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Inside-out Ca(2+) signalling prompted by STIM1 conformational switch.

Ma G, Wei M, He L, Liu C, Wu B, Zhang SL, Jing J, Liang X, Senes A, Tan P, Li S, Sun A, Bi Y, Zhong L, Si H, Shen Y, Li M, Lee MS, Zhou W, Wang J, Wang Y, Zhou Y - Nat Commun (2015)

Bottom Line: The mechanistic underpinnings of this inside-out Ca(2+) signalling were largely undefined.Importantly, we further identify critical residues within the cytoplasmic domain of STIM1 (STIM1-CT) that entail autoinhibition.On the basis of these findings, we propose a model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas 77030, USA.

ABSTRACT
Store-operated Ca(2+) entry mediated by STIM1 and ORAI1 constitutes one of the major Ca(2+) entry routes in mammalian cells. The molecular choreography of STIM1-ORAI1 coupling is initiated by endoplasmic reticulum (ER) Ca(2+) store depletion with subsequent oligomerization of the STIM1 ER-luminal domain, followed by its redistribution towards the plasma membrane to gate ORAI1 channels. The mechanistic underpinnings of this inside-out Ca(2+) signalling were largely undefined. By taking advantage of a unique gain-of-function mutation within the STIM1 transmembrane domain (STIM1-TM), here we show that local rearrangement, rather than alteration in the oligomeric state of STIM1-TM, prompts conformational changes in the cytosolic juxtamembrane coiled-coil region. Importantly, we further identify critical residues within the cytoplasmic domain of STIM1 (STIM1-CT) that entail autoinhibition. On the basis of these findings, we propose a model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels.

No MeSH data available.


Identification of novel gain-of-function mutations within STIM1-TM.(a) Ca2+ influx in HEK ORAI1-CFP stable cells expressing WT and mutant STIM1-TM constructs (n=30–60) monitored by Fura-2 fluorescence ratio. Store depletion was induced by 2.5 μM ionomycin. Shown are representative traces from three independent experiments. The solid bar above the curves indicates 1 mM Ca2+ in the external medium. (b) Effects of tryptophan substitution of STIM1-TM residues on constitutive Ca2+ entry in HEK ORAI1-CFP stable cells. The level of constitutive Ca2+ entry (n=3) was quantified as the difference of mean Fura-2 fluorescence ratio between the peak value in the presence of 1 mM Ca2+ and the basal value without externally added Ca2+. The size of letters beneath the x axis represents the probability of conserved residues at each position across species including humans, mice, worms and flies. Inset, helical wheel projection of an ideal STIM-TM helix. (c,d) Confocal images of footprint of HeLa cells co-transfected with mCherry-ORAI1 and WT (c) or C227W-EGFP-STIM1 (d) constructs. Thapsigargin (TG), 1 μM, was added to trigger store depletion. Scale bar, 5 μm. (e–g) Constitutive activation of CRAC channels by overexpression of STIM1-C227W. (e) Two representative time courses of normalized inward currents (pA/pF), measured at −100 mV in HEK293 cells co-expressing ORAI1+WT STIM1 (blue) or ORAI1+STIM1-C227W (red), respectively. Strongly pre-activated CRAC currents were observed in the cells transfected with ORAI1+STIM1-C227W. (f) Corresponding leak-subtracted I–V curves taken from the break-in time points (t=0 s). (g) Normalized break-in currents averaged from cells transfected with ORAI1+WT STIM1 (n=5) or ORAI1+STIM1-C227W (n=6). (h) Confocal images of GFP-NFAT1 stable HeLa cells (left panels) transiently transfected with WT (right panel, top) or C227W (right panel, bottom) CFP-STIM1 constructs without store depletion. Note that cells expressing CFP-STIM1-C227W (arrow), but not WT CFP-STIM1, led to constitutive GFP-NFAT nuclear translocation. Scale bar, 5 μm. (i) Quantification of NFAT-dependent luciferase activity in HEK293T cells or Jurkat (NFAT-Luc) cells transfected with WT or C227W STIM1 constructs. PMA, phorbol 12-myristate 13-acetate. **P<0.01, ***P<0.001, Student's T-test. Error bars in all panels are defined as s.e.m.
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f1: Identification of novel gain-of-function mutations within STIM1-TM.(a) Ca2+ influx in HEK ORAI1-CFP stable cells expressing WT and mutant STIM1-TM constructs (n=30–60) monitored by Fura-2 fluorescence ratio. Store depletion was induced by 2.5 μM ionomycin. Shown are representative traces from three independent experiments. The solid bar above the curves indicates 1 mM Ca2+ in the external medium. (b) Effects of tryptophan substitution of STIM1-TM residues on constitutive Ca2+ entry in HEK ORAI1-CFP stable cells. The level of constitutive Ca2+ entry (n=3) was quantified as the difference of mean Fura-2 fluorescence ratio between the peak value in the presence of 1 mM Ca2+ and the basal value without externally added Ca2+. The size of letters beneath the x axis represents the probability of conserved residues at each position across species including humans, mice, worms and flies. Inset, helical wheel projection of an ideal STIM-TM helix. (c,d) Confocal images of footprint of HeLa cells co-transfected with mCherry-ORAI1 and WT (c) or C227W-EGFP-STIM1 (d) constructs. Thapsigargin (TG), 1 μM, was added to trigger store depletion. Scale bar, 5 μm. (e–g) Constitutive activation of CRAC channels by overexpression of STIM1-C227W. (e) Two representative time courses of normalized inward currents (pA/pF), measured at −100 mV in HEK293 cells co-expressing ORAI1+WT STIM1 (blue) or ORAI1+STIM1-C227W (red), respectively. Strongly pre-activated CRAC currents were observed in the cells transfected with ORAI1+STIM1-C227W. (f) Corresponding leak-subtracted I–V curves taken from the break-in time points (t=0 s). (g) Normalized break-in currents averaged from cells transfected with ORAI1+WT STIM1 (n=5) or ORAI1+STIM1-C227W (n=6). (h) Confocal images of GFP-NFAT1 stable HeLa cells (left panels) transiently transfected with WT (right panel, top) or C227W (right panel, bottom) CFP-STIM1 constructs without store depletion. Note that cells expressing CFP-STIM1-C227W (arrow), but not WT CFP-STIM1, led to constitutive GFP-NFAT nuclear translocation. Scale bar, 5 μm. (i) Quantification of NFAT-dependent luciferase activity in HEK293T cells or Jurkat (NFAT-Luc) cells transfected with WT or C227W STIM1 constructs. PMA, phorbol 12-myristate 13-acetate. **P<0.01, ***P<0.001, Student's T-test. Error bars in all panels are defined as s.e.m.

