Dynamic Coupling of the Putative Coiled-coil Domain of ORAI1 with STIM1 Mediates ORAI1 Channel Activation
2008; Elsevier BV; Volume: 283; Issue: 12 Linguagem: Inglês
10.1074/jbc.m708898200
ISSN1083-351X
AutoresMartin Muik, Irene Frischauf, Isabella Derler, Marc Fahrner, Judith Bergsmann, Petra Eder, Rainer Schindl, Clemens Hesch, Bernhard Polzinger, Reinhard Fritsch, Heike Kahr, Josef Madl, Hermann J. Gruber, Klaus Groschner, Christoph Romanin,
Tópico(s)Herbal Medicine Research Studies
ResumoSTIM1 and ORAI1 (also termed CRACM1) are essential components of the classical calcium release-activated calcium current; however, the mechanism of the transmission of information of STIM1 to the calcium release-activated calcium/ORAI1 channel is as yet unknown. Here we demonstrate by Förster resonance energy transfer microscopy a dynamic coupling of STIM1 and ORAI1 that culminates in the activation of Ca2+ entry. Förster resonance energy transfer imaging of living cells provided insight into the time dependence of crucial events of this signaling pathway comprising Ca2+ store depletion, STIM1 multimerization, and STIM1-ORAI1 interaction. Accelerated store depletion allowed resolving a significant time lag between STIM1-STIM1 and STIM1-ORAI1 interactions. Store refilling reversed both STIM1 multimerization and STIM1-ORAI1 interaction. The cytosolic STIM1 C terminus itself was able, in vitro as well as in vivo, to associate with ORAI1 and to stimulate channel function, yet without ORAI1-STIM1 cluster formation. The dynamic interaction occurred via the C terminus of ORAI1 that includes a putative coiled-coil domain structure. An ORAI1 C terminus deletion mutant as well as a mutant (L273S) with impeded coiled-coil domain formation lacked both interaction as well as functional communication with STIM1 and failed to generate Ca2+ inward currents. An N-terminal deletion mutant of ORAI1 as well as the ORAI1 R91W mutant linked to severe combined immune deficiency syndrome was similarly impaired in terms of current activation despite being able to interact with STIM1. Hence, the C-terminal coiled-coil motif of ORAI1 represents a key domain for dynamic coupling to STIM1. STIM1 and ORAI1 (also termed CRACM1) are essential components of the classical calcium release-activated calcium current; however, the mechanism of the transmission of information of STIM1 to the calcium release-activated calcium/ORAI1 channel is as yet unknown. Here we demonstrate by Förster resonance energy transfer microscopy a dynamic coupling of STIM1 and ORAI1 that culminates in the activation of Ca2+ entry. Förster resonance energy transfer imaging of living cells provided insight into the time dependence of crucial events of this signaling pathway comprising Ca2+ store depletion, STIM1 multimerization, and STIM1-ORAI1 interaction. Accelerated store depletion allowed resolving a significant time lag between STIM1-STIM1 and STIM1-ORAI1 interactions. Store refilling reversed both STIM1 multimerization and STIM1-ORAI1 interaction. The cytosolic STIM1 C terminus itself was able, in vitro as well as in vivo, to associate with ORAI1 and to stimulate channel function, yet without ORAI1-STIM1 cluster formation. The dynamic interaction occurred via the C terminus of ORAI1 that includes a putative coiled-coil domain structure. An ORAI1 C terminus deletion mutant as well as a mutant (L273S) with impeded coiled-coil domain formation lacked both interaction as well as functional communication with STIM1 and failed to generate Ca2+ inward currents. An N-terminal deletion mutant of ORAI1 as well as the ORAI1 R91W mutant linked to severe combined immune deficiency syndrome was similarly impaired in terms of current activation despite being able to interact with STIM1. Hence, the C-terminal coiled-coil motif of ORAI1 represents a key domain for dynamic coupling to STIM1. The most widespread and perhaps primordial route for Ca2+ entry across the cell membrane is through store-operated channels (SOCs), 5The abbreviations used are: SOCstore-operated channelFRETFörster resonance energy transferCRACCa2+-release-activated Ca2+TGthapsigarginERendoplasmic reticulumGSTglutathione S-transferaseCFPcyan fluorescent proteinYFPyellow fluorescent proteinHEKhuman embryonic kidney. 