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

Abstract

tBax Inhibitor-1 (BI-1) is an evolutionarily conserved six-transmembrane domain endoplasmic reticulum(ER)-localized protein that protects against ER stress-induced apoptotic cell death. This function is closelyconnected to its ability to lower steady-state ER Ca2+levels. Recently, we elucidated BI-1’s Ca2+-channelpore in the C-terminal part of the protein and identified the critical amino acids of its pore. Based on theseinsights, a Ca2+-channel pore-dead mutant BI-1 (BI-1D213R) was developed. We determined whether BI-1behaves as a bona fide H+/Ca2+antiporter or as an ER Ca2+-leak channel by investigating the effect of pHon unidirectional Ca2+-efflux rates. At pH 6.8, wild-type BI-1 expression in BI-1−/−cells increased the ERCa2+-leak rate, correlating with its localization in the ER compartment. In contrast, BI-1D231Rexpressionin BI-1−/−, despite its ER localization, did not increase the ER Ca2+-leak rate. However, at pH < 6.8, theBI-1-mediated ER Ca2+leak was blocked. Finally, a peptide representing the Ca2+-channel pore of BI-1promoting Ca2+flux from the ER was used. Lowering the pH from 6.8 to 6.0 completely abolished theability of the BI-1 peptide to mediate Ca2+flux from the ER. We propose that this pH dependence isdue to two aspartic acid residues critical for the function of the Ca2+-channel pore and located in the ERmembrane-dipping domain, which facilitates the protonation of these residues.