Mentions: Ca2+ influx through the ORAI1 channel is often conveniently triggered through passive store depletion with thapsigargin (TG) or the Ca2+ ionophore ionomycin in model cellular systems such as HeLa or HEK293 cells16. However, mutations causing loss of Ca2+-sensing capability (for example, D76A)17 or perturbing the structural integrity of the cytosolic domain (for example, L251S and R304W)1819 can bypass this manipulation and cause constitutive STIM1 puncta formation and Ca2+ influx through CRAC channels. These types of ‘gain-of-function' mutations are thought to largely represent activated conformations of STIM1, but the existence of such mutations in the STIM1 transmembrane domain has yet to be explored. Given this unique structural and functional feature, we set out to screen constitutively activating mutations throughout STIM1-TM by taking a tryptophan-scanning approach2021, with the hope of introducing steric hindrance at the contact interface to affect STIM1-TM helix packing and thus cause functional abnormality. Our efforts led to the discovery of at least two previously unrecognized ‘gain-of-function' mutations (that is, I220W and C227W), both of which could be mapped to the same side of a projected STIM1-TM helical wheel (Fig. 1a,b). These mutations did not seem to alter Ca2+ release from ER stores or the second phase of ionomycin-triggered Ca2+ influx in the typical ‘Ca2+ add-back' experiment22 (Fig. 1a; Supplementary Fig. 1a,b). The gain-of-function phenotype, albeit at varying degrees of constitutive activation, could also be recapitulated by substituting C227 with several other amino acids, including asparagine, glutamine, histidine, glutamate, tyrosine and aspartate (Supplementary Fig. 1c), or in HEK293 cells without overexpression of ORAI1 (Supplementary Fig. 1d). Since C227W exhibited the highest potency to elicit constitutive Ca2+ influx, we focused our following experiments on this unique mutation.