5The abbreviations used are: SOCstore-operated channelFRETFörster resonance energy transferCRACCa2+-release-activated Ca2+TGthapsigarginERendoplasmic reticulumGSTglutathione S-transferaseCFPcyan fluorescent proteinYFPyellow fluorescent proteinHEKhuman embryonic kidney. which serve essential functions from secretion to gene expression and cell growth (1Berridge M.J. Bootman M.D. Roderick H.L. Nat. Rev. Mol. Cell Biol. 2003; 4: 517-529Crossref PubMed Scopus (4116) Google Scholar). The prototypic SOC is the Ca2+ release-activated Ca2+ (CRAC) channel. It is activated by depletion of intracellular Ca2+ stores, which is induced by the second messenger inositol 1,4,5-triphosphate (2Parekh A.B. Nature. 2006; 441: 163-165Crossref PubMed Scopus (19) Google Scholar, 3Parekh A.B. Nat. Cell Biol. 2006; 8: 655-656Crossref PubMed Scopus (9) Google Scholar, 4Parekh A.B. Putney Jr., J.W. Physiol. Rev. 2005; 85: 757-810Crossref PubMed Scopus (1784) Google Scholar, 5Spassova M.A. Soboloff J. He L.P. Hewavitharana T. Xu W. Venkatachalam K. van Rossum D.B. Patterson R.L. Gill D.L. Biochim. Biophys. Acta. 2004; 1742: 9-20Crossref PubMed Scopus (93) Google Scholar, 6Dutta D. J. Biosci. 2000; 25: 397-404Crossref PubMed Scopus (29) Google Scholar, 7Chakrabarti R. Chakrabarti R. J. Cell Biochem. 2006; 99: 1503-1516Crossref PubMed Scopus (33) Google Scholar). The signaling events leading to the activation of CRAC/SOC as well as the composition of this channel have been a long standing mystery. Recently the use of function-based genetic screen by systematic RNA interference provided solid evidence for a key role of STIM (stromal interaction molecule) and ORAI (also termed CRACM) as essential components of SOC (8Liou J. Kim M.L. Heo W.D. Jones J.T. Myers J.W. Ferrell Jr., J.E. Meyer T. Curr. Biol. 2005; 15: 1235-1241Abstract Full Text Full Text PDF PubMed Scopus (1726) Google Scholar, 9Roos J. DiGregorio P.J. Yeromin A.V. Ohlsen K. Lioudyno M. Zhang S. Safrina O. Kozak J.A. Wagner S.L. Cahalan M.D. Velicelebi G. Stauderman K.A. J. Cell Biol. 2005; 169: 435-445Crossref PubMed Scopus (1497) Google Scholar, 10Feske S. Gwack Y. Prakriya M. Srikanth S. Puppel S.H. Tanasa B. Hogan P.G. Lewis R.S. Daly M. Rao A. Nature. 2006; 441: 179-185Crossref PubMed Scopus (1824) Google Scholar).STIM1 has been identified as a Ca2+ sensor and regulator of the store-operated Ca2+ influx and CRAC function. These proteins are associated in the ER membrane and sense Ca2+ via an EF hand Ca2+-binding site located in the lumen of the ER. Store depletion triggers a rapid redistribution of STIM1 into ER puncta close to the plasma membrane suggested to activate CRAC/SOC channels (11Smyth J.T. Dehaven W.I. Jones B.F. Mercer J.C. Trebak M. Vazquez G. Putney Jr., J.W. Biochim. Biophys. Acta. 2006; 1763: 1147-1160Crossref PubMed Scopus (192) Google Scholar, 12Baba Y. Hayashi K. Fujii Y. Mizushima A. Watarai H. Wakamori M. Numaga T. Mori Y. Iino M. Hikida M. Kurosaki T. