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. 2013 Sep;33(18):3715-23.
doi: 10.1128/MCB.00554-13. Epub 2013 Jul 22.

Mechanisms of STIM1 activation of store-independent leukotriene C4-regulated Ca2+ channels

Xuexin Zhang  1 , José C González-Cobos, Rainer Schindl, Martin Muik, Brian Ruhle, Rajender K Motiani, Jonathan M Bisaillon, Wei Zhang, Marc Fahrner, Margarida Barroso, Khalid Matrougui, Christoph Romanin, Mohamed Trebak
Affiliations

Mechanisms of STIM1 activation of store-independent leukotriene C4-regulated Ca2+ channels

Xuexin Zhang et al. Mol Cell Biol. 2013 Sep.

Abstract

We recently showed, in primary vascular smooth muscle cells (VSMCs), that the platelet-derived growth factor activates canonical store-operated Ca(2+) entry and Ca(2+) release-activated Ca(2+) currents encoded by Orai1 and STIM1 genes. However, thrombin activates store-independent Ca(2+) selective channels contributed by both Orai3 and Orai1. These store-independent Orai3/Orai1 channels are gated by cytosolic leukotriene C4 (LTC4) and require STIM1 downstream LTC4 action. However, the source of LTC4 and the signaling mechanisms of STIM1 in the activation of this LTC4-regulated Ca(2+) (LRC) channel are unknown. Here, we show that upon thrombin stimulation, LTC4 is produced through the sequential activities of phospholipase C, diacylglycerol lipase, 5-lipo-oxygenease, and leukotriene C4 synthase. We show that the endoplasmic reticulum-resident STIM1 is necessary and sufficient for LRC channel activation by thrombin. STIM1 does not form sustained puncta and does not colocalize with Orai1 either under basal conditions or in response to thrombin. However, STIM1 is precoupled to Orai3 and Orai3/Orai1 channels under basal conditions as shown using Forster resonance energy transfer (FRET) imaging. The second coiled-coil domain of STIM1 is required for coupling to either Orai3 or Orai3/Orai1 channels and for LRC channel activation. We conclude that STIM1 employs distinct mechanisms in the activation of store-dependent and store-independent Ca(2+) entry pathways.

