Structural and Functional Characterization of Transmembrane Segment IV of the NHE1 Isoform of the Na+/H+ Exchanger

The Na+/H+ exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals. We characterized the structural and functional aspects of the critical transmembrane (TM) segment IV. Each residue was mutated to cysteine in cysteine-less NHE1. TM IV was exquisitely sensitive to mutation with 10 of 23 mutations causing greatly reduced expression and/or activity. The Phe161 → Cys mutant was inhibited by treatment with the water-soluble sulfhydryl-reactive compounds [2-(trimethylammonium)ethyl]methanethiosulfonate and [2-sulfonatoethyl]methanethiosulfonate, suggesting it is a pore-lining residue. The structure of purified TM IV peptide was determined using high resolution NMR in a CD3OH:CDCl3:H2O mixture and in Me2SO. In CD3OH: CDCl3:H2O, TM IV was structured but not as a canonical α-helix. Residues Asp159–Leu162 were a series of β-turns; residues Leu165–Pro168 showed an extended structure, and residues Ile169–Phe176 were helical in character. These three structured regions rotated quite freely with respect to the others. In Me2SO, the structure was much less defined. Our results demonstrate that TM IV is an unusually structured transmembrane segment that is exquisitely sensitive to mutagenesis and that Phe161 is a pore-lining residue. The Na+/H+ exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals. We characterized the structural and functional aspects of the critical transmembrane (TM) segment IV. Each residue was mutated to cysteine in cysteine-less NHE1. TM IV was exquisitely sensitive to mutation with 10 of 23 mutations causing greatly reduced expression and/or activity. The Phe161 → Cys mutant was inhibited by treatment with the water-soluble sulfhydryl-reactive compounds [2-(trimethylammonium)ethyl]methanethiosulfonate and [2-sulfonatoethyl]methanethiosulfonate, suggesting it is a pore-lining residue. The structure of purified TM IV peptide was determined using high resolution NMR in a CD3OH:CDCl3:H2O mixture and in Me2SO. In CD3OH: CDCl3:H2O, TM IV was structured but not as a canonical α-helix. Residues Asp159–Leu162 were a series of β-turns; residues Leu165–Pro168 showed an extended structure, and residues Ile169–Phe176 were helical in character. These three structured regions rotated quite freely with respect to the others. In Me2SO, the structure was much less defined. Our results demonstrate that TM IV is an unusually structured transmembrane segment that is exquisitely sensitive to mutagenesis and that Phe161 is a pore-lining residue. The mammalian Na+/H+ exchanger isoform 1 (NHE1) 1The abbreviations used are: NHE1–8, Na+/H+ exchanger isoforms 1–8; cNHE1, cysteine-less NHE1; Me2SO, dimethyl sulfoxide; DSS, sodium 2,2-dimethyl-2-silapentane-5-sulfonate; HA, hemagglutinin; HNHA, 15N-edited 1HN-1Hα correlation spectroscopy; HSQC, heteronuclear single quantum coherence spectroscopy; MALDI-MS, matrix assisted laser-desorption ionization mass spectrometry; MTSES ([2-sulfonatoethyl]methanethiosulfonate); MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonate; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement spectroscopy; r.m.s.d., root-mean-square deviation; TM, transmembrane segment; TOCSY, total correlation spectroscopy; HPLC, high pressure liquid chromatography. is a ubiquitously expressed integral membrane protein that mediates the removal of one intracellular proton in exchange for one extracellular sodium ion (1Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar). NHE1 thereby protects cells from intracellular acidification (2Grinstein S. Rotin D. Mason M.J. Biochim. Biophys. Acta. 1989; 988: 73-97Crossref PubMed Scopus (675) Google Scholar, 3Pouyssegur J. Sardet C. Franchi A. L'Allemain G. Paris S. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4833-4837Crossref PubMed Scopus (439) Google Scholar); and stimulation of its activity promotes cell growth and differentiation (2Grinstein S. Rotin D. Mason M.J. Biochim. Biophys. Acta. 1989; 988: 73-97Crossref PubMed Scopus (675) Google Scholar) and regulates sodium fluxes and cell volume after osmotic shrinkage (2Grinstein S. Rotin D. Mason M.J. Biochim. Biophys. Acta. 1989; 988: 73-97Crossref PubMed Scopus (675) Google Scholar, 3Pouyssegur J. Sardet C. Franchi A. L'Allemain G. Paris S. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4833-4837Crossref PubMed Scopus (439) Google Scholar). The Na+/H+ exchanger also plays an important role in the damage that occurs to the human myocardium during ischemia and reperfusion, and it has been shown that inhibition of the exchanger has beneficial effects on the myocardium under these conditions (4Fliegel L. Basic Res. Cardiol. 2001; 96: 301-305Crossref PubMed Scopus (50) Google Scholar). Amiloride and its derivatives are inhibitors of the NHE1 isoform of the Na+/H+ exchanger, and a new generation of Na+/H+ exchanger inhibitors is being developed for clinical treatment of heart disease (5Mentzer Jr., R.M. Lasley R.D. Jessel A. Karmazyn M. Ann. Thorac. Surg. 2003; 75: S700-S708Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Although the activity of NHE1 has been extensively examined in many tissues, only recently is information starting to be elucidated on how this antiporter actually binds and transports Na+ ions and protons. NHE1 is composed of two domains as follows: an N-terminal membrane domain of ∼500 amino acids and a C-terminal regulatory domain of about 315 amino acids (1Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar, 4Fliegel L. Basic Res. Cardiol. 2001; 96: 301-305Crossref PubMed Scopus (50) Google Scholar) (Fig. 1). The N-terminal membrane domain is responsible for ion movement, and it is reported to have 12 transmembrane (TM) segments and 3 membrane-associated segments (6Wakabayashi S. Pang T. Su X. Shigekawa M. J. Biol. Chem. 2000; 275: 7942-7949Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Transmembrane segment four (TM IV; residues 155–177) has been implicated in the ion transport and inhibitor binding properties of NHE1 (7Counillon L. Franchi A. Pouyssegur J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4508-4512Crossref PubMed Scopus (142) Google Scholar, 8Counillon L. Noel J. Reithmeier R.A.F. Pouyssegur J. Biochemistry. 1997; 36: 2951-2959Crossref PubMed Scopus (65) Google Scholar, 9Touret N. Poujeol P. Counillon L. Biochemistry. 2001; 40: 5095-5101Crossref PubMed Scopus (48) Google Scholar). The sequence of human TM IV of NHE1 is 155FLQSDVFFLFLLPPIILDAGYFL177. The underlined residues have been shown to affect Na+ affinity or the inhibitor resistance of mammalian NHE1 (7Counillon L. Franchi A. Pouyssegur J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4508-4512Crossref PubMed Scopus (142) Google Scholar, 8Counillon L. Noel J. Reithmeier R.A.F. Pouyssegur J. Biochemistry. 1997; 36: 2951-2959Crossref PubMed Scopus (65) Google Scholar, 9Touret N. Poujeol P. Counillon L. Biochemistry. 2001; 40: 5095-5101Crossref PubMed Scopus (48) Google Scholar). Recently, we have shown that prolines 167 and 168 are critical forNHE1 function, targeting, and expression (10Slepkov E.R. Chow S. Lemieux M.J. Fliegel L. J. PubMed Scopus Google Scholar). These a for the of many amino residues of TM IV in the ion structure, and transport properties of NHE1. In this we structural and functional aspects of TM IV of the NHE1 isoform of the Na+/H+ We mutagenesis to amino acids are important in and are pore-lining residues of TM IV. the structure of TM the segment was expressed and and we its structure in two membrane structured in the segment as a a single The of conditions a to a membrane has quite for structural of transmembrane or and has of protein segments with in the protein Biochemistry. PubMed Scopus Google Scholar, M. Biophys. J. 2001; 81: Full Text Full Text PDF PubMed Scopus Google Scholar, G. Biochemistry. 