Inside-out Ca(2+) signalling prompted by STIM1 conformational switch.

Ma G, Wei M, He L, Liu C, Wu B, Zhang SL, Jing J, Liang X, Senes A, Tan P, Li S, Sun A, Bi Y, Zhong L, Si H, Shen Y, Li M, Lee MS, Zhou W, Wang J, Wang Y, Zhou Y - Nat Commun (2015)

Identification of novel gain-of-function mutations within STIM1-TM.(a) Ca2+ influx in HEK ORAI1-CFP stable cells expressing WT and mutant STIM1-TM constructs (n=30–60) monitored by Fura-2 fluorescence ratio. Store depletion was induced by 2.5 μM ionomycin. Shown are representative traces from three independent experiments. The solid bar above the curves indicates 1 mM Ca2+ in the external medium. (b) Effects of tryptophan substitution of STIM1-TM residues on constitutive Ca2+ entry in HEK ORAI1-CFP stable cells. The level of constitutive Ca2+ entry (n=3) was quantified as the difference of mean Fura-2 fluorescence ratio between the peak value in the presence of 1 mM Ca2+ and the basal value without externally added Ca2+. The size of letters beneath the x axis represents the probability of conserved residues at each position across species including humans, mice, worms and flies. Inset, helical wheel projection of an ideal STIM-TM helix. (c,d) Confocal images of footprint of HeLa cells co-transfected with mCherry-ORAI1 and WT (c) or C227W-EGFP-STIM1 (d) constructs. Thapsigargin (TG), 1 μM, was added to trigger store depletion. Scale bar, 5 μm. (e–g) Constitutive activation of CRAC channels by overexpression of STIM1-C227W. (e) Two representative time courses of normalized inward currents (pA/pF), measured at −100 mV in HEK293 cells co-expressing ORAI1+WT STIM1 (blue) or ORAI1+STIM1-C227W (red), respectively. Strongly pre-activated CRAC currents were observed in the cells transfected with ORAI1+STIM1-C227W. (f) Corresponding leak-subtracted I–V curves taken from the break-in time points (t=0 s). (g) Normalized break-in currents averaged from cells transfected with ORAI1+WT STIM1 (n=5) or ORAI1+STIM1-C227W (n=6). (h) Confocal images of GFP-NFAT1 stable HeLa cells (left panels) transiently transfected with WT (right panel, top) or C227W (right panel, bottom) CFP-STIM1 constructs without store depletion. Note that cells expressing CFP-STIM1-C227W (arrow), but not WT CFP-STIM1, led to constitutive GFP-NFAT nuclear translocation. Scale bar, 5 μm. (i) Quantification of NFAT-dependent luciferase activity in HEK293T cells or Jurkat (NFAT-Luc) cells transfected with WT or C227W STIM1 constructs. PMA, phorbol 12-myristate 13-acetate. **P<0.01, ***P<0.001, Student's T-test. Error bars in all panels are defined as s.e.m.
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Related In: Results  -  Collection

