SUMMARY The stromal interaction molecule, STIM1 regulates Ca2+ entry via Orai1 channels in response to decreased concentration of ER luminal Ca2+. In search of a mechanism that switches the cytoplasmic aspect of STIM1 from an inactive to an active state, we identified an acidic motif within the STIM1 coiled-coil region that keeps the Ca2+ activation domain (CAD/SOAR) inactive. The STIM1 acidic motif shows significant homology to the C-terminal coiled-coil segment of Orai1, the postulated site of interaction with STIM1. Mutations within the acidic region make STIM1 constitutively active while those within a short basic segment of CAD/SOAR prevent Orai1 activation. We propose that during STIM1 activation, the CAD/SOAR domain is released from an intramolecular clamp allowing the basic segment to activate Orai1 channels. This evolutionary conserved activation mechanism of STIM1 resembles the regulation of protein kinases by intramolecular silencing with pseudosubstrate binding.

Sohn et al. show that plasma membrane PI(4,5)P2 controls the level of its precursor, PI4P, by regulating PI4P/PS exchange activity of ORP5/8. This control is achieved via regulation of ORP5/8 interaction with the plasma membrane by both of these phosphoinositides.

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Calcium depletion of the endoplasmic reticulum (ER) induces oligomerisation, puncta formation and translocation of the ER Ca2+ sensor proteins, STIM1 and −2 into plasma membrane (PM)-adjacent regions of the ER, where they activate the Orai1, −2 or −3 proteins present in the opposing PM. These proteins form ion channels through which store-operated Ca2+ influx (SOC) occurs. Calcium ions exert negative feed-back on SOC. Here we examined whether subplasmalemmal mitochondria, which reduce this feed-back by Ca2+ uptake, are located within or out of the high-Ca2+ microdomains (HCMDs) formed between the ER and plasmalemmal Orai1 channels. For this purpose, COS-7 cells were co-transfected with Orai1, STIM1 labelled with YFP or mRFP and the mitochondrially targeted Ca2+ sensitive fluorescent protein inverse Pericam. Depletion of ER Ca2+ with ATP + thapsigargin (in Ca2+-free medium) induced the appearance of STIM1 puncta in the ≤100 nm wide subplasmalemmal space, as examined with TIRF. Mitochondria were located either in the gaps between STIM1-tagged puncta or in remote, STIM1-free regions. After addition of Ca2+ mitochondrial Ca2+ concentration increased irrespective of the mitochondrion-STIM1 distance. These observations indicate that mitochondria are exposed to Ca2+ diffused laterally from the HCMDs formed between the PM and the subplasmalemmal ER.