Large Store-operated Calcium Selective Currents Due to Co-expression of Orai1 or Orai2 with the Intracellular Calcium Sensor, Stim1
The molecular nature of store-operated Ca2+-selective channels has remained an enigma, due largely to the continued inability to convincingly demonstrate Ca2+-selective store-operated currents resulting from exogenous expression of known genes. Recent findings have implicated two proteins, Stim1 and Orai1, as having essential roles in store-operated Ca2+ entry across the plasma membrane. However, transient overexpression of these proteins on their own results in little or no increase in store-operated entry. Here we demonstrate dramatic synergism between these two mediators; co-transfection of HEK293 cells with Stim1 and Orai1 results in an approximate 20-fold increase in store-operated Ca2+ entry and Ca2+-selective current. This demonstrates that these two proteins are limiting for both the signaling and permeation mechanisms for Ca2+-selective store-operated Ca2+ entry. There are three mammalian homologs of Orai1, and in expression experiments they all produced or augmented store-operated Ca2+ entry with efficacies in the order Orai1 > Orai2 > Orai3. Stim1 apparently initiates the signaling process by acting as a Ca2+ sensor in the endoplasmic reticulum. This results in rearrangement of Stim1 within the cell and migration toward the plasma membrane to regulate in some manner Orai1 located in the plasma membrane. However, we demonstrate that Stim1 does not incorporate in the surface membrane, and thus likely regulates or interacts with Orai1 at sites of close apposition between the plasma membrane and an intracellular Stim1-containing organelle. The molecular nature of store-operated Ca2+-selective channels has remained an enigma, due largely to the continued inability to convincingly demonstrate Ca2+-selective store-operated currents resulting from exogenous expression of known genes. Recent findings have implicated two proteins, Stim1 and Orai1, as having essential roles in store-operated Ca2+ entry across the plasma membrane. However, transient overexpression of these proteins on their own results in little or no increase in store-operated entry. Here we demonstrate dramatic synergism between these two mediators; co-transfection of HEK293 cells with Stim1 and Orai1 results in an approximate 20-fold increase in store-operated Ca2+ entry and Ca2+-selective current. This demonstrates that these two proteins are limiting for both the signaling and permeation mechanisms for Ca2+-selective store-operated Ca2+ entry. There are three mammalian homologs of Orai1, and in expression experiments they all produced or augmented store-operated Ca2+ entry with efficacies in the order Orai1 > Orai2 > Orai3. Stim1 apparently initiates the signaling process by acting as a Ca2+ sensor in the endoplasmic reticulum. This results in rearrangement of Stim1 within the cell and migration toward the plasma membrane to regulate in some manner Orai1 located in the plasma membrane. However, we demonstrate that Stim1 does not incorporate in the surface membrane, and thus likely regulates or interacts with Orai1 at sites of close apposition between the plasma membrane and an intracellular Stim1-containing organelle. Store-operated Ca2+ (SOC) 3The abbreviations used are: SOC, store-operated Ca2+; EYFP, enhanced yellow fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; siRNA, small inhibitory RNA; IP3, inositol 1,4,5-trisphosphate; Icrac, calcium-release-activated calcium current; TRP, transient receptor potential; ER, endoplasmic reticulum; HEK, human embryonic kidney; RNAi, RNA interference; PBS, phosphate-buffered saline; FACS, fluorescent-activated cell sorter; GFP, green fluorescent protein. influx is the major mechanism for Ca2+ entry in many non-excitable cell types. Despite more than two decades of research, little is known about the activation mechanism for the channels responsible for this type of Ca2+ entry. Recently, based primarily on RNAi screens from either Drosophila or mammalian cells, two proteins have been identified as essential components in SOC influx: Stim1 (1Roos 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 (1519) Google Scholar, 2Liou 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 (1753) Google Scholar), and Orai1 (3Feske 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 (1854) Google Scholar, 4Vig 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 (1153) Google Scholar). Stim1 is thought to act as a sensor for Ca2+ in the endoplasmic reticulum, or in that compartment of the endoplasmic reticulum responsible for signaling to store-operated channels. Zhang et al. (5Zhang S.L. Yu Y. Roos J. Kozak J.A. Deerinck T.J. Ellisman M.H. Stauderman K.A. Cahalan M.D. Nature. 