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The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers.

Penna A, Demuro A, Yeromin AV, Zhang SL, Safrina O, Parker I, Cahalan MD - Nature (2008)

Bottom Line: In contrast, co-expression of GFP-tagged Orai with the carboxy terminus of Stim as a cytosolic protein to activate the Orai channel without inducing Ca(2+) store depletion or clustering of Orai into punctae yielded mostly four-step photobleaching, consistent with a tetrameric stoichiometry of the active Orai channel.Interaction with the C terminus of Stim thus induces Orai dimers to dimerize, forming tetramers that constitute the Ca(2+)-selective pore.This represents a new mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of California Irvine, California 92697-4561, USA.

ABSTRACT
Ca(2+)-release-activated Ca(2+) (CRAC) channels underlie sustained Ca(2+) signalling in lymphocytes and numerous other cells after Ca(2+) liberation from the endoplasmic reticulum (ER). RNA interference screening approaches identified two proteins, Stim and Orai, that together form the molecular basis for CRAC channel activity. Stim senses depletion of the ER Ca(2+) store and physically relays this information by translocating from the ER to junctions adjacent to the plasma membrane, and Orai embodies the pore of the plasma membrane calcium channel. A close interaction between Stim and Orai, identified by co-immunoprecipitation and by Förster resonance energy transfer, is involved in the opening of the Ca(2+) channel formed by Orai subunits. Most ion channels are multimers of pore-forming subunits surrounding a central channel, which are preassembled in the ER and transported in their final stoichiometry to the plasma membrane. Here we show, by biochemical analysis after cross-linking in cell lysates and intact cells and by using non-denaturing gel electrophoresis without cross-linking, that Orai is predominantly a dimer in the plasma membrane under resting conditions. Moreover, single-molecule imaging of green fluorescent protein (GFP)-tagged Orai expressed in Xenopus oocytes showed predominantly two-step photobleaching, again consistent with a dimeric basal state. In contrast, co-expression of GFP-tagged Orai with the carboxy terminus of Stim as a cytosolic protein to activate the Orai channel without inducing Ca(2+) store depletion or clustering of Orai into punctae yielded mostly four-step photobleaching, consistent with a tetrameric stoichiometry of the active Orai channel. Interaction with the C terminus of Stim thus induces Orai dimers to dimerize, forming tetramers that constitute the Ca(2+)-selective pore. This represents a new mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule.

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GFP-Orai forms dimers in the basal state and predominantly tetramers when co-expressed with C-Stima, b Representative examples of single-molecule bleaching records obtained from oocytes expressing, respectively, GFP-Orai alone and GFP-Orai together with C-Stim. c, Histogram shows percentages of spots that showed 1, 2, 3, and 4 bleaching steps in oocytes expressing GFP-Orai alone (open bars) and GFP-Orai plus C-Stim (filled bars). Errors bars indicate ± 1 SEM. Data for GFP-Orai were obtained from 400 spots, 11 imaging records, 6 oocytes; and data for GFP-Orai + C-Stim from 278 spots, 5 imaging records, 3 oocytes. Comparison of bleaching step distributions with and without C-Stim yielded a Chi square value of 590; p < 0.001. This cannot be attributed to an increased likelihood of two GFP-Orai dimers happening to lie indistinguishably close to each other because of increased expression level or C-Stim-induced clustering because fluorescence spots in both conditions showed similar random distributions and densities (respectively, 37±6 and 41±4 spots in a 40×40 μm2 region), and we did not observe spots with >4 bleaching steps as might be expected for a macro-molecular clustering.
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Figure 4: GFP-Orai forms dimers in the basal state and predominantly tetramers when co-expressed with C-Stima, b Representative examples of single-molecule bleaching records obtained from oocytes expressing, respectively, GFP-Orai alone and GFP-Orai together with C-Stim. c, Histogram shows percentages of spots that showed 1, 2, 3, and 4 bleaching steps in oocytes expressing GFP-Orai alone (open bars) and GFP-Orai plus C-Stim (filled bars). Errors bars indicate ± 1 SEM. Data for GFP-Orai were obtained from 400 spots, 11 imaging records, 6 oocytes; and data for GFP-Orai + C-Stim from 278 spots, 5 imaging records, 3 oocytes. Comparison of bleaching step distributions with and without C-Stim yielded a Chi square value of 590; p < 0.001. This cannot be attributed to an increased likelihood of two GFP-Orai dimers happening to lie indistinguishably close to each other because of increased expression level or C-Stim-induced clustering because fluorescence spots in both conditions showed similar random distributions and densities (respectively, 37±6 and 41±4 spots in a 40×40 μm2 region), and we did not observe spots with >4 bleaching steps as might be expected for a macro-molecular clustering.