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f1: Identification of novel gain-of-function mutations within STIM1-TM.(a) Ca2+ influx in HEK ORAI1-CFP stable cells expressing WT and mutant STIM1-TM constructs (n=30–60) monitored by Fura-2 fluorescence ratio. Store depletion was induced by 2.5 μM ionomycin. Shown are representative traces from three independent experiments. The solid bar above the curves indicates 1 mM Ca2+ in the external medium. (b) Effects of tryptophan substitution of STIM1-TM residues on constitutive Ca2+ entry in HEK ORAI1-CFP stable cells. The level of constitutive Ca2+ entry (n=3) was quantified as the difference of mean Fura-2 fluorescence ratio between the peak value in the presence of 1 mM Ca2+ and the basal value without externally added Ca2+. The size of letters beneath the x axis represents the probability of conserved residues at each position across species including humans, mice, worms and flies. Inset, helical wheel projection of an ideal STIM-TM helix. (c,d) Confocal images of footprint of HeLa cells co-transfected with mCherry-ORAI1 and WT (c) or C227W-EGFP-STIM1 (d) constructs. Thapsigargin (TG), 1 μM, was added to trigger store depletion. Scale bar, 5 μm. (e–g) Constitutive activation of CRAC channels by overexpression of STIM1-C227W. (e) Two representative time courses of normalized inward currents (pA/pF), measured at −100 mV in HEK293 cells co-expressing ORAI1+WT STIM1 (blue) or ORAI1+STIM1-C227W (red), respectively. Strongly pre-activated CRAC currents were observed in the cells transfected with ORAI1+STIM1-C227W. (f) Corresponding leak-subtracted I–V curves taken from the break-in time points (t=0 s). (g) Normalized break-in currents averaged from cells transfected with ORAI1+WT STIM1 (n=5) or ORAI1+STIM1-C227W (n=6). (h) Confocal images of GFP-NFAT1 stable HeLa cells (left panels) transiently transfected with WT (right panel, top) or C227W (right panel, bottom) CFP-STIM1 constructs without store depletion. Note that cells expressing CFP-STIM1-C227W (arrow), but not WT CFP-STIM1, led to constitutive GFP-NFAT nuclear translocation. Scale bar, 5 μm. (i) Quantification of NFAT-dependent luciferase activity in HEK293T cells or Jurkat (NFAT-Luc) cells transfected with WT or C227W STIM1 constructs. PMA, phorbol 12-myristate 13-acetate. **P<0.01, ***P<0.001, Student's T-test. Error bars in all panels are defined as s.e.m.
Mentions: Ca2+ influx through the ORAI1 channel is often conveniently triggered through passive store depletion with thapsigargin (TG) or the Ca2+ ionophore ionomycin in model cellular systems such as HeLa or HEK293 cells16. However, mutations causing loss of Ca2+-sensing capability (for example, D76A)17 or perturbing the structural integrity of the cytosolic domain (for example, L251S and R304W)1819 can bypass this manipulation and cause constitutive STIM1 puncta formation and Ca2+ influx through CRAC channels. These types of ‘gain-of-function' mutations are thought to largely represent activated conformations of STIM1, but the existence of such mutations in the STIM1 transmembrane domain has yet to be explored. Given this unique structural and functional feature, we set out to screen constitutively activating mutations throughout STIM1-TM by taking a tryptophan-scanning approach2021, with the hope of introducing steric hindrance at the contact interface to affect STIM1-TM helix packing and thus cause functional abnormality. Our efforts led to the discovery of at least two previously unrecognized ‘gain-of-function' mutations (that is, I220W and C227W), both of which could be mapped to the same side of a projected STIM1-TM helical wheel (Fig. 1a,b). These mutations did not seem to alter Ca2+ release from ER stores or the second phase of ionomycin-triggered Ca2+ influx in the typical ‘Ca2+ add-back' experiment22 (Fig. 1a; Supplementary Fig. 1a,b). The gain-of-function phenotype, albeit at varying degrees of constitutive activation, could also be recapitulated by substituting C227 with several other amino acids, including asparagine, glutamine, histidine, glutamate, tyrosine and aspartate (Supplementary Fig. 1c), or in HEK293 cells without overexpression of ORAI1 (Supplementary Fig. 1d). Since C227W exhibited the highest potency to elicit constitutive Ca2+ influx, we focused our following experiments on this unique mutation.

Bottom Line: The mechanistic underpinnings of this inside-out Ca(2+) signalling were largely undefined.Importantly, we further identify critical residues within the cytoplasmic domain of STIM1 (STIM1-CT) that entail autoinhibition.On the basis of these findings, we propose a model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas 77030, USA.

ABSTRACT
Store-operated Ca(2+) entry mediated by STIM1 and ORAI1 constitutes one of the major Ca(2+) entry routes in mammalian cells. The molecular choreography of STIM1-ORAI1 coupling is initiated by endoplasmic reticulum (ER) Ca(2+) store depletion with subsequent oligomerization of the STIM1 ER-luminal domain, followed by its redistribution towards the plasma membrane to gate ORAI1 channels. The mechanistic underpinnings of this inside-out Ca(2+) signalling were largely undefined. By taking advantage of a unique gain-of-function mutation within the STIM1 transmembrane domain (STIM1-TM), here we show that local rearrangement, rather than alteration in the oligomeric state of STIM1-TM, prompts conformational changes in the cytosolic juxtamembrane coiled-coil region. Importantly, we further identify critical residues within the cytoplasmic domain of STIM1 (STIM1-CT) that entail autoinhibition. On the basis of these findings, we propose a model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels.

No MeSH data available.