2001; 40: PubMed Scopus Google Scholar). Our that TM IV is only a of the segment is In we Phe161 as a pore-lining residue in NHE1. and NMR were from was from was from and MTSES were from in TM IV were to an expression a human NHE1 isoform of the Na+/H+ The the of the of NHE1 with 10 cysteine residues mutated to as (10Slepkov E.R. Chow S. Lemieux M.J. Fliegel L. J. PubMed Scopus Google Scholar). Each residue of TM IV was mutated to cysteine using the cysteine-less as a mutagenesis was using with by of the mutagenesis as by the were to a new for in the of the and cells were used to Na+/H+ exchanger expression and activity. These cells an Na+/H+ cell were of by with to the as (10Slepkov E.R. Chow S. Lemieux M.J. Fliegel L. J. PubMed Scopus Google Scholar). cells were using and cell for were from and and NHE1 expression was used on from total cell of were as (10Slepkov E.R. Chow S. Lemieux M.J. Fliegel L. J. PubMed Scopus Google Scholar). of total were on The was a membrane and using and The and was used to of was using of expression was as (10Slepkov E.R. Chow S. Lemieux M.J. Fliegel L. J. PubMed Scopus Google Scholar). cells were with and was used to cell Na+/H+ of the total and were by and was as The of NHE1 on the cell was by the and the of NHE1 in of the total and are shown as with determined by a Na+/H+ and exchange activity was using a or a The of of pH was after using (6Wakabayashi S. Pang T. Su X. Shigekawa M. J. Biol. Chem. 2000; 275: 7942-7949Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) for 3 was used to an and the in the of was as (10Slepkov E.R. Chow S. Lemieux M.J. Fliegel L. J. PubMed Scopus Google Scholar). were in of cell as by the of acidification by are shown as and was determined using a the of and MTSES on activity of the NHE1 we used the Na+/H+ exchanger with acidification of the In this cells were two as a acidification and or MTSES was to a of 10 for 10 in The cells were three in to the acidification and 1 was and of TM TM IV as a protein with the binding domain of protein the residues of human NHE1 amino acids was in the of the of Biochemistry. were to the protein sequence with residues and to the to that in The were and The were to have and as to the A. D. Sci. 2003; PubMed Scopus Google Scholar, Sci. 1997; PubMed Scopus Google Scholar). The were the and in and for protein The were by and were with and to the protein as A. D. Sci. 2003; PubMed Scopus Google Scholar). of the were from a single and in of were with 1 for were in of with The protein was that a the C-terminal was used for of the protein affinity as by the The protein was from the using 1 the using an and in were by using a in 10 pH and were to the transmembrane segment IV of were a purified of peptide was of in to in in with and to in for The mixture was with an volume of and was used to the peptide of the and the TM IV segment were and to The was determined by mass TM IV. TM IV for of the peptide these cells were in a pH The was with 1 of NMR and of TM IV peptide were in CD3OH:CDCl3:H2O and Me2SO. that for NMR were used to were to with for J. J. PubMed Scopus Google Scholar). NMR were on a a of for for extended were in and in CD3OH:CDCl3:H2O peptide and HSQC, and were on the for correlation and were on or using the 15N-edited and were on a were using or S. G. J. A. J. PubMed Scopus Google Scholar); was with 3 D. and D. G. of was in P. J. M. T. Biol. PubMed Scopus Google Scholar) using from the and from the as by and A. J. Chem. 1993; Scopus Google Scholar). and were in and were using with of the in with the not a and a was used a was from to a in the of These were used to one of or with an two were to the as a starting was to and the using a to that of J. Chem. Scopus Google Scholar). of were by using the and and were and in of the were examined in these were and were or this were extended to a as J. Chem. Scopus Google Scholar). were the were the J. J. Scopus Google Scholar) using as by and A. J. Chem. 1993; Scopus Google Scholar) for the for the structured CD3OH: of was to the and The residue was structure using a residue structure to an N-terminal by with with by in 3 on Scholar) was used to and segments of the peptide were determined by the of the of with M.J. 1997; PubMed Scopus Google Scholar); structural of the were using Sci. PubMed Scopus Google Scholar). The of have been in the with the for the CD3OH:CDCl3:H2O and of NHE1 1 a of the Na+/H+ exchanger (Fig. and a TM IV (Fig. amino acids of TM IV were critical for the activity of the Na+/H+ exchanger and amino acids were we used the cysteine-less Na+/H+ exchanger Each residue in TM IV of was mutated to a cysteine residue. examined these mutant of the Na+/H+ exchanger activity. TM IV was exquisitely sensitive to the effects of mutation of amino acids of TM IV to cysteine on activity. of of the amino acids with cysteine in TM IV in of the activity of the protein in In residues and less of the activity. These were not used for of activity. We have that is not critical for of NHE1 (10Slepkov E.R. Chow S. Lemieux M.J. Fliegel L. J. PubMed Scopus Google Scholar). 