Store-operated Ca2+ influx and subplasmalemmal mitochondria

Abstract

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.

Mitochondrial Ca2+ uptake and not mitochondrial motility is required for STIM1-Orai1-dependent store-operated Ca2+ entry

“…pointing to lateral diffusion of Ca 2+ in the subplasmalemmal area (Korzeniowski et al, 2009). Altogether, our data do not support the general concept of local mitochondrial Ca 2+ buffering as a mechanism of their contribution to STIM1-Orai1-dependent SOCE.…”

The STIM1–ORAI1 microdomain

“…Mitochondrial Ca 2+ uptake can occur at these modestly elevated levels of Ca 2+ [reviewed in 78], and would, if anything, be more effective under the conditions of enhanced Ca 2+ entry normally employed to study slow Ca 2+ -dependent inactivation. There is a lingering question, though, whether mitochondria are specifically positioned to take up Ca 2+ at ER-plasma membrane junctions [79]. It may be that respiring mitochondria act mainly as a check on the global cytoplasmic Ca 2+ concentration and its additive contribution to junctional Ca 2+ concentration.…”

Mitochondrial organization and Ca2+ uptake

“…Fluorescently labelled mitochondria and STIM-1 (stromal-interaction molecule 1) did not co-localize either before or after Ca 2 + store depletion in COS-7 cells [79]. The Ca 2 + concentration at the OMM, as measured by targeted GFP (green fluorescent protein)-based Ca 2 + sensors, was similar to global [Ca 2 + ] c , suggesting that mitochondria were not exposed to high [Ca 2 + ] c microdomains during SOCE [28].…”

“…Similarly, although mitochondrial Ca 2 + uptake occurs during SOCE, mitochondria may not be localized particularly close to these sites [28,79]. Fluorescently labelled mitochondria and STIM-1 (stromal-interaction molecule 1) did not co-localize either before or after Ca 2 + store depletion in COS-7 cells [79].…”