Dimeric Structure of Human Na+/H+ Exchanger Isoform 1 Overproduced in Saccharomyces cerevisiae

The Na+/H+ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H+ in exchange for one extracellular Na+. The human NHE1 isoform is involved in heart disease and cell growth and proliferation. Although details of NHE1 regulation and transport are being revealed, there is little information available on the structure of the intact protein. In this report, we demonstrate overexpression, purification, and characterization of the human NHE1 (hNHE1) protein in Saccharomyces cerevisiae. Overproduction of the His-tagged protein followed by purification via nickel-nitrilotriacetic acid-agarose chromatography yielded 0.2 mg of pure protein/liter of cell culture. Reconstitution of hNHE1 in proteoliposomes demonstrated that the protein was active and responsive to an NHE1-specific inhibitor. Circular dichroism spectroscopy of purified hNHE1 revealed that the protein contains 41% α-helix, 23% β-sheet, and 36% random coil. Size exclusion chromatography indicated that the protein-detergent micelle was in excess of 200 kDa, consistent with an hNHE1 dimer. Electron microscopy and single particle reconstruction of negatively stained hNHE1 confirmed that the protein was a dimer, with a compact globular domain assigned to the transmembrane region and an apical ridge assigned to the cytoplasmic domain. The transmembrane domain of the hNHE1 reconstruction was clearly dimeric, where each monomer had a size and shape consistent with the predicted 12 membrane-spanning segments for hNHE1. The Na+/H+ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H+ in exchange for one extracellular Na+. The human NHE1 isoform is involved in heart disease and cell growth and proliferation. Although details of NHE1 regulation and transport are being revealed, there is little information available on the structure of the intact protein. In this report, we demonstrate overexpression, purification, and characterization of the human NHE1 (hNHE1) protein in Saccharomyces cerevisiae. Overproduction of the His-tagged protein followed by purification via nickel-nitrilotriacetic acid-agarose chromatography yielded 0.2 mg of pure protein/liter of cell culture. Reconstitution of hNHE1 in proteoliposomes demonstrated that the protein was active and responsive to an NHE1-specific inhibitor. Circular dichroism spectroscopy of purified hNHE1 revealed that the protein contains 41% α-helix, 23% β-sheet, and 36% random coil. Size exclusion chromatography indicated that the protein-detergent micelle was in excess of 200 kDa, consistent with an hNHE1 dimer. Electron microscopy and single particle reconstruction of negatively stained hNHE1 confirmed that the protein was a dimer, with a compact globular domain assigned to the transmembrane region and an apical ridge assigned to the cytoplasmic domain. The transmembrane domain of the hNHE1 reconstruction was clearly dimeric, where each monomer had a size and shape consistent with the predicted 12 membrane-spanning segments for hNHE1. The Na+/H+ exchanger isoform-1 (NHE1) 4The abbreviations used are: NHE1Na+/H+ exchanger isoform 1LPCl-α-lysophosphatidylcholineDDMn-dodecyl β-d-maltosideFCFos-cholineMES4-morpholineethanesulfonic acidNTAnitrilotriacetic acidhNHE1human NHE1. is a ubiquitously expressed plasma membrane glycoprotein. It extrudes a single intracellular proton in exchange for one extracellular sodium and thereby functions to protect cells from intracellular acidification while facilitating extracellular Na+ entry into the cytosol (1Fliegel L. Int. J. Biochem. Cell Biol. 2005; 37: 33-37Crossref PubMed Scopus (109) Google Scholar). NHE1 was the first isoform discovered and a total of nine isoforms of the Na+/H+ exchanger have been identified to date, designated NHE1-NHE9 (2Sardet C. Franchi A. Pouysségur J. Cell. 1989; 56: 271-280Abstract Full Text PDF PubMed Scopus (671) Google Scholar). While NHE1 appears to be the housekeeping isoform, isoforms NHE2-NHE9 have more limited tissue distributions and some have predominantly intracellular localization (reviewed in Ref. 3Slepkov E.R. Rainey J.K. Sykes B.D. Fliegel L. Biochem. J. 2007; 401: 623-633Crossref PubMed Scopus (196) Google Scholar). The Na+/H+ exchanger consists of two domains, a membrane domain of ∼500 amino acids and a 315 amino acid C-terminal cytosolic domain. The membrane domain carries out ion transport and is regulated by the cytosolic domain in response to phosphorylation and several accessory proteins that bind to this domain. Phosphorylation occurs within the last 178 amino acids (4Wakabayashi S. Bertrand B. Shigekawa M. Fafournoux P. Pouyssegur J. J. Biol. Chem. 1994; 269: 5583-5588Abstract Full Text PDF PubMed Google Scholar, 5Moor A.N. Fliegel L. J. Biol. Chem. 1999; 274: 22985-22992Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and a number of proteins bind throughout the cytosolic domain and regulate NHE1 activity (reviewed in Ref. 6Karmazyn M. Sawyer M. Fliegel L. Curr. Drug Targets Cardiovasc. Haematol. Disord. 