3 a of total cell from cells the single cysteine the mutant and the of with a that the of the Na+/H+ exchanger and a that an of the exchanger that is not (10Slepkov E.R. Chow S. Lemieux M.J. Fliegel L. J. PubMed Scopus Google Scholar). the and the cysteine-less NHE1 showed of and a NHE1. The of is in to the and and have of cell expressed or a high of the of the and cell also expressed much less of the These and We examined the to or MTSES of the mutant Na+/H+ that activity of the cysteine-less NHE1 (Fig. the Na+/H+ only was by treatment with or in in Na+/H+ exchanger activity of and that Phe161 was a critical we the mutant a this in the NHE1 mutant only of activity (Fig. reduced expression and reduced to NHE1 the of Phe161 in NHE1 of in cells of cysteine-less NHE1 and TM IV Na+/H+ exchanger reduced in to reduced in to reduced in to reduced in to reduced in to reduced in to in a new of the and Na+/H+ have that mutation of amino acids of the Na+/H+ exchanger the protein to be to an intracellular (10Slepkov E.R. Chow S. Lemieux M.J. Fliegel L. J. PubMed Scopus Google Scholar, Fliegel L. J. 2001; PubMed Scopus Google Scholar). We used of the intracellular of NHE1 were with and and and were to of the total cell and was by using by with to NHE1 of the and the results In the total and intracellular the NHE1 protein in these The intracellular was that of the Na+/H+ exchanger that not to the NHE1 and of the mutant exchanger were on the membrane in Na+/H+ exchanger and were reduced in expression on the membrane of the the activity of these for in (Fig. that a of the in activity was of of these less of the activity of the cysteine-less NHE1 for not greatly the for activity of with the being in activity of about for and and of TM the structure of TM we it as a protein with the protein as a of in was less the of We the of the protein by mass suggesting that the protein with a of many membrane The of the IV protein was of treatment to TM it was purified by We the of TM IV by mass the the mass a C-terminal is mass and the mass in mass mass from to also and mass were in less of the total from to to the the the mass was and mass was mass mass and mass were in less of the total from 1 to to the was to be for of the after of the of NMR the structure of TM we The and the CD3OH:CDCl3:H2O mixture or Me2SO, in that of were in the and regions of not the peptide of of these NMR In the of to not structural were in CD3OH:CDCl3:H2O and These peptide with as the are the of were NMR of and Google using and correlation with by for and to the of and for the N-terminal and proton The C-terminal residue was as a its and to to The of NMR Scholar). was by the by of the purified peptide the that residues are not to the this is reported to be the peptide with conditions J. 96: Scholar). to by in the a not be In not be and for residues and and of were In Me2SO, were and in the of and were also that of and was with and and with peptide and and to be of and of the C-terminal in CD3OH:CDCl3:H2O and of in have been in the were used to protons. of was examined under not In of the the of to this was for volume of and were in In Me2SO, and were In CD3OH: CDCl3:H2O, were and were to be in the conditions to the and proton In the TM IV showed two regions with 3 of were not in (Fig. The of these regions is with a of have not been the structural a to or in be from 15N-edited were used to in during the the peptide was less structured in in as by the greatly reduced of (Fig. we in only the CD3OH:CDCl3:H2O structural The of the of are in for the of of the for in a new of TM segment four (TM IV; residues 155–177) of the NHE1 isoform of the Na+/H+ exchanger has been implicated in the ion transport and inhibitor binding properties of the protein (7Counillon L. Franchi A. Pouyssegur J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4508-4512Crossref PubMed Scopus (142) Google Scholar, 8Counillon L. Noel J. Reithmeier R.A.F. Pouyssegur J. Biochemistry. 