2005; 437: 902-905Crossref PubMed Scopus (1134) Google Scholar) proposed a mechanism for Stim1-mediated SOC influx by which Stim1, normally an ER membrane resident protein, is transported to and inserted into the plasma membrane upon Ca2+ store depletion. However, others have suggested that Stim1 may re-localize near the plasma membrane without inserting into the membrane upon Ca2+-store depletion (2Liou 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 (1753) Google Scholar). Mammalian cells may also express a homolog of Stim1, Stim2 (1Roos 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 (1519) Google Scholar, 2Liou 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 (1753) Google Scholar, 6Williams R.T. Manji S.S. Parker N.J. Hancock M.S. Van S.L. Eid J.P. Senior P.V. Kazenwadel J.S. Shandala T. Saint R. Smith P.J. Dziadek M.A. Biochem. J. 2001; 357: 673-685Crossref PubMed Scopus (267) Google Scholar), although currently its function in SOC entry is uncertain. Orai1 was first identified by Feske et al. (3Feske 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 (1854) Google Scholar) through a combined RNAi screen and analysis of gene mutations in patients suffering from severe combined immunodeficiency. The protein appears to be resident in the plasma membrane (3Feske 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 (1854) Google Scholar, 4Vig 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 (1153) Google Scholar), and its molecular function has not yet been defined. However, Vig et al. (4Vig 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 (1153) Google Scholar) suggest that Orai1 (which they designated as CRACM1) could function in the plasma membrane either as a component of the calcium release-activated calcium (CRAC) channel, or as a regulator of CRAC channels. In the current study, we have sought to determine if Stim1 and Orai1 functionally interact by co-expressing them in HEK293 cells. Surprisingly, we find that the combination of Stim1 and Orai1 results in a substantial increase in SOC entry, suggesting that these proteins are limiting for, and may actually comprise both the activation as well as permeation mechanism for SOC influx. This is to our knowledge the first demonstration of an Icrac-like current produced by the ectopic expression of known genes. In addition to Orai1, Orai2 expression with Stim1 also enhanced SOC entry in these cells, however to a lesser extent. Although Orai3 alone or with Stim1 showed no elevation in current or Ca2+ entry, it did rescue the knockdown of Orai1 in HEK293 cells. In addition, we have investigated the movements and distribution of Stim1 and conclude that this protein translocates to the vicinity of the plasma membrane, where it presumably interacts with and activates Orai1, but Stim1 does not incorporate into the plasma membrane. Cell Culture—HEK293 cells, obtained from ATCC, in Dulbecco's modified Eagle's with and and in a at In for or cells to and to to In for Ca2+ cells as a cell and to for a of was to the and the cells in for an in Ca2+ Stim1, Orai1, and Orai3 from in the and and and The mutations in both the Stim1 from as well as Stim1 with the fluorescent protein to the obtained from mutations to the of Stim1 and as well as a by with the The mutations all The was obtained from of cells in a on cells with Stim1 or Orai1 and as a The of the Stim1 for Orai1 it a the the cells was with and in cells with for Stim1 with EYFP, or as cells to in for Ca2+ as which on or cells in a on cells with for EYFP, Orai1, or Stim1 from and of The have a in the of the Stim1 a the the cells was with and in cells to in for Ca2+ as which on or In the of used was for of used in some of the experiments with Orai2 and as Cell Ca2+ in HEK293 cells with the as (2Liou 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 (1753) Google Scholar). cells on and in a in with of at in the for cells in and with by at with no of the cells and with a in intracellular Ca2+ are by and as the of of at and the of cells the by cells at a and with to cells In all have been for by which is cells with and cell currents investigated at in HEK293 cells the in the cell The and to with the as Ca2+ and and was Ca2+ was also the as no Ca2+ was from with with and to with In some inositol was to the intracellular and or was to the a to the cell to of to the and the currents based on cell currents by an current the store-operated currents and this from all was between and The currents and and experiments the intracellular distribution of Stim1, HEK293 cells, or for in a as for Ca2+ on and in a on the of a with a of obtained with from an and with a with a at experiments surface expression of Stim1, HEK293 cells that been with or in combination with Orai1 and to with to addition of either or Ca2+ with a cells by the addition of to a of at and with cells for with with by with to with to three The was to to analysis on a for was by the the where in the was in HEK293 cells with the was at this the the was the was the for all was an on an as J.T. G. Jr., J.W. J. Biol. 2005; Full Text Full Text PDF Scopus