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 16704-16709Crossref PubMed Scopus (271) Google Scholar, 13Huang G.N. Zeng W. Kim J.Y. Yuan J.P. Han L. Muallem S. Worley P.F. Nat. Cell Biol. 2006; 8: 1003-1010Crossref PubMed Scopus (560) Google Scholar).RNA interference-based screen as well as analysis of single nucleotide polymorphism arrays of patients with severe combined immune deficiency syndrome who are defective in CRAC function have identified the protein ORAI1. Severe combined immune deficiency syndrome patients are homozygous for single missense mutation in ORAI1, i.e. ORAI1R91W, leading to the loss of store-operated Ca2+ entry (10Feske S. Gwack Y. Prakriya M. Srikanth S. Puppel S.H. Tanasa B. Hogan P.G. Lewis R.S. Daly M. Rao A. Nature. 2006; 441: 179-185Crossref PubMed Scopus (1824) Google Scholar). ORAI1 is a plasma membrane protein with four predicted transmembrane segments containing a putative coiled-coil motif (14Zhang S.L. Yeromin A.V. Zhang X.H. Yu Y. Safrina O. Penna A. Roos J. Stauderman K.A. Cahalan M.D. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9357-9362Crossref PubMed Scopus (736) Google Scholar, 15Cahalan M.D. Zhang S.L. Yeromin A.V. Ohlsen K. Roos J. Stauderman K.A. Cell Calcium. 2007; 42: 133-144Crossref PubMed Scopus (141) Google Scholar), a common protein interaction domain, in the C terminus.Coexpression of STIM1 and ORAI1 revealed their colocalization at ER-plasma membrane junctions (10Feske S. Gwack Y. Prakriya M. Srikanth S. Puppel S.H. Tanasa B. Hogan P.G. Lewis R.S. Daly M. Rao A. Nature. 2006; 441: 179-185Crossref PubMed Scopus (1824) Google Scholar, 14Zhang S.L. Yeromin A.V. Zhang X.H. Yu Y. Safrina O. Penna A. Roos J. Stauderman K.A. Cahalan M.D. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9357-9362Crossref PubMed Scopus (736) Google Scholar, 16Vig M. Peinelt C. Beck A. Koomoa D.L. Rabah D. Koblan-Huberson M. Kraft S. Turner H. Fleig A. Penner R. Kinet J.P. Science. 2006; 312: 1220-1223Crossref PubMed Scopus (1138) Google Scholar, 17Prakriya M. Lewis R.S. J. Gen. Physiol. 2002; 119: 487-507Crossref PubMed Scopus (265) Google Scholar, 18Xu P. Biochem. Biophys. Res. Commun. 2006; 350: 969-976Crossref PubMed Scopus (199) Google Scholar, 19Yeromin A.V. Zhang S.L. Jiang W. Yu Y. Safrina O. Cahalan M.D. Nature. 2006; 443: 226-229Crossref PubMed Scopus (687) Google Scholar, 20Peinelt C. Vig M. Koomoa D.L. Beck A. Nadler M.J. Koblan-Huberson M. Lis A. Fleig A. Penner R. Kinet J.P. Nat. Cell Biol. 2006; 8: 771-773Crossref PubMed Scopus (509) Google Scholar, 21Luik R.M. Wu M.M. Buchanan J. Lewis R.S. J. Cell Biol. 2006; 174: 815-825Crossref PubMed Scopus (534) Google Scholar, 22Soboloff J. Spassova M.A. Tang X.D. Hewavitharana T. Xu W. Gill D.L. J. Biol. Chem. 2006; 281: 20661-20665Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar), which comes along with the activation of Ca2+ currents representing similar biophysical and pharmacological properties following Ca2+ store depletion like endogenous CRAC currents in RBL mast and Jurkat T cells (15Cahalan M.D. Zhang S.L. Yeromin A.V. Ohlsen K. Roos J. Stauderman K.A. Cell Calcium. 2007; 42: 133-144Crossref PubMed Scopus (141) Google Scholar). Based on permeability studies of different point mutations in transmembrane regions 1 and 3 of ORAI1, it is suggested to form the pore of store-operated channels (15Cahalan M.D. Zhang S.L. Yeromin A.V. Ohlsen K. Roos J. Stauderman K.A. Cell Calcium. 2007; 42: 133-144Crossref PubMed Scopus (141) Google Scholar, 23Prakriya M. Feske S. Gwack Y. Srikanth S. Rao A. Hogan P.G. Nature. 