2005; 5: 323-335Crossref PubMed Scopus (96) Google Scholar). Na+/H+ exchanger isoform 1 l-α-lysophosphatidylcholine n-dodecyl β-d-maltoside Fos-choline 4-morpholineethanesulfonic acid nitrilotriacetic acid human NHE1. Mammalian NHE1 plays a key role in regulation of cell pH, cell volume, cell proliferation, and metastasis in some types of tumor cells (1Fliegel L. Int. J. Biochem. Cell Biol. 2005; 37: 33-37Crossref PubMed Scopus (109) Google Scholar, 6Karmazyn M. Sawyer M. Fliegel L. Curr. Drug Targets Cardiovasc. Haematol. Disord. 2005; 5: 323-335Crossref PubMed Scopus (96) Google Scholar, 7Cardone R.A. Casavola V. Reshkin S.J. Nat. Rev. Cancer. 2005; 5: 786-795Crossref PubMed Scopus (726) Google Scholar, 8Avkiran M. Basic Res. Cardiol. 2001; 96: 306-311Crossref PubMed Scopus (88) Google Scholar, 9Zeymer U. Suryapranata H. Monassier J.P. Opolski G. Davies J. Rasmanis G. Linssen G. Tebbe U. Schroder R. Tiemann R. Machnig T. Neuhaus K.L. J. Am. Coll. Cardiol. 2001; 38: 1644-1650Crossref PubMed Google Scholar, 10Lazdunski M. Frelin C. Vigne P. J. Mol. Cell. Cardiol. 1985; 17: 1029-1042Abstract Full Text PDF PubMed Scopus (528) Google Scholar). The NHE1 isoform of the Na+/H+ exchanger plays a critical role in several forms of heart disease, mediating the damage that occurs with ischemia/reperfusion of the heart (8Avkiran M. Basic Res. Cardiol. 2001; 96: 306-311Crossref PubMed Scopus (88) Google Scholar, 9Zeymer U. Suryapranata H. Monassier J.P. Opolski G. Davies J. Rasmanis G. Linssen G. Tebbe U. Schroder R. Tiemann R. Machnig T. Neuhaus K.L. J. Am. Coll. Cardiol. 2001; 38: 1644-1650Crossref PubMed Google Scholar, 10Lazdunski M. Frelin C. Vigne P. J. Mol. Cell. Cardiol. 1985; 17: 1029-1042Abstract Full Text PDF PubMed Scopus (528) Google Scholar) and being an important mediator of myocardial hypertrophy (6Karmazyn M. Sawyer M. Fliegel L. Curr. Drug Targets Cardiovasc. Haematol. Disord. 2005; 5: 323-335Crossref PubMed Scopus (96) Google Scholar). Clinical trials are attempting to develop NHE1 inhibitors for treatment of various forms of heart disease (11Avkiran M. Marber M.S. J. Am. Coll. Cardiol. 2002; 39: 747-753Crossref PubMed Scopus (220) Google Scholar), though serious concerns have been raised about the isoform specificity of the inhibitors in some clinical trials (12Mentzer Jr., R.M. Circulation. 2003; 108 (abstract): 2723Google Scholar). This suggests that an improved knowledge of NHE1 structure might be desirable in the design of improved inhibitors. The lack of a natural source of NHE1 protein required the development of an overproduction system to begin structural studies. Though we have had some success in high level expression and structural analysis of isolated transmembrane segments of the mammalian NHE1 protein (13Slepkov E.R. Rainey J.K. Li X. Liu Y. Cheng F.J. Lindhout D.A. Sykes B.D. Fliegel L. J. Biol. Chem. 2005; 280: 17863-17872Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), this has remained a difficult undertaking and Escherichia coli seems resistant to expression of larger transmembrane fragments of eukaryotic Na+/H+ exchangers (14Dibrov P. Young P.G. Fliegel L. Mol. Cell Biochem. 1998; 182: 125-132Crossref Scopus (3) Google Scholar). The successful use of other microbial expressions systems include x-ray crystallographic structures of a rat brain voltage-dependent potassium channel overproduced in Pichia pastoris (15Long S.B. Campbell E.B. Mackinnon R. Science. 2005; 309: 897-903Crossref PubMed Scopus (1858) Google Scholar, 16Parcej D.N. Eckhardt-Strelau L. J. Mol. Biol. 2003; 333: 103-116Crossref PubMed Scopus (39) Google Scholar) and a rabbit sarcoplasmic reticulum Ca2+-ATPase overproduced in Saccharomyces cerevisiae (17Jidenko M. Nielsen R.C. Sorensen T.L. Moller J.V. le Maire M. Nissen P. Jaxel C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 11687-11691Crossref PubMed Scopus (86) Google Scholar, 18Lenoir G. Menguy T. Corre F. Montigny C. Pedersen P.A. Thines D. le Maire M. Falson P. Biochim. Biophys. Acta. 2002; 1560: 67-83Crossref PubMed Scopus (50) Google Scholar). In addition, mammalian Na+/H+ exchanger isoforms have been previously expressed