1997; 36: 2951-2959Crossref PubMed Scopus (65) Google Scholar, 9Touret N. Poujeol P. Counillon L. Biochemistry. 2001; 40: 5095-5101Crossref PubMed Scopus (48) Google Scholar). We have recently shown (10Slepkov E.R. Chow S. Lemieux M.J. Fliegel L. J. PubMed Scopus Google Scholar) that the and is critical for the of NHE1. were to be critical in the of an structure of TM IV and for NHE1 transport to the functional role of amino acids of a transmembrane segment of a membrane protein is mutagenesis in with with mutagenesis of the that the is the functional in a protein G. S. G. S. Biol. 1993; PubMed Scopus Google Scholar). has been used to pore-lining residues in membrane Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the of M. Biochemistry. 1993; PubMed Scopus Google the M. A. PubMed Scopus Google the human and the human exchanger isoform 1 M. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). mutagenesis the to the of of amino sulfhydryl-reactive A. Biochemistry. PubMed Scopus Google Scholar) that are used in these are and are M. G. PubMed Scopus Google Scholar, J. 2000; Full Text Full Text PDF PubMed Scopus Google with pore-lining residues by and residues the of the amino acids in TM IV of cysteine-less NHE1 with cysteine We that TM IV was exquisitely sensitive to of the cysteine less of the activity of the that in the in activity was to a of expression of the protein (Fig. in for the of membrane protein and not the of activity. of the were expressed as and many but not of these greatly reduced activity. We have that mutation of amino acids greatly affect the of the We examined these mutations the of the Our that many of the mutations of TM IV targeting, or activity are to the results with the human exchanger M. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and with TM of M. Biochemistry. 1993; PubMed Scopus Google Scholar). In for the of T. S. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and in J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google it was to the amino acids of a TM segment and activity. to mutation to not only but also transmembrane segments of the TM of sensitive amino acids M. Biochemistry. 1993; PubMed Scopus Google TM only one J. Biochemistry. PubMed Scopus Google Scholar). be of the of the residues in the and of the of the segment to the structure and of the In it was that was a for many of the amino acids of TM and this be of structural and functional with and MTSES that Phe161 is to and with these sulfhydryl-reactive (Fig. The for this is an a that the ion The the and ion transport M. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Phe161 in TM IV is the residue to be as the ion transport of NHE1. of Phe161 to in of of NHE1 activity (Fig. that this is a critical amino of TM examined the structure of TM IV in CD3OH:CDCl3:H2O and in with many by NMR J. Chem. Scopus Google Scholar, Sci. 2001; PubMed Scopus Google Scholar) the TM IV segment as a a single the peptide of these to the TM IV segment In a of the NHE1 a single be by the TM IV the that we the of this TM segment in a a and a protein it is to that also be the of the NHE1 a of have of or protein segments in membrane to by NMR in or to membrane determined by Biochemistry. PubMed Scopus Google Scholar, M. Biophys. J. 2001; 81: Full Text Full Text PDF PubMed Scopus Google Scholar, G. Biochemistry. 