Bax Inhibitor-1-mediated Ca²⁺ leak is decreased by cytosolic acidosis
2013
Kiviluoto, Santeri | Luyten, Tomas | Schneider, Lars | Lisak, Dmitrij | Rojas-Rivera, Diego | Welkenhuyzen, Kirsten | Missaen, Ludwig | De Smedt, Humbert | Parys, Jan B. | Hetz, Claudio | Methner, Axel | Bultynck, Geert
Bax 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 closely connected to its ability to lower steady-state ER Ca²⁺ levels. Recently, we elucidated BI-1's Ca²⁺-channel pore in the C-terminal part of the protein and identified the critical amino acids of its pore. Based on these insights, a Ca²⁺-channel pore-dead mutant BI-1 (BI-1ᴰ²¹³ᴿ) was developed. We determined whether BI-1 behaves as a bona fide H⁺/Ca²⁺ antiporter or as an ER Ca²⁺-leak channel by investigating the effect of pH on unidirectional Ca²⁺-efflux rates. At pH 6.8, wild-type BI-1 expression in BI-1⁻/⁻ cells increased the ER Ca²⁺-leak rate, correlating with its localization in the ER compartment. In contrast, BI-1ᴰ²³¹ᴿ expression in BI-1⁻/⁻, despite its ER localization, did not increase the ER Ca²⁺-leak rate. However, at pH<6.8, the BI-1-mediated ER Ca²⁺ leak was blocked. Finally, a peptide representing the Ca²⁺-channel pore of BI-1 promoting Ca²⁺ flux from the ER was used. Lowering the pH from 6.8 to 6.0 completely abolished the ability of the BI-1 peptide to mediate Ca²⁺ flux from the ER. We propose that this pH dependence is due to two aspartic acid residues critical for the function of the Ca²⁺-channel pore and located in the ER membrane-dipping domain, which facilitates the protonation of these residues.
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