2006; 443: 230-233Crossref PubMed Scopus (1093) Google Scholar). Subsequently both proteins are supposed to manifest the main components of CRAC; however, the detailed mechanism between STIM1 multimerization and ORAI1 activation is still unknown.To elucidate the communication between STIM1 and ORAI1, we utilized Förster resonance energy transfer (FRET) microscopy to reveal a dynamic coupling of STIM1 and ORAI1 within a range of <10 nm, which results in the activation of Ca2+ entry. STIM1-STIM1 multimerization is followed by STIM1-ORAI1 interaction with a significant delay in time, and both processes could be reversed by store refilling. This association was also observed with the C terminus of STIM1 alone, but in a constitutive manner that resulted in channel activation yet without ORAI1-STIM1 cluster formation. A putative coiled-coil structure in the C terminus of ORAI1 was identified as the relevant domain for this functional protein-protein coupling, whereas the N terminus together with the critical position of Arg91 controlled rather the gating than the interaction with STIM1.EXPERIMENTAL PROCEDURESMolecular Cloning and Mutagenesis—Human ORAI1 (ORAI1; accession number NM_032790) was kindly provided by A. Rao's lab (Harvard Medical School). C-terminally tagged pECFP-N1 and pEYFP-N1/ORAI1 constructs were cloned using the XhoI and BamHI sites of the contemplated vectors. N-terminally tagged ORAI1 constructs were cloned via SalI and SmaI restriction sites of pECFP-C1 and pEYFP-C1 expression vectors (Clontech). An N-terminal deletion mutant (ΔN-term-ORAI1, amino acids 89-301) was cloned via PCR into the T/A site of pCDNA3.1V5-His TOPO expression vector (Invitrogen) and recloned into pECFP-C1 and pEYFP-C1 internal restriction sites KpnI and XbaI. A C-terminal deletion mutant (ORAI1-ΔCtermin, amino acids 1-260) was constructed similarly, and ECFP-C1 as well as EYFP-C1 N-terminally labeled ORAI1-ΔCtermin constructs were prepared as described above. pECFP/pEYFP-C1/ORAI1 served as a template for the generation of the coiled-coil mutant L273S and the ORAI1 R91W mutant. Suitable primers exchanged the corresponding codon from GAG to TCG (L273S) or from CGG to TGG (R91W) using a QuikChange XL site-directed mutagenesis kit (Stratagene). Human STIM1 (STIM1, accession number NM_003156) N-terminally ECFP- and EYFP-tagged was kindly provided by T. Meyer's lab (Stanford University). C-terminally EYFP-tagged STIM1 was purchased from GeneCopoeia™ (catalog number EX-S0521-M02). STIM1 C terminus (amino acids 233-685) was cloned into the T/A site of pcDNA3.1V5 His TOPO by PCR and subcloned into pECFP-C1 and pEYFP-C1 via their internal restriction sites KpnI and XbaI. For pulldown assays the STOP of STIM1 C terminus on position 685 was substituted by serine to obtain a STIM1 C-terminal/His6 fusion protein. GST fusion proteins (GST ORAI1-N-term and GST-ORAI1 C-term) were cloned in frame with the GST gene into pGEX4T-1 (Clontech) via its internal restriction sites BamHI and XhoI. The integrity of all resulting clones was confirmed by sequence analysis.His Tag Pulldown Assay—His6-STIM1 C terminus and ORAI1 were synthesized in separate reactions using the TnT reticulocyte lysate (Promega) in the presence of 35S. His6-STIM1 C terminus was coupled to magnetic (MagZ) beads, specific for His tag proteins purified from the TnT system (Promega), by shaking at 15 min at room temperature. STIM1 C terminus-coupled beads were washed three times and diluted in binding buffer (20 mm NaH2PO4, 500 mm NaCl, pH 7.4). For the binding assay, these beads were incubated with 20 μl of the ORAI1 reaction for 1 h at room temperature in binding buffer including protease inhibitors, washed with increasing concentrations of imidazol (10-40 mm), diluted in 2× Laemmli buffer, and boiled. The proteins were separated on a 12% polyacrylamide gel which was fixed, dried, and exposed to an x-ray film overnight. As a negative control, beads without His6-STIM1 C terminus were incubated with ORAI1.GST Tag Pulldown Assay—These assays were carried out using glutathione-Sepharose to collect GST fusion proteins (ORAI1 N terminus and ORAI1 C terminus). His-tagged STIM1 C terminus employed as a prey was tested for its ability to interact with prebound ORAI1 fragments. GST alone served as a negative control to detect possible nonspecific