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Dear Editor, Bax Inhibitor-1 (BI-1) is an evolutionary conserved endoplasmic reticulum (ER)-located protein that protects against ER stress-induced apoptosis.1 This function has been closely related to its ability to permeate Ca2+ from the ER2 and to lower the steady-state [Ca2+]ER.3 BI-1 may function as an H+/Ca2+-antiporter2 or Ca2+ channel.4 Recently, BI-1 was proposed as a negative regulator of autophagy through IRE1α.5 However, recent findings indicate that BI-1 may promote autophagy.6 The latter required the presence of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R). The observations were explained through BI-1-enhanced IP3R activity, which lowered steady-state [Ca2+]ER, reducing ER-mitochondrial Ca2+ transfer and decreasing mitochondrial bio-energetics.7 However, direct evidence that BI-1 binds to IP3Rs and sensitizes IP3-induced Ca2+ release (IICR) is lacking. Therefore, we studied the regulation of IP3R function by BI-1 (see Supplementary Information for Methods). We constructed a 5xMyc-BI-1-expression plasmid, allowing the detection and purification of ectopically expressed BI-1 from transfected HeLa cells using anti-Myc-agarose beads (Figure 1a). Using isoform-specific IP3R antibodies, we demonstrated the co-immunoprecipitation of IP3R1 and IP3R3 with 5xMyc-BI-1 from HeLa cell lysates. Next, we screened for the subdomain of BI-1 responsible for IP3R interaction. We found that a synthetic Flag-tagged peptide containing BI-1’s Ca2+-channel pore domain (CTP1; amino acids 198–217 of human BI-1) interacted with IP3R1 (Figure 1b). Lysates not exposed to Flag-CTP1 served as negative control. Moreover, proteolytic fragments of the IP3R containing its C terminus (indicated as IP3R1-Cterm in Figure 1b) were immunoprecipitated with Flag-CTP1. These C-terminal fragments were recognized by our antibody (Rbt03) that has its epitope in the last 15 C-terminal amino acids of the IP3R1.8 These fragments include the Ca2+-channel pore of the IP3R1, indicating that the Ca2+-channel pore domain of BI-1 interacted with the Ca2+-channel pore domain of IP3R1. Next, we examined the effect of BI-1 on IP3R function. Therefore, we used BI-1−/− mouse embryonic fibroblasts (MEF) and stably and ectopically overexpressed either empty vector (RFP-only), wild-type BI-1 or BI-1D213R with a bi-cistronic C-terminal IRES-RFP reporter. BI-1D213R is a mutant, in which the Asp213 critical for BI-1-mediated Ca2+ flux is altered into an Arg and which fails to lower [Ca2+]ER.4 BI-1-mRNA expression was detected using specific primers, and similar expression levels were found for wild-type BI-1 and BI-1D213R, while no signal was observed in vector-expressing BI-1−/− MEF cells (inset Figure 1c). Wild-type BI-1, but not BI-1D213R, overexpression significantly improved cell survival after thapsigargin exposure, an irreversible SERCA inhibitor, which kills cells through ER stress (empty vector: 33.65±4.48%; wild-type BI-1: 44.39±5.31%*; BI-1D213R: 34.14±4.19% surviving cells after 48 h, 20 nM thapsigargin normalized to vehicle-treated cells expressing empty vector. Mean±S.E.M. of four pooled experiments done in triplicates is shown, *P<0.05 Student’s t-test). These data indicate that BI-1’s Ca2+-flux properties are essential for BI-1’s anti-apoptotic function. Next, we analyzed the direct effect of ectopically expressed BI-1 on IP3R function in the absence of endogenous BI-1 (Figure 1c). We used a unidirectional 45Ca2+-flux assay in saponin-permeabilized BI-1−/− MEF cells, allowing direct ER access and an accurate analysis of IP3R function in the absence of plasmalemmal Ca2+ fluxes, SERCA activity or mitochondrial Ca2+ uptake.8 Cells ectopically overexpressing BI-1 displayed a sensitized IICR and concomitant decrease in EC50 from 3.57 μM to 2.25 μM IP3. To exclude that Ca2+ flux mediated by BI-1 indirectly sensitized IP3Rs through Ca2+-induced Ca2+ release, we examined the effect of BI-1D213R overexpression on IP3R function. BI-1D213R also sensitized IICR and concomitantly decreased the EC50 from 3.57 μM to 1.98 μM IP3. This correlates with the ability of BI-1D213R to co-immunoprecipitate with IP3Rs (Figure 1a). Collectively, these data indicate a direct sensitizing effect of BI-1 on IP3Rs, which may contribute to a decrease in steady-state [Ca2+]ER and mitochondrial bioenergetics and subsequent induction of basal autophagy. The authors declare no conflict of interest This work was supported by Deutsche Forschungsgemeinschaft Grant ME1922/9-1 and the Forschungskommission of the Heinrich-Heine University Dusseldorf (to AM), by Research Foundation-Flanders (FWO) grants G.0604.07N (to HDS), G.0788.11N (to GB) and G.0724.09 (to LM), by the Research Council of the KU Leuven via the Concerted Actions program (GOA/09/012) and an OT START (STRT/10/044) and by Interuniversity Attraction Poles Program Belgian Science Policy P6/28 (to HDS, JBP and LM). We thank Dr. JC Reed (Sanford-Burnham Medical Research Institute, La Jolla, CA) for providing the BI-1−/− mice. Supplementary Information accompanies the paper on Cell Death and Disease website

Bax Inhibitor-1 is a novel IP3 receptor-interacting and -sensitizing protein