Sensor, channel, pump and leak - TMBIM6 assembles a multi-protein complex that governs the Ca2+ content of the endoplasmic reticulum

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Ca2+ release from the endoplasmic reticulum (ER) controls a vast array of cellular functions and is limited by the activity of sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA) which pump back Ca2+ against a steep concentration gradient. Besides this function as a Ca2+ store, the ER also plays a key role in the synthesis, folding and sorting of proteins destined for the secretory pathway. In the event of dysfunction of the ER, the number of misfolded proteins increases which triggers the unfolded protein response, a mechanism that coordinates an increase in the ER folding capacity and a decrease in folding load via the stress sensor inositol-requiring protein 1a (IRE1a). Dysregulated release of ER Ca2+, reduced SERCA activity or an excess of misfolded proteins all cause cell death.TMBIM6 (Transmembrane Bax inhibitor motif containing 6) is an evolutionarily conserved pH-dependent ER Ca2+ leak channel that interacts with ligand-induced Ca2+ channels inositol 1,4,5-trisphosphate receptors 1 and 3 (IP3R1/3) and IRE1a at the ER membrane. TMBIM6 therefore connects the Ca2+ store and protein-folding functions of the ER. We recently reported that lymphocytes from mice lacking the loop domain of TMBIM6, alleged TMBIM6 knockout mice, have increased cytosolic and ER Ca2+ levels that critically attenuate their function and increase their susceptibility to cell death. While an increased ER Ca2+ concentration can be expected for cells lacking an important ER Ca2+ leak channel, it remains unclear why lack of TMBIM6 should result in an increased cytosolic Ca2+ concentration. This project is based on preliminary data that suggest that the evolutionarily conserved N-terminus of TMBIM6, which is still expressed in the above-mentioned mice, inhibits the ubiquitous SERCA2b pump by direct interaction. Such a physical integration of SERCA activity and Ca2+ leakage by TMBIM6 and IP3R1/3 probably results in faster refilling of the store under physiological conditions while the interaction with IRE1a allows fine-tuning of the ER Ca2+ content in a way that increases the ER folding capacity. Here, we will clarify how and when SERCA2b, IRE1a and TMBIM6 are present in the same protein complex. We will study Ca2+ leakage and SERCA activity in cells lacking full-length TMBIM6 and not just the loop domain. We will further refine the inhibitory activity of the N-terminal part of TMBIM6 on these mechanisms to identify amino acids or motifs necessary for activity and generate TMBIM6 variants lacking these motifs. By comparing the effect of these TMBIM6 variants on protein interaction, Ca2+ homeostasis and ER stress with those of a previously identified point-mutant lacking channel activity, we will advance our understanding of important aspects of cell biology and human disease caused by misfolded proteins.