Bax inhibitor-1 regulates endoplasmic reticulum Ca²⁺ homeostasis downstream of Bcl-2-family proteins

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Bax Inhibitor-1 (BI-1) is an evolutionarily conserved multi-transmembrane protein that resides in the endoplasmic reticulum (ER) and that has documented cytoprotective functions in both animals and plants. Recent studies indicate that BI-1 shares in common with Bcl-2/Bax-family proteins the ability to regulate the amounts of Ca²⁺ that can be released from the ER by agents such as the ER-Ca²⁺ATPase (SERCA) inhibitor Thapsigargin (TG). Using an ER-targeted, Ca²⁺ indicator (“cameleon”), with characteristics optimized for measuring ER Ca²⁺ ([Ca²⁺]_er), we studied the effects of BI-1 on [Ca²⁺]_er in resting and TG-treated cells. Similar to cells over-expressing anti-apoptotic Bcl-2 or Bcl-X_L, over-expression of BI-1 resulted in lower resting [Ca²⁺]_er, with concomitantly less Ca²⁺ released into the cytosol upon stimulation by TG and with a higher rate of Ca²⁺ leakage from the ER. Co-expression of SERCA restored levels of [Ca²⁺]_er to normal, showing opposing actions of the ER-Ca²⁺ATPase and BI-1 on ER Ca²⁺ homeostasis. Conversely, cells with deficient BI-1 have increased [Ca²⁺]_er, and release more Ca²⁺ into the cytosol when challenged with TG. In BI-1-deficient cells, Bcl-X_L fails to reduce [Ca²⁺]_er, indicating that BI-1 functions downstream of Bcl-X_L. In bax^-/-bak^-/- double knock-out cells, both BI-1 and Bcl-X_L retained their ability to reduce [Ca²⁺]_er, suggesting that BI-1 and Bcl-X_L operate downstream of or parallel to Bax/Bak. The findings reveal a hierarchy of functional interactions of BI-1 with Bcl-2/Bax-family proteins in regulating ER Ca²⁺ homeostasis. (Supported by NIH AG-15393.)