in S. cerevisiae, though only in relatively small amounts with NHE1 expressed either as a functionally inactive or mistargeted protein (19Flegelova H. Haguenauer-Tsapis R. Sychrova H. Biochim. Biophys. Acta. 2006; 1760: 504-516Crossref PubMed Scopus (22) Google Scholar, 20Flegelova H. Sychrova H. FEBS Lett. 2005; 579: 4733-4738Crossref PubMed Scopus (7) Google Scholar, 21Montero-Lomeli M. Okorokova Facanha A.L. Biochem. Cell Biol. 1999; 77: 25-31Crossref PubMed Google Scholar). These results suggested that S. cerevisiae might be a suitable host for large scale NHE1 overexpression. Herein we describe the expression and characterization of the NHE1 isoform of the Na+/H+ exchanger in this microbial system, following an approach similar to that used for overproduction of an anion exchanger (AE1 or Band 3) in S. cerevisiae (22Sekler I. Kopito R. Casey J.R. J. Biol. Chem. 1995; 270: 21028-21034Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Our results show that this system can be used to produce milligram of human NHE1 where the protein is purified from is in and is suitable for structural studies. particle microscopy of the purified hNHE1 protein a compact These the first structural into hNHE1 and from was from and from for of S. cerevisiae from was from and 200 from The n-dodecyl β-d-maltoside and Fos-choline and from l-α-lysophosphatidylcholine and from from was from was from Int. The S. cerevisiae and the expression by and of hNHE1 was from the the region (13Slepkov E.R. Rainey J.K. Li X. Liu Y. Cheng F.J. Lindhout D.A. Sykes B.D. Fliegel L. J. Biol. Chem. 2005; 280: 17863-17872Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). was with followed by use of the as by the to a for use in The was with the to the amino acid to into the expression the in the of hNHE1 in was by the and The was by with the and and and a C-terminal followed by a This was into the of the and and the The was of the and the and was a high Curr. 1989; PubMed Scopus Google Scholar) and on large scale cells into with and was into and cells with an of was The expression of hNHE1 was by of to a of for cells cells of culture. of from of membrane and purified protein out or on cells by for The was in and The was in pH 1 1 1 with 1 1 a of a with The was for to a and The was in an of and for to a to cell and a The was for The was with the and for The and and for in a The high the membrane in hNHE1. The membrane in pH a protein of The of was about mg of total protein of culture. in and and of to and hNHE1 purification, membrane of membrane proteins by with high This was for of the protein. to and in pH The was for in a The was in of the used the and Fos-choline and a protein of and of to was out for followed by for The of hNHE1 was from and by analysis and with large scale of 1 of was to an of or in pH The was for with was by for purification of the from was to a in pH The was first with of pH and and with of hNHE1 was with in with a 0.2 This purification be out the or The hNHE1 from this purification was used for structure and with the of the dichroism spectroscopy studies. the protein was purified a the the was The was a 1 with a of with of the protein was with of a of 0.2 and of exclusion chromatography was used to the of hNHE1. of purified hNHE1 on a 200 in pH The was by the of proteins of Reconstitution of NHE1 into proteoliposomes followed S. Y. H. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). mg of was to a and with pH with the pH of and of purified hNHE1 to a of The was to a that was with The was for with mg of to excess The was to a that was with The proteoliposomes hNHE1 for Na+/H+ exchanger activity via a with an of and an of hNHE1 and followed by into a pH to a pH was to Na+/H+ was by the in The hNHE1 by of a region of the the in was from to a in of to hNHE1 purified hNHE1 protein was into pH by was on a D. Fliegel L. Biochem. Cell Biol. 1998; PubMed Scopus Google Scholar). from to in cells of as the of was by amino acid analysis and the in was to and Sci. PubMed Scopus Google Scholar). and protein for and on as Fliegel L. Am. J. 2002; PubMed Scopus Google Scholar). and of hNHE1 was with the to the a was used as the as Fliegel L. Am. J. 2002; PubMed Scopus Google Scholar) and was used as a with an and for membrane a and for purified protein an Pedersen Biochem. 1985; PubMed Scopus Google Scholar, C. Biochem. 