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

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.