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Figures

Fig 1
Fig 1
Thrombin-activated Ca2+ entry requires the sequential catalytic activities of PLC, DAG lipase, 5-LO, and LTC4S. (A) Schematic representation of the pharmacological approach employed to study the pathway downstream thrombin receptor (crosses, drugs that inhibited thrombin-activated Ca2+ entry; checkmarks, drugs that had no effect; upward arrows, drugs that potentiated thrombin-activated Ca2+ entry). (B and C) Representative Ca2+ imaging traces from cells pretreated with either the vehicle control or various drugs at the concentrations indicated. (D) Statistical summary of effects of all drugs employed on thrombin-activated Ca2+ entry (data shown as x, y, where x = number of independent runs and y = total number of cells). Throughout the figures, *, **, and *** indicate P values of <0.05, 0.01, and 0.001, respectively.
Fig 2
Fig 2
ER-resident STIM1 is required for activation of thrombin-activated Ca2+ entry. (A) Schematic representation of various versions of STIM1 employed in erase-and-replace experiments (see Materials and Methods). (B) Western blot analysis confirmed successful endogenous STIM1 knockdown and validated the erase-and-replace approach. Endogenous STIM1 is detected by Western blotting (lane 1), and knockdown was achieved using specific siRNA against STIM1 (lane 2). STIM1 rescue was achieved by transfecting 1.5 μg of plasmid DNA per 106 cells. Plasmids expressing full-length STIM1 (lane 3), STIM1 N131Q N171Q glycosylation mutant (lane 4; note the lower molecular mass of this mutant), and eYFP-STIM1 (lane 5). Please note that while Western blotting assays represent total protein expression in a mixed population of cells, Ca2+ imaging experiments were conducted only in cells that were successfully transfected as evidenced by eYFP-STIM1 or cotransfected GFP (for untagged plasmids) fluorescence. Positions of protein molecular mass markers are shown on the left. (C) Representative Ca2+ imaging traces from cells subjected to the erase-and-replace approach showing rescue of thrombin-activated Ca2+ entry with all STIM1 constructs. (D) Summary of Ca2+ imaging data is shown.
Fig 3
Fig 3
Thrombin-activated Ca2+ entry and currents are not blocked by drugs that inhibit STIM1 punctum formation. Ca2+ signals in VSMCs were measured in response to either thrombin (Th, 100 nM) (A) or thapsigargin (TG, 2 μM) (B). Pharmacological agents known to inhibit SOCE (50 μM 2-APB and 50 μM ML-9) completely abrogated thapsigargin-induced Ca2+ entry (B) with no effect in thrombin-activated Ca2+ signals (A). Whole-cell patch clamp electrophysiology confirmed that thrombin-activated Ca2+ (measured in Ca2+-containing bath solutions) and Na+ (measured in DVF bath solutions) currents (C) are not sensitive to 2-APB (Na+ I/V depicted; n = 5) (D). However, store depletion (20 mM BAPTA in pipette)-activated Ca2+ and Na+ CRAC currents (F) are sensitive to 2-APB (Na+ I/V depicted; n = 5) (G). Statistics on LRC and CRAC current data are shown in panels E and H, respectively, as mean/range.
Fig 4
Fig 4
Thrombin stimulation failed to cause sustained STIM1 puncta and STIM1/Orai1 colocalization. (A) Representative confocal time-lapse images of VSMCs expressing 0.75 μg of eYFP-STIM1 and nonstimulated (Control; panels 1, 4, and 7) or upon stimulation with maximal concentrations of thapsigargin (TG, 4 μM; panels 2 and 3; n = 5) (see also Movie S1 in the supplemental material), PDGF (100 ng/ml; panels 5 and 6; n = 3) (see also Movie S2 in the supplemental material), and thrombin (100 nM; panels 8 and 9; n = 8) (see also Movie S3 in the supplemental material) showing sustained puncta only in response to thapsigargin and PDGF. Images were acquired every 30 s, and stimuli were added at t = 120 s. (B and C) Confocal time-lapse images of VSMCs cotransfected with eYFP-STIM1 (0.5 μg) and CFP-Orai1 (3 μg) before and after stimulation with thapsigargin (B) (TG, 2 μM; n = 3) (see also Movie S4 in the supplemental material) or thrombin (C) (100 nM; n = 3) (see also Movie S5 in the supplemental material). Images were acquired every 20 s. Intensity profiles for STIM1 (green) and Orai1 (red) showed that while thapsigargin stimulation caused these proteins to colocalize in areas close to the plasma membrane, thrombin stimulation failed to cause STIM1/Orai1 colocalization. Scale bars for panels B and C are 15 μm.
Fig 5
Fig 5
STIM1 colocalizes with Orai3, not Orai1, under basal conditions. (A) Representative confocal z-stack images of VSMCs coexpressing eYFP-STIM1 (green; 0.5 μg) (panel 1) and CFP-Orai1 (red; 3 μg) (panel 2) acquired under basal conditions (in Hanks balanced salt solution [HBSS] containing 2 mM Ca2+; n = 4) show no significant colocalization (panel 3). Three-dimensional surface generation for visualization of protein distribution (see Movie S6 in the supplemental material) shows minimal stochastic basal colocalization (panels 4 and 5). (B) However, coexpression of eYFP-STIM1 (green; 0.5 μg) (panel 1) and CFP-Orai3 (red; 3 μg) (panel 2) shows significant basal colocalization (panel 3) that is confirmed by three-dimensional surface generation (panel 4) and STIM1/Orai3 surface interactions (colocalization channel) (panel 5) (see also Movie S7 in the supplemental material) (PCCV = 0.56 ± 0.07; n = 10). All images were acquired under basal conditions in the absence of agonists. Scale bars for panels A and B are 15 μm.
Fig 6
Fig 6
Orai3/STIM1 basal precoupling is not associated with constitutive current activity. (A) Representative confocal images of HEK293 cells overexpressing eYFP-STIM1/CFP-Orai1 (panels 1 to 3; n = 15), eYFP-STIM1/CFP-Orai3 (panels 4 to 6; n = 3), and eYFP-STIM1/CFP-Orai3-Orai1 tandem (panels 7 to 9; n = 6) acquired under basal conditions. (B) Confocal images showing colocalization of STIM1 (green) and Orai1 (red) protein coexpressed in HEK293 cells acquired under basal conditions (panels 1 to 3) and upon store depletion with 60 μM BHQ (panels 5 to 7). Net FRET values confirmed the minimal basal interaction of Orai1/STIM1 (panel 4) that is induced in response to store depletion (60 μM BHQ for 5 min) (panel 8). (C) However, confocal images showing colocalization of STIM1 (green) and Orai3 (red) protein coexpressed in HEK293 cells showed marked colocalization (panel 3) without store depletion. Net FRET values confirm strong interaction of Orai3/STIM1 (panel 4) that is marginally enhanced in response to store depletion (panel 8). (D) Statistical analysis of FRET experiments from several experiments similar to those in panels B (n = 5) and C (n = 4). (E) Representative confocal images of HEK293 cells coexpressing STIM1-A376K/Orai1 (panels 1 to 3; n = 15), STIM1-A376K/Orai3 (panels 4 to 6; n = 3), and STIM1-A376K/Orai3-Orai1 tandem (panels 7 to 9; n = 6) confirmed loss of basal colocalization (panels 6 and 9). Scale bars for panels A to C and E represent 5 μm.
Fig 7
Fig 7
The second coiled-coil domain of ER-STIM1 is important for activation of thrombin-activated Ca2+ entry. (A) Schematic representations of mutant versions of eYFP-STIM1 employed in erase-and-replace assays. (B) Western blot shows STIM1 expression in siRNA control-transfected VSMC (lane 1) and successful endogenous STIM1 knockdown with siRNA (lane 2). Rescue of STIM1 expression was achieved using wild-type human eYFP-STIM1 (lane 5) and two eYFP-STIM1 coiled-coil mutants (lanes 3 and 4). EYFP-STIM1 plasmids were transfected at 1.5 μg per 106 cells. Positions of protein molecular mass markers are shown on the left. (C) Among plasmids expressing either STIM1-A376K (lane 3), STIM1-L373S A376S (lane 4), or eYFP-STIM1 (lane 5), only wild-type eYFP-STIM1 could rescue thrombin-activated Ca2+ entry. (D) Statistical analysis of the Ca2+ imaging data from several independent runs similar to those for panel C.
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