Polyunsaturated fatty acids inhibit stimulated coupling between the ER Ca2+ sensor STIM1 and the Ca2+ channel protein Orai1 in a process that correlates with inhibition of stimulated STIM1 oligomerization

Abstract

Polyunsaturated fatty acids (PUFAs) have been found to be effective inhibitors of cell signaling in numerous contexts, and we find that acute addition of micromolar PUFAs such as linoleic acid are effective inhibitors of Ca2+ responses in mast cells stimulated by antigen-mediated crosslinking of FcεRI or by the SERCA pump inhibitor, thapsigargin. In contrast, the saturated fatty acid, stearic acid, with the same carbon chain length as linoleic acid does not inhibit these responses. Consistent with this inhibition of store-operated Ca2+ entry (SOCE), linoleic acid inhibits antigen-stimulated granule exocytosis to a similar extent. Using the fluorescently labeled plasma membrane Ca2+ channel protein, AcGFP-Orai1, together with the labeled ER Ca2+ sensor protein, STIM1-mRFP, we monitor stimulated coupling of these proteins that is essential for SOCE with a novel spectrofluorimetric resonance energy transfer method. We find effective inhibition of this stimulated coupling by linoleic acid that accounts for the inhibition of SOCE. Moreover, we find that linoleic acid induces some STIM1-STIM1 association, while inhibiting stimulated STIM1 oligomerization that precedes STIM1-Orai1 coupling. We hypothesize that linoleic acid and related PUFAs inhibit STIM1-Orai1 coupling by a mechanism that involves perturbation of ER membrane structure, possibly by disrupting electrostatic interactions important in STIM1 oligomerization.

Molecular anatomy of the early events in STIM1 activation – oligomerization or conformational change?

“…10 6 cells were stirred in an acrylic cuvette at 37°C in Ca 2+ -free Krebs-Ringer buffer supplemented with 2 mM Ca 2+ , and the time course of acceptor over donor fluorescence (mApple/AcGFP with excitation at 485 nm, and emission at 515 nm for donor and 580 nm long pass for acceptor) was monitored in response to Ca 2+ store depletion with ATP and Tg addition (50 µM and 200 nM, respectively). (Holowka et al, 2014).…”

Defective lipid metabolism in neurodegeneration with brain iron accumulation (NBIA) syndromes: not only a matter of iron

“…Notably, another relevant role of iPLA2β is the modulation of capacitative Ca 2+ entry (CCE), a type of Ca 2+ influx into the cells that is activated when ER Ca 2+ stores are depleted. It has been demonstrated that iPLA 2 β,t h eE Rc a l c i u ms e n s o r STIM1 and the plasma membrane pore-forming subunit Orai1 are equally required for CCE (Smani et al 2004;Bolotina 2008), and a very recent study has shown that PUFAs, like linoleic acid, inhibit STIM1-Orai1 coupling by a mechanism that involves perturbation of ER membrane structure (Holowka et al 2014). Relevant to INAD pathology, using two different mouse models of the disease, the group of Georg Reiser demonstrated a severe disturbance in astrocyte ATPstimulated Ca 2+ signalling in vivo (Strokin et al 2012).…”

Roles for Ca2+ mobilization and its regulation in mast cell functions: recent progress

“…In this study, we developed an effective fluorimetry-based fluorescence resonance energy transfer (FRET) method for monitoring stimulated association of fluorescently tagged STIM1 and Orai1 in transfected COS7 and RBL cells, and we found that micromolar concentrations of linoleic acid disrupt the physical coupling between these proteins in parallel with inhibition of SOCE. Saturated fatty acids of the same acyl chain length do not inhibit STIM1/Orai1 coupling, and the mechanism for this inhibition is downstream of FcεRI-mediated tyrosine phosphorylation [19]. Our current evidence suggests that PUFAs inhibit STIM1/Orai1 coupling by disrupting ER membrane organization, possibly by electrostatic interference with oligomerization of STIM1 dimers or their conformational transition that is normally activated by depletion of Ca 2+ from ER stores.…”