Subunit stoichiometry of human Orai1 and Orai3 channels in closed and open states

We applied single-molecule photobleaching to investigate the stoichiometry of human Orai1 and Orai3 channels tagged with eGFP and expressed in mammalian cells. Orai1 was detected predominantly as dimers under resting conditions and as tetramers when coexpressed with C-STIM1 to activate Ca2+ influx. Orai1 was also found to be tetrameric when coexpressed with STIM1 and evaluated following fixation. We show that fixation rapidly causes release of Ca2+, redistribution of STIM1 to the plasma membrane, and STIM1/Orai1 puncta formation, and may cause the channel to be in the activated state. Consistent with this possibility, Orai1 was found predominantly as a dimer when coexpressed with STIM1 in living cells under resting conditions. We further show that Orai3, like Orai1, is dimeric under resting conditions and is predominantly tetrameric when activated by C-STIM1. Interestingly, a dimeric Orai3 stoichiometry was found both before and during application of 2-aminoethyldiphenyl borate (2-APB) to activate a nonselective cation conductance in its STIM1-independent mode. We conclude that the human Orai1 and Orai3 channels undergo a dimer-to-tetramer transition to form a Ca2+-selective pore during store-operated activation and that Orai3 forms a dimeric nonselective cation pore upon activation by 2-APB.

[1]  Richard S Lewis,et al.  Stoichiometric requirements for trapping and gating of Ca2+ release-activated Ca2+ (CRAC) channels by stromal interaction molecule 1 (STIM1) , 2011, Proceedings of the National Academy of Sciences.

[2]  A. Patapoutian,et al.  Temperature-dependent STIM1 activation induces Ca2+ influx and modulates gene expression , 2011, Nature chemical biology.

[3]  Tao Xu,et al.  Graded activation of CRAC channel by binding of different numbers of STIM1 to Orai1 subunits , 2011, Cell Research.

[4]  K. Chandy,et al.  The functional network of ion channels in T lymphocytes , 2009, Immunological reviews.

[5]  D. Steel,et al.  Determination of the oligomer size of amyloidogenic protein beta-amyloid(1-40) by single-molecule spectroscopy. , 2009, Biophysical journal.

[6]  S. Feske,et al.  A minimal regulatory domain in the C terminus of STIM1 binds to and activates ORAI1 CRAC channels. , 2009, Biochemical and biophysical research communications.

[7]  M. Cahalan.  STIMulating store-operated Ca2+ entry , 2009, Nature Cell Biology.

[8]  C. Romanin,et al.  A Cytosolic Homomerization and a Modulatory Domain within STIM1 C Terminus Determine Coupling to ORAI1 Channels* , 2009, Journal of Biological Chemistry.

[9]  O. Mignen,et al.  The Orai1 Severe Combined Immune Deficiency Mutation and Calcium Release-activated Ca2+ Channel Function in the Heterozygous Condition* , 2009, Journal of Biological Chemistry.

[10]  Elizabeth D. Covington,et al.  STIM1 Clusters and Activates CRAC Channels via Direct Binding of a Cytosolic Domain to Orai1 , 2009, Cell.

[11]  Joseph P. Yuan,et al.  SOAR and the polybasic STIM1 domains gate and regulate the Orai channels , 2009, Nature Cell Biology.

[12]  B. Hille,et al.  Functional stoichiometry of the unitary calcium-release-activated calcium channel , 2008, Proceedings of the National Academy of Sciences.

[13]  Shenyuan L. Zhang,et al.  The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers , 2008, Nature.

[14]  Rebecca R. Boyles,et al.  Complex Actions of 2-Aminoethyldiphenyl Borate on Store-operated Calcium Entry* , 2008, Journal of Biological Chemistry.

[15]  R. Penner,et al.  2‐Aminoethoxydiphenyl borate directly facilitates and indirectly inhibits STIM1‐dependent gating of CRAC channels , 2008, The Journal of physiology.

[16]  Andriy V. Yeromin,et al.  Store-dependent and -independent Modes Regulating Ca2+ Release-activated Ca2+ Channel Activity of Human Orai1 and Orai3* , 2008, Journal of Biological Chemistry.

