhibitor-1, a mammalian apoptosis suppressor identified by functional screening in yeast. Mol Cell 1, 337–346. 2 Chae HJ, Kim HR, Xu C, Bailly-Maitre B, Krajewska M, Krajewski S, Banares S, Cui J, Digicaylioglu M, Ke N et al. (2004) BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum stress. Mol Cell 15, 355–366. 3 Bailly-Maitre MB, Fondevila C, Kaldas F, Droin N, Luciano F, Ricci JE, Croxton R, Krajewska M, Zapata JM, Kupiecweglinski JW et al. (2006) Cytoprotective gene bi-1 is required for intrinsic protection from endo- plasmic reticulum stress and ischemia–reperfusion injury. Proc Natl Acad Sci USA 103, 2809–2814. 4Hu ¨ ckelhoven R (2004) BAX Inhibitor-1, an ancient cell death suppressor in animals and plants with prokaryotic relatives. Apoptosis 9 , 299–307. 5 Chae HJ, Ke N, Kim HR, Chen S, Godzik A, Dickman M & Reed JC (2003) Evolutionarily conserved cytopro- tection provided by Bax inhibitor-1 homologs from ani- mals, plants, and yeast. Gene 323, 101–113. 6 Kawai-Yamada M, Ohori Y & Uchimiya H (2004) Dis- section of Arabidopsis Bax inhibitor-1 suppressing Bax-, hydrogen peroxide-, and salicylic acid-induced cell death. Plant Cell 16, 21–32. Ca 2+ ⁄ H + antiporter activity of Bax inhibitor-1 T. Ahn et al. 2290 FEBS Journal 276 (2009) 2285–2291 ª 2009 The Authors Journal compilation ª 2009 FEBS 7 Baek D, Nam J, Koo YD, Kim DH, Lee J, Jeong JC, Kwak SS, Chung WS, Lim CO, Bahk JD et al. (2004) Bax-induced cell death of Arabidopsis is meditated through reactive oxygen-dependent and -independent processes. Plant Mol Biol 56, 15–27. 8 Lee GH, Kim HK, Chae SW, Kim DS, Ha KC, Cuddy M, Kress C, Reed JC, Kim HR & Chae HJ (2007) Bax inhibitor-1 regulates endoplasmic reticulum stress-associated reactive oxygen species and heme oxygenase-1 expression. J Biol Chem 282, 21618– 21628. 9 Kim HR, Lee GH, Ha KC, Ahn T, Moon JY, Lee BJ, Cho SG, Kim S, Seo YR, Shin YJ et al. (2008) Bax inhibitor-1 is a pH-dependent regulator of Ca 2+ chan- nel activity in the endoplasmic reticulum. J Biol Chem 283, 15946–15955. 10 Taylor CW & Laude AJ (2002) IP 3 receptors and their regulation by calmodulin and cytosolic Ca 2+ . Cell Cal- cium 32, 321–334. 11 Miyakawa T, Maeda A, Yamazawa T, Hirose K, Kuro- saki T & Iino M (1999) Encoding of Ca 2+ signals by differential expression of IP 3 receptor subtypes. EMBO J 18, 1303–1308. 12 Bezprozvanny I, Watras J & Ehrlich BE (1991) Bell-shaped calcium-response curve of Ins(1,4,5)P 3 - and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature 351, 751–754. 13 Finch EA, Turner TJ & Goldin SM (1991) Calcium as a coagonist of inositol 1,4,5-trisphosphate-induced calcium release. Science 252, 443–446. 14 Thomas AP, Bird GS, Hajnoczky G, Robb-Gaspers LD & Putney JW Jr (1996) Spatial and temporal aspects of cellular calcium signaling. FASEB J 10, 1505–1517. 15 Berridge MJ (1998) Neuronal calcium signaling. Neuron 21, 13–26. 16 Yoo SH & Jeon CJ (2000) Inositol 1,4,5-trisphosphate receptor ⁄ Ca 2+ channel modulatory role of chromogra- nin A, a Ca 2+ storage protein of secretory granules. J Biol Chem 275, 15067–15073. 17 Kloda A, Ghazi A & Martinac B (2006) C-terminal charged cluster of MscL, RKKEE, functions as a pH sensor. Biophys J 90, 1992–1998. 18 Bolduc N, Ouellet M, Pitte F & Brisson LF (2003) Molecular characterization of two plant BI-1 homo- logues which suppress Bax-induced apoptosis in human 292 cells. Planta 216, 377–386. 19 Shikano S & Li M (2003) Membrane receptor traffick- ing: Evidence of proximal and distal zones conferred by two independent endoplasmic reticulum localization sig- nals. Proc Natl Acad Sci USA 100, 5783–5788. 20 Xu C, Xu W, Palmer AE & Reed JC (2008) BI-1 regu- lates endoplasmic reticulum Ca 2+ homeostasis down- stream of Bcl-2 family proteins. J Biol Chem 283, 11477–11484. 21 Vaskovsky VE, Kostetsky EY & Vasendin IM (1975) A universal reagent for phospholipid analysis. J Chroma- togr 114, 129–141. 22 Meyer T, Wensel T & Stryer L (1990) Kinetics of cal- cium channel opening by inositol 1,4,5-trisphosphate. Biochemistry 29, 32–37. T. Ahn et al. Ca 2+ ⁄ H + antiporter activity of Bax inhibitor-1 FEBS Journal 276 (2009) 2285–2291 ª 2009 The Authors Journal compilation ª 2009 FEBS 2291 . Ca 2+ ⁄ H + antiporter-like activity of human recombinant Bax inhibitor-1 reconstituted into liposomes Taeho Ahn 1 , Chul-Ho Yun 2 , Ho Zoon Chae 2 ,. February 2009) doi:10.1111/j.1742-4658.2009.06956.x We investigated the functional activity of recombinant Bax inhibitor-1 reconstituted into liposomes. When proteoliposomes were suspended in acidic solutions, encapsulated. internalized Ca 2+ . These results suggest that reconstituted Bax inhibitor-1 has a Ca 2+ ⁄ H + antiporter-like activity. Abbreviations BI-1, Bax inhibitor-1; ER, endoplasmic reticulum; InsP 3 ,