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

The endoplasmic reticulum (ER) serves as the major intracellular Ca²⁺ store and has a role in the synthesis and folding of proteins. BAX (BCL2-associated X protein) inhibitor-1 (BI-1) is a Ca²⁺ leak channel also implicated in the response against protein misfolding, thereby connecting the Ca²⁺ store and protein-folding functions of the ER. We found that BI-1-deficient mice suffer from leukopenia and erythrocytosis, have an increased number of splenic marginal zone B cells and higher abundance and nuclear translocation of NF-κB (nuclear factor-κ light-chain enhancer of activated B cells) proteins, correlating with increased cytosolic and ER Ca²⁺ levels. When put into culture, purified knockout T cells and even more so B cells die spontaneously. This is preceded by increased activity of the mitochondrial initiator caspase-9 and correlated with a significant surge in mitochondrial Ca²⁺ levels, suggesting an exhausted mitochondrial Ca²⁺ buffer capacity as the underlying cause for cell death in vitro. In vivo, T-cell-dependent experimental autoimmune encephalomyelitis and B-cell-dependent antibody production are attenuated, corroborating the ex vivo results. These results suggest that BI-1 has a major role in the functioning of the adaptive immune system by regulating intracellular Ca²⁺ homeostasis in lymphocytes.The endoplasmic reticulum (ER) serves as the major intracellular calcium (Ca²⁺) store, the release of which controls a vast array of cellular functions from short-term responses such as contraction and secretion to long-term regulation of cell growth and proliferation.¹ Dysregulated release of ER Ca²⁺, in contrast, initiates programmed cell death by several mechanisms including mitochondrial Ca²⁺ overload, depolarization, ATP loss and cytochrome c release.² Besides this, the ER also has a key role in the synthesis, folding and sorting of proteins destined for the secretory pathway. The deleterious consequences of an increase in unfolded proteins is called ER stress and can be antagonized by the unfolded protein response (UPR), a mechanism that coordinates a simultaneous increase in the ER folding capacity and a decrease in folding load. In the case of insufficient adaptation to ER stress, cells undergo apoptosis.³BAX (BCL2-associated X protein) inhibitor-1 (BI-1) is an evolutionarily conserved protein that bridges both the Ca²⁺ homeostasis and UPR functions of the ER.⁴ BI-1 was first identified in a screen for human proteins capable of inhibiting BAX-mediated cell death in yeast.⁵ In mammalian cells, BI-1''s antiapoptotic function is most pronounced in paradigms of ER stress⁶ and involves changes in the amount of Ca²⁺ that can be released from intracellular stores.^{6, 7} BI-1 is a highly hydrophobic protein that forms a Ca²⁺ pore responsible for its Ca²⁺ leak properties⁸ and is the founding member of a family of six proteins with similar properties.⁹ The increase in the ER Ca²⁺ leak mediated by BI-1 is blocked at a more acidic pH¹⁰ – a function recently corroborated by a structural analysis of a bacterial homolog of BI-1.¹¹Despite its evolutionarily conserved role in important functions such as ER stress and Ca²⁺ regulation, bi-1−/− mice were reported to have no phenotypic abnormalities but increased infarct volumes in a stroke model, and increased sensitivity to tunicamycin-induced kidney toxicity.⁶ Moreover, livers from BI-1-deficient mice regenerate faster than those from wild-type (WT) mice and this correlates with increased nuclear translocation of nuclear factor of activated T cells (NFATs)¹², a Ca²⁺-dependent process. BI-1 knockout (KO) mice also express more of the spliced form of X-box-binding protein-1 (sXBP-1) in their liver and kidney,¹³ which is generated by the endoribonuclease activity of inositol requiring enzyme 1 (IRE1), and is considered an indicator of increased UPR activity. This was later reproduced and attributed to an inhibitory function of BI-1 on IRE1α mediated via a direct interaction of the two proteins.¹⁴In our study, we found that bi-1−/− mice are more obese and suffer from leukopenia. T and B cells from these mice show significant changes in cellular Ca²⁺ homeostasis and dynamics, and are more prone to spontaneous death in culture but, surprisingly, demonstrate no signs of ongoing ER stress within the homeostatic system of the living animal. These changes lead to an attenuated functioning of the adaptive immune system in vivo. Our results suggest that a major role of BI-1 in vivo involves its effects on the intracellular Ca²⁺ homeostasis in lymphocytes in line with its function as an ER Ca²⁺ leak channel.