interaction of His-tagged STIM1 C terminus, which did not occur in any experiment (data not shown). GST fusion proteins were expressed in Escherichia coli and purified with the GST gene fusion system (GE Healthcare). His-tagged STIM1 C terminus was obtained from transiently transfected HEK293 cells and purified using the MagneHis protein purification system (Promega). Glutathione-Sepharose was incubated with 20 μg of GST fusion proteins for 30 min at 4 °C. To eliminate nonspecific binding, the samples were washed three times according to the manufacturer's manual. Purified His-tagged STIM1 C terminus (80 μg) was then incubated with the GST fusion proteins at 4 °C for 80 min. After incubation, Sepharose was washed three times, and bound proteins were eluted by the addition of 360 μl of elution buffer. Eluted proteins (40 μl) were subjected to electrophoretic separation on a 12% SDS-PAGE. To visualize potential interaction of His-tagged STIM1 C terminus and GST fusion proteins, Western blot analysis was carried out using a His5 horseradish peroxidase-conjugated antibody (Qiagen). Inputs of GST fusion proteins were detected reusing the blotted membrane with an anti GST antibody (GE Healthcare).Electrophysiology—Electrophysiological experiments were performed at 20-24 °C, using the patch clamp technique in the whole cell recording configuration. For STIM1/ORAI1 current measurements voltage ramps were usually applied every 5 s from a holding potential of 0 mV, covering a range of -90 to 90 mV over 1s. The internal pipette solution contained 3.5 mm MgCl2, 145 mm cesium methane sulfonate, 8 mm NaCl, 10 mm HEPES, 10 mm EGTA, pH 7.2. Extracellular solution consisted of 145 mm NaCl, 5 mm CsCl, 1 mm MgCl2, 10 mm HEPES, 10 mm glucose, 10 mm CaCl2, pH 7.4.Confocal FRET Fluorescence Microscopy—Confocal FRET microscopy was performed similarly as described in Ref. 24Singh A. Hamedinger D. Hoda J.C. Gebhart M. Koschak A. Romanin C. Striessnig J. Nat. Neurosci. 2006; 9: 1108-1116Crossref PubMed Scopus (107) Google Scholar. In brief, a QLC100 real time confocal system (VisiTech Int.) was used for recording fluorescence images connected to two Photometrics CoolSNAPHQ monochrome cameras (Roper Scientific) and a dual port adapter (dichroic, 505lp; cyan emission filter, 485/30; yellow emission filter, 535/50; Chroma Technology Corp.). This system was attached to an Axiovert 200M microscope (Zeiss) in conjunction with an argon ion multi-wave-length (457, 488, and 514 nm) laser (Spectra Physics). The wavelengths were selected by an Acousto Optical Tuneable Filter (VisiTech Int.). MetaMorph 5.0 software (Universal Imaging Corp.) was used to acquire images and to control the confocal system. Illumination times for CFP/FRET and YFP images that were recorded with a minimum delay consecutively were approximately 900 ms. Prior to the calculation, the images have to be corrected because of cross-talk as well as cross-excitation. For this, the appropriate cross-talk calibration factors were determined for each of the constructs on the day the FRET experiments were performed. The corrected FRET image (NFRET) was calculated after background subtraction and threshold determination using a custom-made software (25Derler I. Hofbauer M. Kahr H. Fritsch R. Muik M. Kepplinger K. Hack M.E. Moritz S. Schindl R. Groschner K. Romanin C. J. Physiol. 