56: PubMed Scopus Google Scholar) with as Electron hNHE1 was with from an of 0.2 to a of following a for The was with one of and to on a of for 1 The excess was with and the was to on a in the and the of The was 200 and on a of with a from to with a with a of followed by to a of and reconstruction out S.J. J. Biol. 1999; PubMed Scopus Google Scholar) and J. M. P. J. Li Y. M. A. J. Biol. PubMed Scopus Google Scholar) from with a size of The for the into and The by and into a of of was assigned to in in and in These to and and in The was as a for of in and of of the reconstruction was by the two in of the reconstruction with the predicted for hNHE1 indicated that the structure an hNHE1 dimer. was and was to the reconstruction and in of NHE1 in S. cerevisiae and of suitable amounts of protein for structural we expressed hNHE1 acids in a expression system S. The of hNHE1 occurs and occurs within the first extracellular L. Pouyssegur J. R.A. 1994; PubMed Scopus Google Scholar). a to we a in the was It has previously been that of hNHE1 is required for either exchange activity or of the protein L. Pouyssegur J. R.A. 1994; PubMed Scopus Google Scholar, Fliegel L. Biochem. J. PubMed Scopus Google Scholar). In to this a was the of hNHE1 to protein a His-tagged hNHE1 was into the expression D. B. A. P. PubMed Google Scholar) with a This expression system has previously been to be suitable for the expression of rabbit Ca2+-ATPase G. Menguy T. Corre F. Montigny C. Pedersen P.A. Thines D. le Maire M. Falson P. Biochim. Biophys. Acta. 2002; 1560: 67-83Crossref PubMed Scopus (50) Google Scholar) and the plasma membrane T. J. B. B. C. C. J. Mol. Biol. 2001; 309: PubMed Scopus Google Scholar). was used to membrane that in hNHE1 and analysis of the the of hNHE1 in the various The expressed hNHE1 on the is in with the predicted of for the protein. The to reticulum and of the hNHE1 while the and hNHE1. of of membrane a protein of used the membrane for and purification of the purification of hNHE1 expressed in S. are for 1 of cell are for 1 of cell in a and of to we the of high treatment to membrane proteins and to the membrane for the hNHE1 protein. The of with to of the protein while more of the hNHE1 was to the this This to an of and this to be in the purification by ion this was we that from the of the of this hNHE1 was on the and high with and are to the and of membrane the The of with proteins and of a suitable is important in protein and in protein a of and for to hNHE1 from of by the of hNHE1. The results are in The of the hNHE1 protein with the and The was and with and and the the of Fos-choline that be important in the of hNHE1. and suitable that be used in the and of hNHE1. of to followed by and to the purification The of hNHE1 protein was on the and the with either or only amounts of the hNHE1. in hNHE1 with a of The purification a for hNHE1 following a single of and from the The protein this chromatography was of to by and of purification of hNHE1 was via This was to of the of a on the C-terminal of NHE1 B. S. T. Pouyssegur J. Shigekawa M. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). In this the in hNHE1 and with in the of followed by with This purification in of and a more protein on by of the purification was used relatively amounts of purified protein we to the of the hNHE1 by with NHE1 expressed in mammalian Our hNHE1 a similar to the or of NHE1 in cells (13Slepkov E.R. Rainey J.K. Li X. Liu Y. Cheng F.J. Lindhout D.A. Sykes B.D. Fliegel L. J. Biol. Chem. 2005; 280: 17863-17872Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). This is consistent with on the that the of from the hNHE1. Na+/H+ that hNHE1 was and purified hNHE1 was into in the of and the pH has been to the pH R. J.P. C. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, F.J. J.P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). of the proteoliposomes into in acid of the to of of H+ in exchange for Na+ was by the of of The activity that proteoliposomes with a of exchange by hNHE1 The of the the and in of with the a NHE1 J. Rainey J.K. C. Sykes B.D. Fliegel L. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar), exchange activity of the hNHE1 proteoliposomes was in a The for hNHE1 by was is similar to the for hNHE1 expressed in mammalian cells J. Rainey J.K. C. Sykes B.D. Fliegel L. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). the indicated that we hNHE1 we the of transport to the of protein. was a of and in the and of the The NHE1-specific transport was in the transport in the and of the inhibitor. NHE1 carries out and proton potassium with sodium for transport and is an for NHE1 Am. J. Google Scholar, T. Frelin C. Vigne P. P. M. J. Biol. Chem. 1985; Full Text PDF PubMed Google Scholar). Our hNHE1 transport that with and transport transport activity transport activity transport the was there was These results that hNHE1 is with an and that is of this and of hNHE1 of purified hNHE1 was by chromatography on a 200 hNHE1 in was to a with the The of two a small with the and a large with a of a in excess of 200 hNHE1 as by analysis the micelle number of from the for an hNHE1 is kDa, consistent with the larger in the in into the structure of purified hNHE1 we used The two and of a high Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of the indicated that the hNHE1 contains 41% α-helix, 23% β-sheet, and 36% random coil. These are consistent with of NHE1 that 12 transmembrane S. T. X. Shigekawa M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Electron and of of negatively stained hNHE1 revealed a and of that in yielded that by and in and to the of to an This was used as a for of and followed by in and The indicated a of with a of particle used in the reconstruction The in to a is the for an hNHE1 the reconstruction that hNHE1 is a dimer. This size is consistent with on chromatography as as that NHE1 is a L. Biochem. J. PubMed Scopus Google Scholar, P. J. Pouysségur J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, T. T. Shigekawa M. S. PubMed Scopus Google Scholar). The hNHE1 reconstruction can be into two a globular domain that we to the transmembrane domain of hNHE1 and an apical ridge that we to the cytoplasmic domain of hNHE1. The size of is consistent with and kDa, the globular region of the reconstruction into two and The size and shape of each of with the of 12 transmembrane from hNHE1 The cytoplasmic domain of the hNHE1 reconstruction forms an ridge that the transmembrane domain. The size of this ridge is consistent with a by hNHE1 cytoplasmic domains, each of amino the transmembrane the apical ridge a single domain. This suggests cytoplasmic in the hNHE1 dimer. The cytoplasmic in the hNHE1 reconstruction is in with a that the region of the cytoplasmic acids of hNHE1 has to T. T. Shigekawa M. S. PubMed Scopus Google Scholar). NHE1 is an important for the clinical of heart disease, and a number of inhibitors of hNHE1 have been for this (11Avkiran M. Marber M.S. J. Am. Coll. Cardiol. 2002; 39: 747-753Crossref PubMed Scopus (220) Google Scholar). The structure of hNHE1 the development of this structure While the Na+/H+ exchangers high are in in mammalian has been to amounts of proteins from for structural While we have expressed single transmembrane segments of hNHE1 in coli and by spectroscopy E.R. Rainey J.K. Sykes B.D. Fliegel L. Biochem. J. 2007; 401: 623-633Crossref PubMed Scopus (196) Google Scholar), larger segments two or more transmembrane have to In addition, we have been successful in the of a Na+/H+ in coli (14Dibrov P. Young P.G. Fliegel L. Mol. Cell Biochem. 1998; 182: 125-132Crossref Scopus (3) Google Scholar). In an to we used a expression system to the hNHE1 protein in amounts suitable for characterization of the protein. S. cerevisiae as an expression system has successful with transport proteins of similar and G. Menguy T. Corre F. Montigny C. Pedersen P.A. Thines D. le Maire M. Falson P. Biochim. Biophys. Acta. 2002; 1560: 67-83Crossref PubMed Scopus (50) Google Scholar, I. Kopito R. Casey J.R. J. Biol. Chem. 1995; 270: 21028-21034Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, T. J. B. B. C. C. J. Mol. 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Full Text Full Text PDF PubMed Scopus Google Scholar, C. M. A. H. 2005; PubMed Scopus Google Scholar), as as crystallographic of two Na+/H+ exchanger U. S. J. 1999; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar) and J. 2005; PubMed Scopus Google Scholar). While this that and NHE1 are a of NHE1 has been on the structure of M. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). These used a approach of and to a of NHE1. the of NHE1 the structure of the was by of the as predicted by the NHE1 The of with NHE1 into the of the 12 transmembrane segments in hNHE1 The that from this analysis to hNHE1 the cytoplasmic domain of reconstruction forms an apical ridge that the transmembrane This suggests a compact cytoplasmic that a role in This is in with that have that the region of the NHE1 cytoplasmic domain has a to a T. T. Shigekawa M. S. PubMed Scopus Google Scholar). a for the of on spectroscopy D. Y. H. G. Biophys. J. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar) with hNHE1 reconstruction This about the transmembrane segments that the where transmembrane segments and to the dimer. While for a for studies. for with the and we Liu for

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