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The Orai1 Store-operated Calcium Channel Functions as a Hexamer*

  • Author Footnotes
    1 Both authors contributed equally to this work.
    Xiangyu Cai
    Footnotes
    1 Both authors contributed equally to this work.
    Affiliations
    From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and
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  • Author Footnotes
    1 Both authors contributed equally to this work.
    Yandong Zhou
    Correspondence
    To whom correspondence may be addressed:
    Footnotes
    1 Both authors contributed equally to this work.
    Affiliations
    From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and
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  • Robert M. Nwokonko
    Affiliations
    From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and
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  • Natalia A. Loktionova
    Affiliations
    From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and
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  • Xianming Wang
    Affiliations
    From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and
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  • Ping Xin
    Affiliations
    From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and
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  • Mohamed Trebak
    Affiliations
    From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and
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  • Youjun Wang
    Affiliations
    the Beijing Key Laboratory of Gene Resources and Molecular Development, College of Life Sciences, Beijing Normal University, Beijing 100875, China
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  • Donald L. Gill
    Correspondence
    To whom correspondence may be addressed:
    Affiliations
    From the Department of Cellular and Molecular Physiology, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and
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  • Author Footnotes
    * This work was supported in whole or part by National Institute of Health Grants R01 GM120783 and R01 GM109279. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    ♦ This article was selected as a Paper of the Week.
    This article contains supplemental Figs. S1 and S2.
    1 Both authors contributed equally to this work.
Open AccessPublished:October 25, 2016DOI:https://doi.org/10.1074/jbc.M116.758813

      Abstract

      Orai channels mediate store-operated Ca2+ signals crucial in regulating transcription in many cell types, and implicated in numerous immunological and inflammatory disorders. Despite their central importance, controversy surrounds the basic subunit structure of Orai channels, with several biochemical and biophysical studies suggesting a tetrameric structure yet crystallographic evidence indicating a hexamer. We systematically investigated the subunit configuration of the functional Orai1 channel, generating a series of tdTomato-tagged concatenated Orai1 channel constructs (dimers to hexamers) expressed in CRISPR-derived ORAI1 knock-out HEK cells, stably expressing STIM1-YFP. Surface biotinylation demonstrated that the full-length concatemers were surface membrane-expressed. Unexpectedly, Orai1 dimers, trimers, tetramers, pentamers, and hexamers all mediated similar and substantial store-operated Ca2+ entry. Moreover, each Orai1 concatemer mediated Ca2+ currents with inward rectification and reversal potentials almost identical to those observed with expressed Orai1 monomer. In Orai1 tetramers, subunit-specific replacement with Orai1 E106A “pore-inactive” subunits revealed that functional channels utilize only the N-terminal dimer from the tetramer. In contrast, Orai1 E106A replacement in Orai1 hexamers established that all the subunits can contribute to channel formation, indicating a hexameric channel configuration. The critical Ca2+ selectivity filter-forming Glu-106 residue may mediate Orai1 channel assembly around a central Ca2+ ion within the pore. Thus, multiple E106A substitutions in the Orai1 hexamer may promote an alternative “trimer-of-dimers” channel configuration in which the C-terminal E106A subunits are excluded from the hexameric core. Our results argue strongly against a tetrameric configuration for Orai1 channels and indicate that the Orai1 channel functions as a hexamer.