2006; 577: 31-44Crossref PubMed Scopus (94) Google Scholar) integrated in MatLab 7.0.4 according to the published method (26Xia Z. Liu Y. Biophys. J. 2001; 81: 2395-2402Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). The local ratio between CFP and YFP might vary because of different localizations of diverse protein constructs, which could lead to the calculation of false FRET values (27Berney C. Danuser G. Biophys. J. 2003; 84: 3992-4010Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar). Accordingly, the analysis should be limited to pixels with a CFP:YFP molar ratio between 1:10 and 10:1 (27Berney C. Danuser G. Biophys. J. 2003; 84: 3992-4010Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar) to yield reliable results. This occurred rather seldom in our experiments, and approximately 90% of ratios are between 1:5 and 5:1.Statistics—Significance analysis was performed with the two-tailed Mann-Whitney test. The mean ± S.E. values are shown throughout the manuscript.RESULTS AND DISCUSSIONCoupling between STIM1 and ORAI1—Association of ORAI1 and STIM1 in a signaling complex is a controversial issue (19Yeromin A.V. Zhang S.L. Jiang W. Yu Y. Safrina O. Cahalan M.D. Nature. 2006; 443: 226-229Crossref PubMed Scopus (687) Google Scholar, 28Vig M. Beck A. Billingsley J.M. Lis A. Parvez S. Peinelt C. Koomoa D.L. Soboloff J. Gill D.L. Fleig A. Kinet J.P. Penner R. Curr. Biol. 2006; 16: 2073-2079Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar, 29Gwack Y. Srikanth S. Feske S. Cruz-Guilloty F. Oh-hora M. Neems D.S. Hogan P.G. Rao A. J. Biol. Chem. 2007; 282: 16232-16243Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). To evaluate the possibility of a dynamic, physical interaction as a concept of communication between STIM1 and ORAI1, we utilized confocal FRET microscopy for imaging of living human embryonic kidney 293 cells that expressed both proteins in fluorescent-labeled form. These proteins either N- or C-terminally tagged with CFP (donor) or YFP (acceptor) displayed similar behavior as native proteins (data not shown), evident from the activation of CRAC-like currents following ER store depletion (Fig. 1a). Their biophysical characteristics such as time course of activation and maximum current density were not significantly different from native CRAC currents, with a reversal potential larger than +50 mV (Fig. 1b). ORAI1 has been reported to redistribute in response to store depletion from a homogenous to a clustered localization accumulating in the plasma membrane adjacent to clustered STIM1 (18Xu P. Biochem. Biophys. Res. Commun. 2006; 350: 969-976Crossref PubMed Scopus (199) Google Scholar, 21Luik R.M. Wu M.M. Buchanan J. Lewis R.S. J. Cell Biol. 2006; 174: 815-825Crossref PubMed Scopus (534) Google Scholar). Following ER store depletion by thapsigargin, we observed (Fig. 1c, left panel) both proteins labeled C-terminally colocalized in clusters. Such regions exhibited robust FRET values (0.09 ± 0.01, n = 8; Fig. 1g) demonstrating high local proximity of the tagged proteins, compatible with either direct or indirect interaction between STIM1 and ORAI1. In control experiments in which STIM1 was labeled at its N-terminal side, which is located within the ER lumen, no FRET was measured (-0.01 ± 0.01, n = 10; Fig. 1, c, right panel, and g) consistent with distant localization of fluorescence labels on opposite sides of the membrane. It is of note that in this situation a similar STIM1-ORAI1 cluster formation is evident (Fig. 1c, right panel).STIM1 C Terminus Interacts in Vivo as Well as in Vitro with ORAI1—For further investigation of the interaction, we utilized the C terminus of STIM1, which is expected (13Huang G.N. Zeng W. Kim J.Y. Yuan J.P. Han L. Muallem S. Worley P.F. Nat. Cell Biol. 2006; 8: 1003-1010Crossref PubMed Scopus (560) Google Scholar) to constitutively couple to ORAI1 provided that no other processes emerging from ER store depletion are absolutely required for channel activation. Coexpression of ORAI1 with STIM1 C terminus resulted indeed in constitutive currents (Fig. 1d) with current-voltage relationships (Fig. 1e) similar to Fig. 1b. Activation of these currents by STIM1 C terminus occurred to a