2001; 40: PubMed Scopus Google Scholar). we that CD3OH:CDCl3:H2O a peptide structure, the peptide in much less that is a of in membrane peptide in a single not of the structured of four to residues of the TM IV segment and were as regions by M.J. 1997; PubMed Scopus Google Scholar) and by of the of residues and Ile169–Phe176 These are in and is in Each with residues in for the a to the structure in the of The and are and as as for and structural the for these The of are also of the in as follows: and from has a much the residues the peptide are or with but not with the is of and demonstrate in to the of the is shown in as a that the segment a membrane with the on the of a extended as that in is of the one that structure be as only of the the segments of the TM IV as of for and for of the of the of the TM IV peptide and to the structure for that on of and using as using on of and using as using on of and using on of and using on of and using on of and using as using on of and using on of and using on of on of and using on of on of on of on of on of on of and on of on of and as using on of on of and on of on of and on of and on of and on of and on of and as using on of and on of and on of and as using on of and on of and as using on of and on of and using A. 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PubMed Scopus Google Scholar) of the and residues in the of the the three regions by residues are the by these is the residue in one IV and the residue in a is the 3 and 1 residue for these as as the residue in a the Phe161 and in the and and as the 3 residue for this series of in of the to this Leu165–Pro168 are as by these residues to be quite extended (Fig. with or IV in of the residues Ile169–Phe176 the only the transmembrane The helical residues for of the starting and and with one of these of the helical the segment of The of this is by and of as the residue for a of IV in or of the Although resolution in NMR has been using in PubMed Scopus Google an the TM IV peptide with In its this segment of the NHE1 protein and a with a high In the a transmembrane segment has a structure, as a canonical the as an In the we that the about and be greatly this actually be an the peptide structure to the We that the structure in a mixture of a of a of the structured regions of the TM IV be in of using of in for NMR that are by of the Scopus Google Scholar). and of the structural for the in activity for of the be to and exchanger and mutations of these prolines to residues not for a to a Na+/H+ exchanger (10Slepkov E.R. Chow S. Lemieux M.J. Fliegel L. J. PubMed Scopus Google Scholar). from an to a much amino the structural this of prolines (Fig. the of it is that the extended structural of this is in NHE1 the functional The mutation also in activity. is to as the residue in an IV N-terminal to the residue that also of activity. of the the and of and for the of that the of and have a to of to the this of the TM IV these are it that be the with transmembrane segments or the the of these with the less cysteine these thereby the NHE1 structure as a to the in activity. the mutated to be this is not a The mutation for with the sulfhydryl-reactive that Na+/H+ exchanger activity. that residue is the NHE1 this in of the structure of the TM IV segment a Phe161 is actually in the of residues in of the not of the is about (Fig. of the two C-terminal domains to Phe161 be and it was not to this C-terminal amino acids the the of the Phe161 the extracellular of the segment is in the that the TM IV segment in membrane a structure with the for the exchanger as a the of (Fig. the is on the of the it that the also the of the exchanger, it to in during of this amino activity and reduced expression of the with a critical role for this amino We have that residues transmembrane segments of the Na+/H+ exchanger are important in and transport P. Fliegel L. Biochemistry. 36: Scopus Google Scholar, P. Fliegel L. PubMed Scopus Google and the structure of TM IV a role for this residue. Our has the structural and functional information on TM IV of the NHE1 isoform of the Na+/H+ TM IV to be sensitive to in its amino We that it is a structured transmembrane its structure is from a α-helix. this its role in binding and Phe161 and are amino residues that the of the and were the for of We the NMR for in the of the of NMR is by the of and of and the of We also and for the and and T. of the of of for

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