Bax Inhibitor-1 is a Bimodal Regulator of Cellular Ca2+ Homeostasis

For all cells, calcium ions (Ca2+) are extremely toxic and therefore need to be either extruded or sequestered into intracellular stores. The main intracellular store, the endoplasmic reticulum (ER) contains approximately 10 000x more Ca2+ than the surrounding cytosol. By storing Ca2+ in the ER, the cell acquires the possibility to utilize Ca2+ as a messenger and release it at a specific magnitude, time and place to signal downstream pathways. All eukaryotic cells from yeast to plants and animals use Ca2+ to transmit important signals throughout the cell. In animals, Ca2+ signals regulate pathways that control vital functions such as fertilization, cell differentiation, contraction of a muscle, firing of nerve cells, secretion of cellular components, cell metabolism, transcription of genes, cell motility and cell death. The level of free Ca2+ that is available in the ER for signaling is determined by the action of Ca2+ pumps (SERCA) that fill the ER with Ca2+ and an enigmatic leak of Ca2+ through the ER membrane. The level of free Ca2+ in the ER thus determines how the cell will react to activators of Ca2+ release mainly via the IP3 receptor (IP3R), the most widely expressed release channel at the ER membrane. Bax inhibitor-1 is a protein that protects cells from cell death by reducing the amount of Ca2+ in the ER. It confers protection against stroke or ischemia reperfusion (IR)-injury in the liver, kidney and brain where it is abundantly expressed. It also increases aggressiveness of cancer cells and is linked to cancers of the prostate, breast, ovary and lung. Here we have shown that BI-1 contains a short domain which resides near the conserved C-terminus of the protein, which acts as a Ca2+-pore channel domain in cells, independently of other known Ca2+-release mechanisms. We also discovered that reducing pH dramatically changes the Ca2+-pore property of BI-1, and closes the channel at a surrounding pH ≤ 6.2. This indicates that the Ca2+-pore channel of BI-1 can be modulated by conditions that involve mild acidification of the cytosol, such as stroke or IR-injury. We also showed that the ability of BI‑1 to leak Ca2+ is protective to cells when they are subjected to chemical stress that depletes ER Ca2+ (ER stress). Apart from its role as a Ca2+-leak channel in animals, we discovered that BI-1 is able to sensitize IP3Rs to IP3 and that this function is independent of the Ca2+-pore channel of BI-1. We also showed that another member of the same protein family, GRINA was able to suppress IP3R function, presumably by binding to both BI-1 and IP3R. In summary, we have shown that BI-1 can regulate cellular Ca2+ homeostasis in two ways: 1) by functioning as a Ca2+-leak channel that reduces the steady state concentration of Ca2+ at the ER and 2) by sensitizing the main ER Ca2+ channel, the IP3R. Since BI-1 is conserved in evolution up to yeast, we are now studying the most ancient roles of BI‑1 at the ER membrane, namely how it can regulate protein foldingand clearance of misfolded proteins. For this purpose we will use biochemical methods as well as a well-characterized animal model for development, namely the zebrafish. Since protein folding and regulation of ER stress are implicated in the progression of several diseases such as Alzheimer s disease, Parkinson s disease, Creutzfeld‑Jakob s disease, amyotrophic lateral sclerosis, type 2 diabetes and atherosclerosis, it is important to learn about the mechanisms that underlie the basic functioning of the ER and thus cell health.I. INTRODUCTION 1. THE ENDOPLASMIC RETICULUM (ER) 2. REGULATION OF ER CA2+ 2.1. Ca2+-binding proteins 2.2. SERCAs 2.3. Inositol 1,4,5-trisphosphate receptors (IP3Rs) 2.4. Ryanodine receptors (RyRs) 2.5. Ca2+ leak from the ER 3. CA2+ SIGNALS DURING THE LIFE OF A CELL 3.1. Autophagy 3.2. ER stress 3.3. Cell death 4. THE ANCIENT PROTECTORS – THE BAX INHIBITOR-1 (BI-1) FAMILY 4.1. BI-1, the founding member of the family 4.2. Expression patterns of BI-1 5. CYTOPROTECTIVE FUNCTIONS OF BI-1 5.1. BI-1 in cell death 5.2. BI-1 and ER stress 5.3. BI-1 modulates autophagy6. BI-1 AND CA2+ HOMEOSTASIS II. OBJECTIVES 1. IDENTIFICATION OF THE CA2+-CHANNEL PORE DOMAIN OF BI-1 2. REGULATION OF BI-1 CA2+ CHANNEL ACTIVITY BY INTRACELLULAR ACIDOSIS 3. REGULATION OF IP3RS BY BI-1 III. RESULTS 1. THE C-TERMINUS OF BI-1 FORMS A CA2+-PERMEABLE CHANNEL PORE 2. BI-1-MEDIATED CA2+ LEAK IS DECREASED BY CYTOSOLIC ACIDOSIS 3. BI-1 IS A NOVEL IP3 RECEPTOR-INTERACTING AND -SENSITIZING PROTEIN 3.1. Addendum IV. GENERAL DISCUSSION 1. STEADY-STATE ER CA2+ IS REGULATED BY BI-1 2. COPING WITH STRESS – BI-1 AND IP3RS 2.1. Regulation of IICR during ER stress – GRINA steps up 2.2. Dual role of BI-1 during response to stress V. CONCLUSIONS VI. FUTURE PERSPECTIVES VII. SUMMARY VIII. SAMENVATTING IX. BIBLIOGRAPHY X. CURRICULUM VITAEnrpages: 129status: publishe