similar extent when endogenous STIM1 was suppressed by small interfering RNA specific to full-length STIM1 (data not shown). N-terminally tagged STIM1 C terminus displayed, besides some cytosolic localization, clear targeting to the plasma membrane when expressed together with ORAI1 in HEK cells (Fig. 1f) but remained cytosolic when expressed alone (Fig. 1f; lower panel). STIM1 C terminus was colocalized with ORAI1 and yielded robust FRET (0.074 ± 0.007, n = 36). Generation of clusters was not visible when only the C-terminal fragment instead of full-length STIM1 was used (Fig. 1, compare c and f), underscoring the ER localization of the latter as a prerequisite for cluster formation (in this process). Nevertheless, the STIM1 C terminus is principally sufficient as a surrogate of STIM1 in terms of association and channel activation, yet independent of ER store depletion and ORAI1 cluster formation. In vitro pulldown experiments using His6-tagged STIM1 C terminus as bait demonstrated the ability of the C-terminal domain of STIM1 to associate with ORAI1 in cell-free conditions (Fig. 1h, left panel). ORAI1 binding increased significantly (33 ± 4 versus 23 ± 5 arbitrary units, n = 9, p < 0.01) above the relatively high background binding of ORAI1 when His6-tagged STIM1 C terminus was prebound. To strengthen this in vitro approach, we focused on the N and C termini of ORAI1 and employed these fragments as GST proteins to potentially pull down His-tagged STIM1 C terminus (Fig. 1h, right panel). The C terminus of ORAI1 in contrast to its N terminus showed clear interaction with STIM1 C terminus (n = 7), indicating association of the proteins in vitro and suggesting binding as a molecular process involved in channel activation.Dynamic STIM1-ORAI1 Coupling Is Controlled by the Filling State of the ER—To analyze kinetic aspects of this coupling process, we monitored the time dependence of the STIM1-ORAI1 interaction by dynamic FRET measurements in response to ER store depletion by thapsigargin or ionomycin (Fig. 2a). Under resting conditions the STIM1-ORAI1 interaction seemed to be largely reduced (Fig. 2a) based on the low FRET values. Despite some colocalization between STIM1 and ORAI1, clusters were scarcely visible. Following thapsigargin application, formation of STIM1-ORAI1 coclusters developed over a time period of 240 s and was temporally as well as spatially correlated with an increase in FRET (Fig. 2, a and b). This suggested a causal relationship between ER depletion and development of dynamical STIM1-ORAI1 interactions. Consistently, ionomycin (Fig. 2b, see also Fig. 2g), which is known to deplete ER stores more rapidly than thapsigargin, induced both a faster onset and development of FRET over 120 s, further substantiating that the increasing STIM1-ORAI1 interactions are linked to store depletion.FIGURE 2STIM1-ORAI1 coupling and STIM1 multimerization occur reversibly correlated in time with the ER filling state. a, left panel, localization, overlay, and calculated FRET life cell image series of ORAI1-CFP and STIM1-YFP in response to 2 μm TG. Right panel, magnified section as indicated by the white box in the left panel with arrows marking STIM1 clusters and corresponding ORAI1-STIM1 FRET. b, time course of relative FRET between ORAI1-CFP and STIM1-YFP in response to 2 μm TG or 300 nm ionomycin administered at 2 min. To enable comparison, both FRET curves were shifted (TG, by -0.0011; ionomycin, by -0.0233) to 0.0000 at the time point of 2 min. At 4 min the relative FRET values (TG, n = 8; ionomycin, n = 7) are significantly different (p = 0.0041). The numbers included in the time course with TG refer to the numbers given in the left margin of a. c, localization, overlay, and calculated FRET life cell image series of