The C terminus of bax inhibitor-1 forms a Ca 2+-permeable channel pore

Abstract

Bax inhibitor-1 (BI-1) is a multitransmembrane domain-spanning 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 45Ca 2+-flux experiments and identified an α-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 Ca 2+-leak properties of BI-1 are an added function during evolution. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.