Google Scholar). was by a through a and through a by a was not to cells, HEK293 cells with the Ca2+ did not Ca2+ or of with this not was from from of Stim1 and Orai1 in a and in SOC the results of experiments in which HEK293 cells with Orai1, or cells identified by the from or by for cells or cells with SOC entry was by the of the upon of Ca2+ to cells with the reticulum in the of of Ca2+ in a elevation in of little if on Ca2+ entry in this Surprisingly, overexpression of Orai1 SOC entry. of both with Orai1 in a substantial increase in store-operated Ca2+ entry synergism between expression of Stim1 and Orai1 was in experiments as a for Ca2+ The Ca2+ entry resulting from of and Orai1 was by or which is the of store-operated channels Jr., J.W. 2001; Google Scholar). of Stim2 with Orai1 in with Orai1 of entry cell currents in HEK293 cells with these In our HEK293 cells not store-operated and in these currents have been as small and are (4Vig 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 (1153) Google Scholar, A. A. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, C. B. PubMed Scopus Google Scholar). we into HEK293 cells with and IP3, we no of current in cells or in cells with either or Orai1 However, in cells with and Orai1, we of the order of with Ca2+ and The currents with and in the and a with alone currents showed and at as for a The current was by which at this is to store-operated channels J. Scopus Google Scholar, D. Jr., J.W. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of is that of a transient increase in current due to of Ca2+ M. Penner R. J. Scopus Google Scholar) by a process of of Ca2+ from sites A. Lewis R.S. J. PubMed Scopus Google Scholar). in the currents in Orai1 Stim1 cells showed upon of the current was augmented by and was by of this which is a of M. Lewis R.S. J. 2001; Scopus Google Scholar) Although we did not store-operated currents in our HEK293 cells, based on the by Vig et al. (4Vig 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 (1153) Google Scholar) of the Icrac-like currents with of Stim1 and Orai1 suggest an increase in current of at a of of of Orai2 and addition to Orai1, mammalian cells express two Orai2 and Orai3 (3Feske 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 (1854) Google Scholar). determine if these also components of an Icrac-like entry we of these with Stim1 in the manner as for this Orai1, Ca2+ entry on its and Ca2+ entry with Stim1 and the increase in was than in the Orai1 experiments are in for for Orai1, this entry is by or and In the experiments in cells with of the Orai2 a to that used for Orai1 In we that currents with cells in this thus the of Orai2 to which in is actually the used for experiments in and that overexpression of Orai2 and Stim1 results in currents than for Orai1, although with The currents in and The for the Orai2 Stim1 currents showed of Icrac-like currents currents with Orai2 alone of Orai3 alone to SOC entry and of Stim1 and Orai3 to Ca2+ entry, or store-operated with of and The of Orai3 could that this protein has some function than or of Ca2+ entry. it could be or function in with for in with However, Orai3 alone did not Ca2+ entry, we to that Orai3 function in store-operated Ca2+ entry in RNAi rescue in and knockdown of Orai1 by RNAi results in substantial of Ca2+ entry, in of (3Feske 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 (1854) Google Scholar, 4Vig 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 (1153) Google Scholar, S.L. Yeromin A.V. Zhang Yu Y. Safrina O. A. Roos J. Stauderman K.A. Cahalan M.D. S. A. 2006; PubMed Scopus Google Scholar). we Orai3 in cells Orai1 Ca2+ entry was to the and did not Ca2+ currents in these cells, with the that entry was to the The of Orai3 to rescue Ca2+ entry knockdown of Orai1 may that Orai3 is at a more than Orai1, or that it function with experiments be to or the of entry by Orai3 in this demonstrates that this gene also a in the or of store-operated Ca2+ entry. In we find that in expression all three or store-operated Ca2+ entry with efficacies in the order Orai1 > Orai2 > Orai3. this order to their function of expression Stim1 in to Ca2+ sought to the of Stim1 regulate the to have been to et al. (2Liou 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 (1753) Google Scholar), Stim1 within the the plasma membrane, but does not incorporate into the plasma membrane. by Zhang et al. (5Zhang S.L. Yu Y. Roos J. Kozak J.A. Deerinck T.J. Ellisman M.H. Stauderman K.A. Cahalan M.D. Nature. 2005; 437: 902-905Crossref PubMed Scopus (1134) Google Scholar) that Stim1 actually and is into the plasma membrane where it interacts with in the store-operated Ca2+ entry et al. M. J. Dziadek M. D.L. S. 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Biol. 2005; 15: 1235-1241Abstract Full Text Full Text PDF PubMed Scopus (1753) Google Scholar), in S.L. Yu Y. Roos J. Kozak J.A. Deerinck T.J. Ellisman M.H. Stauderman K.A. Cahalan M.D. Nature. 