      Introduction

      The members of the Orai family of ion channels are ubiquitously expressed among cell types and mediate “store-operated” Ca2+ entry signals, crucial in the control of many responses including gene expression, cell growth, secretory events, and cell motility (
      • Prakriya M.
      • Lewis R.S.
      Store-operated calcium channels.
      ,
      • Amcheslavsky A.
      • Wood M.L.
      • Yeromin A.V.
      • Parker I.
      • Freites J.A.
      • Tobias D.J.
      • Cahalan M.D.
      Molecular biophysics of Orai store-operated Ca channels.
      • Shim A.H.
      • Tirado-Lee L.
      • Prakriya M.
      Structural and functional mechanisms of CRAC channel regulation.
      ). Orai channels are highly Ca2+-selective plasma membrane (PM)
      The abbreviations used are: PM, plasma membrane, ER, endoplasmic reticulum, STIM, stromal-interacting molecule, CRAC, Ca2+ release-activating Ca2+, gRNA, guide RNA, BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
      The abbreviations used are: PM, plasma membrane, ER, endoplasmic reticulum, STIM, stromal-interacting molecule, CRAC, Ca2+ release-activating Ca2+, gRNA, guide RNA, BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
      channels activated through an elaborate intermembrane coupling mechanism by the Ca2+-sensing STIM proteins of the endoplasmic reticulum (ER) (
      • Prakriya M.
      • Lewis R.S.
      Store-operated calcium channels.
      • Amcheslavsky A.
      • Wood M.L.
      • Yeromin A.V.
      • Parker I.
      • Freites J.A.
      • Tobias D.J.
      • Cahalan M.D.
      Molecular biophysics of Orai store-operated Ca channels.
      ,
      • Shim A.H.
      • Tirado-Lee L.
      • Prakriya M.
      Structural and functional mechanisms of CRAC channel regulation.
      ,
      • Soboloff J.
      • Rothberg B.S.
      • Madesh M.
      • Gill D.L.
      STIM proteins: dynamic calcium signal transducers.
      ,
      • Hogan P.G.
      The STIM1-ORAI1 microdomain.
      • Derler I.
      • Jardin I.
      • Romanin C.
      Molecular mechanisms of STIM/Orai communication.
      ). Alterations in the function of Orai channels and STIM proteins are implicated in a large number of immunological, muscular, and inflammatory disease states (
      • Prakriya M.
      • Lewis R.S.
      Store-operated calcium channels.
      ,
      • Kar P.
      • Parekh A.
      STIM proteins, Orai1, and gene expression.
      ,
      • Zhou Y.
      • Trebak M.
      • Gill D.L.
      Calcium signals tune the fidelity of transcriptional responses.
      • Feske S.
      • Wulff H.
      • Skolnik E.Y.
      Ion channels in innate and adaptive immunity.
      ). STIM1 undergoes a complex conformational rearrangement in the ER membrane in response to depletion of ER luminal Ca2+, and then translocates into discrete ER-PM junctions where it attaches to the PM surface and is able to directly tether and activate PM Orai1 channels (
      • Prakriya M.
      • Lewis R.S.
      Store-operated calcium channels.
      ,
      • Amcheslavsky A.
      • Wood M.L.
      • Yeromin A.V.
      • Parker I.
      • Freites J.A.
      • Tobias D.J.
      • Cahalan M.D.
      Molecular biophysics of Orai store-operated Ca channels.
      ,
      • Soboloff J.
      • Rothberg B.S.
      • Madesh M.
      • Gill D.L.
      STIM proteins: dynamic calcium signal transducers.
      ,
      • Hogan P.G.
      The STIM1-ORAI1 microdomain.
      • Derler I.
      • Jardin I.
      • Romanin C.
      Molecular mechanisms of STIM/Orai communication.
      ). The structural properties of Orai channels and STIM proteins and details of the molecular coupling they undergo are key to understanding their physiological activation, pharmacological modification, and pathophysiological role in disease states. Better understanding of the molecular coupling interface between STIM1 and Orai1 continues to emerge (
      • Stathopulos P.B.
      • Schindl R.
      • Fahrner M.
      • Zheng L.
      • Gasmi-Seabrook G.M.
      • Muik M.
      • Romanin C.
      • Ikura M.
      STIM1/Orai1 coiled-coil interplay in the regulation of store-operated calcium entry.
      ,
      • Wang X.
      • Wang Y.
      • Zhou Y.
      • Hendron E.
      • Mancarella S.
      • Andrake M.D.
      • Rothberg B.S.
      • Soboloff J.
      • Gill D.L.
      Distinct Orai-coupling domains in STIM1 and STIM2 define the Orai-activating site.
      • Tirado-Lee L.
      • Yamashita M.
      • Prakriya M.
      Conformational changes in the Orai1 C-terminus evoked by STIM1 binding.
      ), and significant advances are being made in discerning the mechanism by which Orai1 channels are gated by STIM1 (
      • Gudlur A.
      • Quintana A.
      • Zhou Y.
      • Hirve N.
      • Mahapatra S.
      • Hogan P.G.
      STIM1 triggers a gating rearrangement at the extracellular mouth of the ORAI1 channel.
