Identification of Sites in the Second Exomembrane Loop and Ninth Transmembrane Helix of the Mammalian Na+/H+ Exchanger Important for Drug Recognition and Cation Translocation

Mammalian Na+/H+exchanger (NHE) isoforms are differentially sensitive to inhibition by several distinct classes of pharmacological agents, including amiloride- and benzoyl guanidinium-based derivatives. The determinants of drug sensitivity, however, are only partially understood. Earlier studies of the drug-sensitive NHE1 isoform have shown that residues within the fourth membrane-spanning helix (M4) (Phe165, Phe166, Leu167, and Gly178) and a 66-amino acid segment encompassing M9 contribute significantly to drug recognition. In this report, we have identified two residues within M9, one highly conserved (Glu350) and the other non-conserved (Gly356), that are major determinants of drug sensitivity. In addition, residues in the second exomembrane loop between M3 and M4 (Gly152, Phe157, and Pro158) were also found to modestly influence drug sensitivity. A double substitution of crucial sites within M4 and M9 of NHE1 with the corresponding residues present in the drug-resistant NHE3 isoform (i.e. L167F/G356A) greatly reduced drug sensitivity in a cooperative manner to levels nearing that of wild type NHE3. The above mutations did not appreciably affect Nao+ affinity but did markedly decrease the catalytic turnover of the transporter. These data suggest that specific sites encompassing M4 and M9 are critical determinants of both drug recognition and cation translocation. Mammalian Na+/H+exchanger (NHE) isoforms are differentially sensitive to inhibition by several distinct classes of pharmacological agents, including amiloride- and benzoyl guanidinium-based derivatives. The determinants of drug sensitivity, however, are only partially understood. Earlier studies of the drug-sensitive NHE1 isoform have shown that residues within the fourth membrane-spanning helix (M4) (Phe165, Phe166, Leu167, and Gly178) and a 66-amino acid segment encompassing M9 contribute significantly to drug recognition. In this report, we have identified two residues within M9, one highly conserved (Glu350) and the other non-conserved (Gly356), that are major determinants of drug sensitivity. In addition, residues in the second exomembrane loop between M3 and M4 (Gly152, Phe157, and Pro158) were also found to modestly influence drug sensitivity. A double substitution of crucial sites within M4 and M9 of NHE1 with the corresponding residues present in the drug-resistant NHE3 isoform (i.e. L167F/G356A) greatly reduced drug sensitivity in a cooperative manner to levels nearing that of wild type NHE3. The above mutations did not appreciably affect Nao+ affinity but did markedly decrease the catalytic turnover of the transporter. These data suggest that specific sites encompassing M4 and M9 are critical determinants of both drug recognition and cation translocation. Na+/H+ exchanger 5- (N-ethyl-N-isopropyl) amiloride Na+/H+ exchangers (NHE)1 are present at the cell surface and various organellar compartments of mammalian cells and mediate the electroneutral exchange of Na+ for H+, a process driven by the relative concentration gradients of the respective cations. To date seven distinct isoforms (NHE1 to NHE7) have been isolated that share ∼20–70% amino acid identity (calculated Mr ranging from ∼74,000 to 93,000) and exhibit similar membrane topologies, with 12 predicted N-terminal membrane-spanning (M) α-helices and a large C-terminal cytoplasmic region (1Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar, 2Wakabayashi S. Shigekawa M. Pouysségur J. Physiol. Rev. 1997; 77: 51-74Crossref PubMed Scopus (563) Google Scholar, 3Yun C.H.C. Tse C.-M. Nath S.K. Levine S.A. Brant S.R. Donowitz M. Am. J. Physiol. 