2005; 437: 902-905Crossref PubMed Scopus (1134) Google Scholar), was in S.L. Yu Y. Roos J. Kozak J.A. Deerinck T.J. Ellisman M.H. Stauderman K.A. Cahalan M.D. Nature. 2005; 437: 902-905Crossref PubMed Scopus (1134) Google Scholar), and with the of produced in and all produced Ca2+ entry and and in with the of which In cell Ca2+ experiments we that the rescue Ca2+ entry in cells, which have been with Stim1 and that the and Ca2+ entry both as well as of the cells by an with a the EYFP, which be and to the if the protein inserted into the plasma membrane S.S. Parker N.J. R.T. Van S.L. Dziadek M. Smith P.J. PubMed Scopus Google Scholar). However, we that in cells with plasma no was not or depletion of intracellular Ca2+ with either or the of Stim1 and This was not by of the to the as cells which with to showed of the with and with cells which been with and with This the of cells from cells based on and of cells that been with the cell receptor which is with with its M. S. M. A. J. PubMed Scopus Google Scholar) with the In of cells that with with the and cells in cells that with in Ca2+ for to Ca2+ to of cells into the the for these cells is not from cells with and which into the and that these cells are likely did a of cells that with the with a of (2Liou 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 (1753) Google Scholar) that results in activation of Ca2+ entry (2Liou 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 (1753) Google Scholar) However, the of cells was for cells and for cells, all cells with this Ca2+ entry. of the cells a substantial of both and and the that a small of to to the and this be to the of intracellular In addition, it is from the in that knockdown of Stim1 by RNAi is and thus a small but of surface Stim1 in these cells. we cells with the combination of and Orai1 and to as in that if plasma membrane Stim1 is a component or for SOC entry, the in SOC entry and current that from overexpression of and Orai1 be by a increase in the of in the plasma membrane. However, the results from in we to of the surface of cells the of the of Ca2+ entry Jr., J.W. Cell PubMed Scopus Google Scholar), has on two the signaling mechanism the depletion of intracellular Ca2+ to plasma membrane Ca2+ and the of the store-operated channels Jr., J.W. Google Scholar, Penner R. 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The between the of these proteins and Orai1 they may have a first to their we these proteins with and without Stim1, as with Orai2 to alone entry was and with Stim1, entry and currents In this Orai2 currents than obtained with we no Ca2+ or currents with However, Orai3 was to rescue Ca2+ entry in cells in which Orai1 was by we conclude that all three homologs are of or store-operated channels. This the that SOC channels may of be to determine their roles in Zhang et al. (5Zhang S.L. Yu Y. Roos J. Kozak J.A. Deerinck T.J. Ellisman M.H. Stauderman K.A. Cahalan M.D. Nature. 2005; 437: 902-905Crossref PubMed Scopus (1134) Google Scholar) surface of Stim1 to determine that it is inserted into the plasma membrane Ca2+ store depletion. This on of cell to proteins located on the cell cell the proteins are from proteins by them and This has been used to proteins that have to the However, an for this is that or cells that have plasma membrane be to the and thus a of proteins in the be intracellular proteins from these cells. This could be the of protein to on the surface is small appears to be the In addition, it is that proteins that are not actually within the plasma membrane, but to plasma membrane proteins be with this J.P. K. J. S.H. M.H. S. Full Text Full Text PDF PubMed Scopus Google Scholar). these we used surface of HEK293 cells, as well as analysis of surface of the on cells on the of both we to of Stim1 in the plasma membrane, and we did not of Stim1 to the surface in to Ca2+ store depletion. we are the distribution of a protein, we the of a small of Stim1 in the plasma membrane, as suggested by et al. M. J. Dziadek M. D.L. S. A. 2006; PubMed Scopus Google Scholar). However, our that of Stim1 from endoplasmic reticulum into the plasma membrane in to Ca2+ store depletion does not and that the plasma membrane for Stim1 is have that which rescue Ca2+ entry in Stim1 cells, and which Ca2+ entry, are at the cell surface of cells by and in a small of cells by In the of the of Stim1, Ca2+ entry is in the of store depletion that if Stim1 of from the is no for its activation no store the that we no in the plasma membrane of HEK293 cells the of channels to have been into for plasma membrane Stim1, or a for of Stim1 within the ER Ca2+ store as suggested by et al. (2Liou 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 (1753) Google Scholar). This is an it the of Stim1 as a of the store-operated channel, and that the of Stim1 likely interacts with an intracellular Orai1, which either regulates or the store-operated Ca2+ for the and the and