      ,
      • Palty R.
      • Stanley C.
      • Isacoff E.Y.
      Critical role for Orai1 C-terminal domain and TM4 in CRAC channel gating.
      • Zhou Y.
      • Cai X.
      • Loktionova N.A.
      • Wang X.
      • Nwokonko R.M.
      • Wang X.
      • Wang Y.
      • Rothberg B.S.
      • Trebak M.
      • Gill D.L.
      The STIM1 binding site nexus remotely controls Orai1 channel gating.
      ). Recent studies have also provided a better understanding of the arrangement, dynamics, and stoichiometry of STIM proteins and Orai channels during their activation and coupling (
      • Li Z.
      • Liu L.
      • Deng Y.
      • Ji W.
      • Du W.
      • Xu P.
      • Chen L.
      • Xu T.
      Graded activation of CRAC channel by binding of different numbers of STIM1 to Orai1 subunits.
      • Hoover P.J.
      • Lewis R.S.
      Stoichiometric requirements for trapping and gating of Ca2+ release-activated Ca2+ (CRAC) channels by stromal interaction molecule 1 (STIM1).
      ,
      • Rothberg B.S.
      • Wang Y.
      • Gill D.L.
      Orai channel pore properties and gating by STIM: implications from the Orai crystal structure.
      ,
      • Wu M.M.
      • Covington E.D.
      • Lewis R.S.
      Single-molecule analysis of diffusion and trapping of STIM1 and Orai1 at endoplasmic reticulum-plasma membrane junctions.
      ,
      • Perni S.
      • Dynes J.L.
      • Yeromin A.V.
      • Cahalan M.D.
      • Franzini-Armstrong C.
      Nanoscale patterning of STIM1 and Orai1 during store-operated Ca2+ entry.
      • Zhou Y.
      • Wang X.
      • Wang X.
      • Loktionova N.A.
      • Cai X.
      • Nwokonko R.M.
      • Vrana E.
      • Wang Y.
      • Rothberg B.S.
      • Gill D.L.
      STIM1 dimers undergo unimolecular coupling to activate Orai1 channels.
      ).
      Despite the many advances in identifying the molecular mechanisms of Orai channel function, considerable uncertainty still exists in some of the fundamental basic structural properties of Orai channels and how they become activated by STIM proteins. In particular, the multimeric assembly of the Orai1 channel, the most commonly expressed of the three-member mammalian Orai channel family, has remained a contentious issue (
      • Prakriya M.
      • Lewis R.S.
      Store-operated calcium channels.
      ,
      • Amcheslavsky A.
      • Wood M.L.
      • Yeromin A.V.
      • Parker I.
      • Freites J.A.
      • Tobias D.J.
      • Cahalan M.D.
      Molecular biophysics of Orai store-operated Ca channels.
      ,
      • Hogan P.G.
      The STIM1-ORAI1 microdomain.
      ,
      • Derler I.
      • Jardin I.
      • Romanin C.
      Molecular mechanisms of STIM/Orai communication.
      ,
      • Hou X.
      • Pedi L.
      • Diver M.M.
      • Long S.B.
      Crystal structure of the calcium release-activated calcium channel Orai.
      ,
      • Thompson J.L.
      • Shuttleworth T.J.
      How many Orai's does it take to make a CRAC channel?.
      ). Recent crystallographic evidence reveals that Drosophila Orai has a hexameric subunit structure, a result reinforced by cross-linking and chromatographic evidence (
      • Hou X.
      • Pedi L.
      • Diver M.M.
      • Long S.B.
      Crystal structure of the calcium release-activated calcium channel Orai.
      ). The four transmembrane domains of Orai channels are exceedingly conserved from Drosophila to human, and the pore-lining residues revealed from the crystal structure coincide closely with residues predicted to lie in the pore from elegant structure-function studies (
      • McNally B.A.
      • Yamashita M.
      • Engh A.
      • Prakriya M.
      Structural determinants of ion permeation in CRAC channels.
      • Zhou Y.
      • Ramachandran S.
      • Oh-Hora M.
      • Rao A.
      • Hogan P.G.
      Pore architecture of the ORAI1 store-operated calcium channel.
      ,
      • Zhang S.L.
      • Yeromin A.V.
      • Hu J.
      • Amcheslavsky A.
      • Zheng H.
      • Cahalan M.D.
      Mutations in Orai1 transmembrane segment 1 cause STIM1-independent activation of Orai1 channels at glycine 98 and channel closure at arginine 91.
      • McNally B.A.
      • Somasundaram A.
      • Yamashita M.
      • Prakriya M.
      Gated regulation of CRAC channel ion selectivity by STIM1.
      ) that preceded the crystallization results. However, despite this congruity of structural understanding, several earlier studies revealed a fundamentally different, tetrameric subunit structure for Orai1. Thus, Shuttleworth and colleagues (
      • Mignen O.
      • Thompson J.L.
      • Shuttleworth T.J.
      Orai1 subunit stoichiometry of the mammalian CRAC channel pore.