Mentions: To determine Orai stoichiometry at the single-molecule level in the native membrane environment of intact cells we used a recently developed method23, employing total internal reflection microscopy (TIRFM) to image bleaching steps of individual GFP-tagged Orai. Xenopus oocytes were injected with cRNA for GFP-Orai with or without coincident injection of cRNA for Stim or C-Stim. When Stim was co-expressed, depletion of the Ca2+ store with TG resulted in clustering of GFP-Orai in punctae (Fig 3a), making single molecules difficult to resolve. However, consistent with the above results in Drosophila S2 cells, oocytes expressing both GFP-Orai and C-Stim showed large Ca2+ influx as assessed by Ca2+ fluorimetry, together with activation of endogenous Ca2+-dependent Cl- current; whereas neither Orai nor C-Stim alone were effective (Fig. 3b). Moreover, TIRFM imaging of GFP-Orai-expressing oocytes revealed numerous, diffraction-limited fluorescent spots (Fig. 3c), that were absent in non-injected oocytes and increased in density with time of expression. Continuous exposure to laser excitation resulted in stepwise decrements of fluorescence at these spots (Supplementary Movies), corresponding to bleaching of individual GFP molecules23. Different spots showed varying numbers of bleaching steps, ranging from one to a maximum of four (Fig. 4a,b). Notably, estimates of the mean number of GFP molecules per spot made in this way differed markedly depending on expression of C-Stim. In oocytes expressing GFP-Orai alone, a majority (∼70%) of spots showed two steps to complete bleaching (Fig. 4a,c), consistent with biochemical observations in S2 cells, whereas with co-expression of C-Stim most spots (∼62%) showed four-step bleaching (Fig. 4b,c). The small proportions of spots that showed one- or three-step bleaching may reflect instances of near-simultaneous stochastic bleaching steps that could not separately resolved or expression of non-fluorescent GFP molecules23. The optical resolution of the microscope (ca. 250 nm) is inadequate to determine whether a spot showing 4 bleaching steps is truly a tetramer or, for example, two distinct dimers linked by C-Stim. We favor the former interpretation based on evidence15 that expression of an Orai1 tandem tetramer construct forms functional CRAC channels, and that CRAC is inhibited when one subunit in the tetramer is replaced by a dominant-negative Orai. We thus conclude that Orai is present in the membrane predominantly as dimers under basal conditions, and that activation by C-Stim induces association to form tetramers.


The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers.

Penna A, Demuro A, Yeromin AV, Zhang SL, Safrina O, Parker I, Cahalan MD - Nature (2008)

GFP-Orai forms dimers in the basal state and predominantly tetramers when co-expressed with C-Stima, b Representative examples of single-molecule bleaching records obtained from oocytes expressing, respectively, GFP-Orai alone and GFP-Orai together with C-Stim. c, Histogram shows percentages of spots that showed 1, 2, 3, and 4 bleaching steps in oocytes expressing GFP-Orai alone (open bars) and GFP-Orai plus C-Stim (filled bars). Errors bars indicate ± 1 SEM. Data for GFP-Orai were obtained from 400 spots, 11 imaging records, 6 oocytes; and data for GFP-Orai + C-Stim from 278 spots, 5 imaging records, 3 oocytes. Comparison of bleaching step distributions with and without C-Stim yielded a Chi square value of 590; p < 0.001. This cannot be attributed to an increased likelihood of two GFP-Orai dimers happening to lie indistinguishably close to each other because of increased expression level or C-Stim-induced clustering because fluorescence spots in both conditions showed similar random distributions and densities (respectively, 37±6 and 41±4 spots in a 40×40 μm2 region), and we did not observe spots with >4 bleaching steps as might be expected for a macro-molecular clustering.
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Related In: Results  -  Collection

