The C Terminus of Bax Inhibitor-1 Forms a Ca2+-permeable Channel Pore

Abstract

Background: Evolutionary conserved Bax inhibitor-1 (BI-1) protects against ER stress-mediated apoptosis. Results: We identified a Ca 2ϩ -permeable channel pore in the C terminus of BI-1. Critical pore properties are an ␣-helical structure and two aspartate residues conserved among animals, but not among plants and yeast. Conclusion: C-terminal domain of BI-1 harbors a Ca 2ϩ -permeable channel pore. Significance: BI-1 has Ca 2ϩ channel properties likely relevant for its function in ER stress and apoptosis. Bax inhibitor-1 (BI-1) is a multitransmembrane domainspanning endoplasmic reticulum (ER)-located protein that is evolutionarily conserved and protects against apoptosis and ER stress. Furthermore, BI-1 is proposed to modulate ER Ca 2؉homeostasis by acting as a Ca 2؉ -leak channel. Based on experimental determination of the BI-1 topology, we propose that its C terminus forms a Ca 2؉ pore responsible for its Ca 2؉ -leak properties. We utilized a set of C-terminal peptides to screen for Ca 2؉ leak activity in unidirectional 45 Ca 2؉ -flux experiments and identified an ␣-helical 20-amino acid peptide causing Ca 2؉ leak from the ER. The Ca 2؉ leak was independent of endogenous ER Ca 2؉ -release channels or other Ca 2؉ -leak mechanisms, namely translocons and presenilins. The Ca 2؉ -permeating property of the peptide was confirmed in lipid-bilayer experiments. Using mutant peptides, we identified critical residues responsible for the Ca 2؉ -leak properties of this BI-1 peptide, including a series of critical negatively charged aspartate residues. Using peptides corresponding to the equivalent BI-1 domain from various organisms, we found that the Ca 2؉ -leak properties were conserved among animal, but not plant and yeast orthologs. By mutating one of the critical aspartate residues in the proposed Ca 2؉ -channel pore in full-length BI-1, we found that Asp-213 was essential for BI-1-dependent ER Ca 2؉ leak. Thus, we elucidated residues critically important for BI-1-mediated Ca 2؉ leak and its potential channel pore. Remarkably, one of these residues was not conserved among plant and yeast BI-1 orthologs, indicating that the ER Ca2؉ -leak properties of BI-1 are an added function during evolution. leak from the ER (9, 10). BI-1 seems to be strongly evolutionarily conserved and BI-1 orthologs from plants can substitute for mammalian BI-1 in regard to its anti-apoptotic function (11). Besides this, other diverse functions of BI-1 have been described. BI-1 is a negative regulator of the ER-stress sensor (12), it interacts with G-actin and increases actin polymerization (13), enhances cancer metastasis by altering glucose metabolism and by activating a sodium-hydrogen exchanger (14), and it reduces production of reactive oxygen species through direct interaction with NADPH-P450 reductase (15), a member of the microsomal monooxygenase system.Recently, the role of BI-1 in Ca 2ϩ signaling has been further explored. The effect of BI-1 on cell death seems to involve changes in the amount of Ca 2ϩ that is releasable from intrace...

BAX inhibitor-1 is a Ca2+ channel critically important for immune cell function and survival

“…These results are well in line with previously reported effects of BI-1 in the control of the ER Ca 2+ content. [6][7][8]10 Also, structural evidence obtained from a bacterial homolog of BI-1 just demonstrated that BI-1 is indeed a Ca 2+ leak channel with an evolutionarily conserved pH sensor. 11 Bar graphs show the mean ± S.E.M., n = 3.…”

“…5 In mammalian cells, BI-1's antiapoptotic function is most pronounced in paradigms of ER stress 6 and involves changes in the amount of Ca 2+ that can be released from intracellular stores. 6,7 BI-1 is a highly hydrophobic protein that forms a Ca 2+ pore responsible for its Ca 2+ leak properties 8 and is the founding member of a family of six proteins with similar properties. 9 The increase in the ER Ca 2+ leak mediated by BI-1 is blocked at a more acidic pH 10 -a function recently corroborated by a structural analysis of a bacterial homolog of BI-1.…”

Bax Inhibitor-1-mediated Ca2+ leak is decreased by cytosolic acidosis

“…Five million MEF cells were lysed in RIPA buffer and 30 g of total protein was loaded on Tris-Acetate gels (Invitrogen) in the presence of urea sample buffer [9]. The proteins were transferred on a PVDF membrane and blotted for IP 3 R1 [25], IP 3 R3 (BD Biosciences), SERCA2b [26] or non-muscle myosin IIA (Abcam) for loading control.…”

“…The assays were performed as previously described [25,27], except that different buffers were used for proper pH control in the efflux medium (imidazole for pH 6.8 and 6.2; MES for pH 5.8 and acetate for pH 5.2). In the experiments in which a peptide corresponding to the C-terminal Ca 2+ -channel pore of BI-1 was used, the peptide (CTP1) was added acutely at a concentration of 40 M, well above the EC 50 for provoking a Ca 2+ flux (32 M, [9]). Purity (>80%) was verified by mass spectrometry (ThermoFisher, Germany).…”

“…In particular, a lowering of the ER Ca 2+ -store content has been implicated as a protective cellular mechanism against apoptosis [3,4]. Recently, a family of transmembrane Bax Inhibitor-1 (BI-1)-containing motif proteins (TMBIM) emerged as conserved and ancient celldeath suppressors by affecting ER-Ca 2+ homeostasis and dynamics [5][6][7][8][9][10][11][12][13].…”