      ) compared the function of concatenated Orai1 channels (dimers, trimers, and tetramers) and concluded that the functional unit of the Orai1 channel is a tetramer. Shortly after, a number of other studies using a variety of approaches, including recovery after photobleaching, cross-linking, and electron microscopy, reported a similar tetrameric subunit structure for Orai channels (
      • Penna A.
      • Demuro A.
      • Yeromin A.V.
      • Zhang S.L.
      • Safrina O.
      • Parker I.
      • Cahalan M.D.
      The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers.
      • Ji W.
      • Xu P.
      • Li Z.
      • Lu J.
      • Liu L.
      • Zhan Y.
      • Chen Y.
      • Hille B.
      • Xu T.
      • Chen L.
      Functional stoichiometry of the unitary calcium-release-activated calcium channel.
      ,
      • Maruyama Y.
      • Ogura T.
      • Mio K.
      • Kato K.
      • Kaneko T.
      • Kiyonaka S.
      • Mori Y.
      • Sato C.
      Tetrameric Orai1 is a teardrop-shaped molecule with a long, tapered cytoplasmic domain.
      ,
      • Madl J.
      • Weghuber J.
      • Fritsch R.
      • Derler I.
      • Fahrner M.
      • Frischauf I.
      • Lackner B.
      • Romanin C.
      • Schütz G.J.
      Resting state Orai1 diffuses as homotetramer in the plasma membrane of live mammalian cells.
      • Demuro A.
      • Penna A.
      • Safrina O.
      • Yeromin A.V.
      • Amcheslavsky A.
      • Cahalan M.D.
      • Parker I.
      Subunit stoichiometry of human Orai1 and Orai3 channels in closed and open states.
      ). Reports have also suggested that Orai channels may exist as dimers, which could associate into tetramers (
      • Penna A.
      • Demuro A.
      • Yeromin A.V.
      • Zhang S.L.
      • Safrina O.
      • Parker I.
      • Cahalan M.D.
      The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers.
      ,
      • Demuro A.
      • Penna A.
      • Safrina O.
      • Yeromin A.V.
      • Amcheslavsky A.
      • Cahalan M.D.
      • Parker I.
      Subunit stoichiometry of human Orai1 and Orai3 channels in closed and open states.
      ) or possibly hexamers (
      • Li P.
      • Miao Y.
      • Dani A.
      • Vig M.
      α-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels.
      ). A seemingly more definitive study from Thompson and Shuttleworth (
      • Thompson J.L.
      • Shuttleworth T.J.
      How many Orai's does it take to make a CRAC channel?.
      ) directly compared the functional channel properties of tetrameric and hexameric concatemers of Orai1. It was reported that only the Orai1 tetramer gave rise to the Ca2+-selective, inwardly rectifying, Ca2+ release-activated Ca2+ current (ICRAC), the hallmark of authentic Orai1 channel function. In contrast, the hexameric concatemer gave an essentially non-selective cation conductance that was distinct from ICRAC, leading the authors to conclude that the hexameric Orai1 channel fails to replicate the function of endogenous CRAC channels (
      • Thompson J.L.
      • Shuttleworth T.J.
      How many Orai's does it take to make a CRAC channel?.
      ).
      Given the controversy in defining the functional Orai1 channel structure, we undertook a systematic investigation of the subunit configuration of the Orai1 channel. We generated a series of tdTomato-tagged concatenated Orai1 channel constructs containing from two to six Orai1 subunits. We expressed these concatemers in CRISPR-derived HEK cells in which endogenous Orai1 expression was eliminated. We confirmed that all of the Orai1 concatemers were PM-expressed and retained their concatemeric structure at the PM. Unexpectedly, the dimeric, trimeric, tetrameric, pentameric, and hexameric Orai1 constructs all mediated substantial store-operated Ca2+ entry. Still more surprisingly, each one of the different concatemers mediated current with inward rectification and reversal potentials virtually identical to those observed with expressed Orai1 monomers. Replacement of specific subunits within each of the concatemers with Orai1 E106A “pore-dead” subunits revealed that the functional channels are predominantly formed from insertion of just the N-terminal pair of Orai1 subunits into the channel structure. The hexameric concatemer appears distinct from shorter concatemers in being able to at least partially form functional channels by itself. Our results argue strongly against a tetrameric configuration for Orai1 channels and provide evidence consistent with the conclusion that the functional Orai1 channel is hexameric.

      Author Contributions

      Y. Z., X. C., and D. L. G. conceived the project, designed the experiments, undertook experiments, and wrote the manuscript. X. C., Y. Z., X. W., N. A. L., R. M. N., and P. X. performed the experiments and analyzed the data. Y. W. and M. T. provided valuable insight and helped write the manuscript. All authors reviewed the results and approved the final version of the manuscript.

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