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Figure 4: GFP-Orai forms dimers in the basal state and predominantly tetramers when co-expressed with C-Stima, b Representative examples of single-molecule bleaching records obtained from oocytes expressing, respectively, GFP-Orai alone and GFP-Orai together with C-Stim. c, Histogram shows percentages of spots that showed 1, 2, 3, and 4 bleaching steps in oocytes expressing GFP-Orai alone (open bars) and GFP-Orai plus C-Stim (filled bars). Errors bars indicate ± 1 SEM. Data for GFP-Orai were obtained from 400 spots, 11 imaging records, 6 oocytes; and data for GFP-Orai + C-Stim from 278 spots, 5 imaging records, 3 oocytes. Comparison of bleaching step distributions with and without C-Stim yielded a Chi square value of 590; p < 0.001. This cannot be attributed to an increased likelihood of two GFP-Orai dimers happening to lie indistinguishably close to each other because of increased expression level or C-Stim-induced clustering because fluorescence spots in both conditions showed similar random distributions and densities (respectively, 37±6 and 41±4 spots in a 40×40 μm2 region), and we did not observe spots with >4 bleaching steps as might be expected for a macro-molecular clustering.
Mentions: To determine Orai stoichiometry at the single-molecule level in the native membrane environment of intact cells we used a recently developed method23, employing total internal reflection microscopy (TIRFM) to image bleaching steps of individual GFP-tagged Orai. Xenopus oocytes were injected with cRNA for GFP-Orai with or without coincident injection of cRNA for Stim or C-Stim. When Stim was co-expressed, depletion of the Ca2+ store with TG resulted in clustering of GFP-Orai in punctae (Fig 3a), making single molecules difficult to resolve. However, consistent with the above results in Drosophila S2 cells, oocytes expressing both GFP-Orai and C-Stim showed large Ca2+ influx as assessed by Ca2+ fluorimetry, together with activation of endogenous Ca2+-dependent Cl- current; whereas neither Orai nor C-Stim alone were effective (Fig. 3b). Moreover, TIRFM imaging of GFP-Orai-expressing oocytes revealed numerous, diffraction-limited fluorescent spots (Fig. 3c), that were absent in non-injected oocytes and increased in density with time of expression. Continuous exposure to laser excitation resulted in stepwise decrements of fluorescence at these spots (Supplementary Movies), corresponding to bleaching of individual GFP molecules23. Different spots showed varying numbers of bleaching steps, ranging from one to a maximum of four (Fig. 4a,b). Notably, estimates of the mean number of GFP molecules per spot made in this way differed markedly depending on expression of C-Stim. In oocytes expressing GFP-Orai alone, a majority (∼70%) of spots showed two steps to complete bleaching (Fig. 4a,c), consistent with biochemical observations in S2 cells, whereas with co-expression of C-Stim most spots (∼62%) showed four-step bleaching (Fig. 4b,c). The small proportions of spots that showed one- or three-step bleaching may reflect instances of near-simultaneous stochastic bleaching steps that could not separately resolved or expression of non-fluorescent GFP molecules23. The optical resolution of the microscope (ca. 250 nm) is inadequate to determine whether a spot showing 4 bleaching steps is truly a tetramer or, for example, two distinct dimers linked by C-Stim. We favor the former interpretation based on evidence15 that expression of an Orai1 tandem tetramer construct forms functional CRAC channels, and that CRAC is inhibited when one subunit in the tetramer is replaced by a dominant-negative Orai. We thus conclude that Orai is present in the membrane predominantly as dimers under basal conditions, and that activation by C-Stim induces association to form tetramers.

Bottom Line: In contrast, co-expression of GFP-tagged Orai with the carboxy terminus of Stim as a cytosolic protein to activate the Orai channel without inducing Ca(2+) store depletion or clustering of Orai into punctae yielded mostly four-step photobleaching, consistent with a tetrameric stoichiometry of the active Orai channel.Interaction with the C terminus of Stim thus induces Orai dimers to dimerize, forming tetramers that constitute the Ca(2+)-selective pore.This represents a new mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of California Irvine, California 92697-4561, USA.

ABSTRACT
Ca(2+)-release-activated Ca(2+) (CRAC) channels underlie sustained Ca(2+) signalling in lymphocytes and numerous other cells after Ca(2+) liberation from the endoplasmic reticulum (ER). RNA interference screening approaches identified two proteins, Stim and Orai, that together form the molecular basis for CRAC channel activity. Stim senses depletion of the ER Ca(2+) store and physically relays this information by translocating from the ER to junctions adjacent to the plasma membrane, and Orai embodies the pore of the plasma membrane calcium channel. A close interaction between Stim and Orai, identified by co-immunoprecipitation and by Förster resonance energy transfer, is involved in the opening of the Ca(2+) channel formed by Orai subunits. Most ion channels are multimers of pore-forming subunits surrounding a central channel, which are preassembled in the ER and transported in their final stoichiometry to the plasma membrane. Here we show, by biochemical analysis after cross-linking in cell lysates and intact cells and by using non-denaturing gel electrophoresis without cross-linking, that Orai is predominantly a dimer in the plasma membrane under resting conditions. Moreover, single-molecule imaging of green fluorescent protein (GFP)-tagged Orai expressed in Xenopus oocytes showed predominantly two-step photobleaching, again consistent with a dimeric basal state. In contrast, co-expression of GFP-tagged Orai with the carboxy terminus of Stim as a cytosolic protein to activate the Orai channel without inducing Ca(2+) store depletion or clustering of Orai into punctae yielded mostly four-step photobleaching, consistent with a tetrameric stoichiometry of the active Orai channel. Interaction with the C terminus of Stim thus induces Orai dimers to dimerize, forming tetramers that constitute the Ca(2+)-selective pore. This represents a new mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule.

Show MeSH
Related in: MedlinePlus