CFP-STIM1 and YFP-STIM1 coexpressed with untagged ORAI1 in response to 2 μm TG. The arrows mark STIM1-STIM1 clusters. d, time course of FRET (NFRET) from ER-targeted cameleon (YC4-ER) and between CFP-STIM1 and YFP-STIM1 in response to 2 μm TG. e, time courses of FRET values for the individual steps of ORAI1 activation machinery from b and d normalized for superposition at 2 and 8 min to 0 and 1, respectively. FRET of CFP-ORAI1 and YFP-ORAI1 coexpressed with untagged STIM1 that did not change following TG application was set to 0 at 2 min. f, time course of normalized FRET of CFP-STIM1 and YFP-STIM1/ORAI1-CFP and STIM1-YFP in response to 10 μm ionomycin. At t = 25 s the relative FRET values (STIM1/STIM1: n = 5, t½ = 5 s; STIM1/ORAI1: n = 9, t½ = 15 s) are significantly different (p = 0.0004). g, time course of normalized FRET of CFP-STIM1 and YFP-STIM1/ORAI1-CFP and STIM1-YFP in response to 100 μm CCH and 20 μm BHQ (for store depletion) and 100 μm atropine + Ca2+ (store repletion) (STIM1/STIM1: n = 6, t½ = 25 s; STIM1/ORAI1: n = 7, t½ = 30 s). The error bars reflect S.E. The numbers of experiments are given in parentheses. Scale bars, 5 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further elucidate this relationship, we imaged individual steps of the STIM1-ORAI1 activation machinery over time at several sequential stages: Ca2+ depletion of ER, STIM1 multimerization, STIM1-ORAI1 interaction, and homomeric ORAI1 complex formation. The time course of ER store depletion was monitored employing ER-targeted cameleon (31Miyawaki A. Llopis J. Heim R. McCaffery J.M. Adams J.A. Ikura M. Tsien R.Y. Nature. 1997; 388: 882-887Crossref PubMed Scopus (2594) Google Scholar) (YC4-ER) and correlated with the multimerization of STIM1 (Fig. 2, c and d). For the latter we employed a FRET based assay (32Liou J. Fivaz M. Inoue T. Meyer T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 9301-9306Crossref PubMed Scopus (511) Google Scholar) utilizing CFP/YFP-tagged STIM1 to monitor STIM1 multimerization that yielded punctuate clusters following thapsigargin application. STIM1 seemed already partially preassociated (32Liou J. Fivaz M. Inoue T. Meyer T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 9301-9306Crossref PubMed Scopus (511) Google Scholar, 33Dziadek M.A. Johnstone L.S. Cell Calcium. 2007; 42: 123-132Crossref PubMed Scopus (87) Google Scholar) based on the elevated FRET values (t = 2 min: 0.12 ± 0.01, n = 12) under resting cell conditions that substantially increased (t = 8 min: 0.19 ± 0.01, n = 12) upon ER store depletion (Fig. 2d). Correlating the decrease in ER Ca2+ concentration with the increase in FRET from homomeric STIM1, multimerization revealed (Fig. 2d) a reciprocal time course that supports the linkage between these two processes. Moreover, superimposing (Fig. 2e) the normalized time courses of the individual steps of the ORAI1 activation machinery indicated a temporal coherence and an overlap of these processes. Accelerated ER store depletion employing 10 μm ionomycin (32Liou J. Fivaz M. Inoue T. Meyer T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 9301-9306Crossref PubMed Scopus (511) Google Scholar) enabled us (Fig. 2f) to resolve a time lag between STIM1-STIM1 (t½ = 5 s, consistent with Ref. 32Liou J. Fivaz M. Inoue T. Meyer T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 9301-9306Crossref PubMed Scopus (511) Google Scholar) and STIM1-ORAI1 (t½ = 15 s) interactions, supporting the concept of a causal chain of aggregation and association events. The STIM1-ORAI1 interaction seemed to occur on a similar time scale as STIM1 puncta formation (t½ = 15 s, data not shown) in the HEK293 cells. Homomeric ORAI1 interactions as derived from substantial FR
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