BI-1 regulates endoplasmic reticulum Ca2+ Homeostasis downstream of bcl-2 family proteins

BI-1 (Bax inhibitor-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 overexpressing antiapoptotic Bcl-2 or Bcl-XL, overexpression 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-XL fails to reduce [Ca2+](er), indicating that BI-1 functions downstream of Bcl-XL. In bax(-/-) bak(-/-) double knock-out cells, both BI-1 and Bcl-XL retained their ability to reduce [Ca2+](er), suggesting that BI-1 and Bcl-XL 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.