[17]  E. Isacoff,et al.  The Voltage-Gated Proton Channel Hv1 Has Two Pores, Each Controlled by One Voltage Sensor , 2008, Neuron.

[18]  H. Kahr,et al.  Dynamic Coupling of the Putative Coiled-coil Domain of ORAI1 with STIM1 Mediates ORAI1 Channel Activation* , 2008, Journal of Biological Chemistry.

[19]  O. Mignen,et al.  Orai1 subunit stoichiometry of the mammalian CRAC channel pore , 2008, The Journal of physiology.

[20]  Joseph P. Yuan,et al.  STIM1 heteromultimerizes TRPC channels to determine their function as store-operated channels , 2007, Nature Cell Biology.

[21]  Y. Gwack,et al.  Biochemical and Functional Characterization of Orai Proteins* , 2007, Journal of Biological Chemistry.

[22]  E. Isacoff,et al.  Subunit counting in membrane-bound proteins , 2007, Nature Methods.

[23]  Tao Xu,et al.  Aggregation of STIM1 underneath the plasma membrane induces clustering of Orai1. , 2006, Biochemical and biophysical research communications.

[24]  J. Buchanan,et al.  The elementary unit of store-operated Ca2+ entry: local activation of CRAC channels by STIM1 at ER–plasma membrane junctions , 2006, The Journal of cell biology.

[25]  Joseph P. Yuan,et al.  STIM1 carboxyl-terminus activates native SOC, Icrac and TRPC1 channels , 2006, Nature Cell Biology.

[26]  Rebecca R. Boyles,et al.  Large Store-operated Calcium Selective Currents Due to Co-expression of Orai1 or Orai2 with the Intracellular Calcium Sensor, Stim1* , 2006, Journal of Biological Chemistry.

[27]  X. Zhang,et al.  Genome-wide RNAi screen of Ca(2+) influx identifies genes that regulate Ca(2+) release-activated Ca(2+) channel activity. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[28]  M. Nadler,et al.  Amplification of CRAC current by STIM1 and CRACM1 (Orai1) , 2006, Nature Cell Biology.

[29]  J. Kinet,et al.  CRACM1 Is a Plasma Membrane Protein Essential for Store-Operated Ca2+ Entry , 2006, Science.

[30]  Bogdan Tanasa,et al.  A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function , 2006, Nature.

[31]  I. Parker,et al.  “Optical Patch-clamping” , 2005, The Journal of general physiology.

[32]  Tobias Meyer,et al.  STIM Is a Ca2+ Sensor Essential for Ca2+-Store-Depletion-Triggered Ca2+ Influx , 2005, Current Biology.

[33]  S. Wagner,et al.  STIM1, an essential and conserved component of store-operated Ca2+ channel function , 2005, The Journal of cell biology.

[34]  J. Putney,et al.  Store-operated calcium channels. , 2005, Physiological reviews.

[35]  Andriy V. Yeromin,et al.  A Store-operated Calcium Channel in Drosophila S2 Cells , 2004, The Journal of general physiology.

[36]  Richard S Lewis,et al.  Potentiation and inhibition of Ca2+ release‐activated Ca2+ channels by 2‐aminoethyldiphenyl borate (2‐APB) occurs independently of IP3 receptors , 2001, The Journal of physiology.

[37]  M. Cahalan,et al.  Differential Ca2+ Influx, KCa Channel Activity, and Ca2+ Clearance Distinguish Th1 and Th2 Lymphocytes1 , 2000, The Journal of Immunology.

[38]  R. Lewis,et al.  Mitogen-regulated Ca2+ current of T lymphocytes is activated by depletion of intracellular Ca2+ stores. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[39]  P. Hogan,et al.  STIM1 gates the store-operated calcium channel ORAI1 in vitro , 2010, Nature Structural &Molecular Biology.

[40]  Tobias Meyer,et al.  STIM Is a Ca 2+ Sensor Essential for Ca 2+ -Store-Depletion-Triggered Ca 2+ Influx , 2005 .

[41]  I. Parker,et al.  “Optical Patch-clamping”: Single-channel Recording by Imaging Ca 2 (cid:1) Flux through Individual Muscle Acetylcholine Receptor Channels , 2005 .

[42]  Supplementary Figures , 2022 .