1995; 269: G1-G11Crossref PubMed Scopus (38) Google Scholar, 4Numata M. Petrecca K. Lake N. Orlowski J. J. Biol. Chem. 1998; 273: 6951-6959Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 5Numata M. Orlowski J. J. Biol. Chem. 2001; 276: 17387-17394Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). They show considerable differences in their patterns of tissue expression, membrane localization, kinetic properties, sensitivity to pharmacological antagonists, and responsiveness to various signaling pathways. Consistent with their molecular diversity, the exchangers participate in a broad spectrum of physiological processes, including the regulation of intracellular pH (pHi), maintenance of cell volume, and transepithelial transport of electrolytes. In addition, activation of certain exchangers appear to facilitate cellular growth and proliferation in response to numerous growth factors and other mitogens and are associated with events leading to apoptosis (6Shrode L.D. Tapper H. Grinstein S. J. Bioenerg. Biomembr. 1997; 29: 393-399Crossref PubMed Scopus (179) Google Scholar, 7Li J. Eastman A. J. Biol. Chem. 1995; 270: 3203-3211Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 8Zhu W.H. Loh T.T. Biochim. Biophys. Acta. 1995; 1269: 122-128Crossref PubMed Scopus (57) Google Scholar). The NHE is a known target for inhibition by the diuretic compound amiloride and its analogues (9Kleyman T.R. Cragoe Jr., E.J. J. Membr. Biol. 1988; 105: 1-21Crossref PubMed Scopus (1065) Google Scholar). Amiloride analogues containing hydrophobic substituents on the 5-amino group of the pyrazine ring, such as 5-(N-ethyl-N-isopropyl) amiloride (EIPA), have higher affinity and specificity for NHE relative to other ion transporters. Comparison of the NHE isoforms in heterologous expression systems show that they have varying affinities for amiloride and its analogues that span more than 2 orders of magnitude, with the following order of sensitivity: NHE1 ≥ NHE2 > NHE5 > NHE3 (10Orlowski J. J. Biol. Chem. 1993; 268: 16369-16377Abstract Full Text PDF PubMed Google Scholar, 11Yu F.H. Shull G.E. Orlowski J. J. Biol. Chem. 1993; 268: 25536-25541Abstract Full Text PDF PubMed Google Scholar, 12Szabó E.Z. Numata M. Shull G.E. Orlowski J. J. Biol. Chem. 2000; 275: 6302-6307Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). NHE4 also has an apparent low affinity for many of these antagonists, but its activity in transfected fibroblasts can only be detected under specialized experimental conditions (13Bookstein C. Musch M.W. DePaoli A. Xie Y. Rabenau K. Villereal M. Rao M.C. Chang E.B. Am. J. Physiol. Cell Physiol. 1996; 271: 1629-1638Crossref PubMed Google Scholar, 14Chambrey R. Achard J.M. Warnock D.G. Am. J. Physiol. Cell Physiol. 1997; 272: 90-98Crossref PubMed Google Scholar) that preclude direct comparisons with other ectopically expressed isoforms. Recently, novel benzoyl guanidinium compounds (e.g. HOE694, HOE642 or cariporide, and EMD85131) have been developed that inhibit the NHE isoforms with a similar rank order but over a larger concentration range (3–4 orders of magnitude) (15Counillon L. Scholz W. Lang H.J. Pouysségur J. Mol. Pharmacol. 1993; 44: 1041-1045PubMed Google Scholar, 16Orlowski J. Kandasamy R.A. J. Biol. Chem. 1996; 271: 19922-19927Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 17Scholz W. Albus U. Counillon L. Gögelein H. Lang H.-J. Linz W. Weichert A. Schölkens B.A. Cardiovasc. Res. 1995; 29: 260-268Crossref PubMed Scopus (346) Google Scholar, 18Gumina R.J. Mizumura T. Beier N. Schelling P. Schultz J.J. Gross G.J. J. Pharmacol. Exp. Ther. 1998; 286: 175-183PubMed Google Scholar). The more selective binding properties of these compounds for NHE1 have been exploited therapeutically as effective agents in the treatment of cardiac ischemia and reperfusion injuries (17Scholz W. Albus U. Counillon L. Gögelein H. Lang H.-J. Linz W. Weichert A. Schölkens B.A. Cardiovasc. Res. 1995; 29: 260-268Crossref PubMed Scopus (346) Google Scholar, 18Gumina R.J. Mizumura T. Beier N. Schelling P. Schultz J.J. Gross G.J. J. Pharmacol. Exp. Ther. 1998; 286: 175-183PubMed Google Scholar, 19Scholz W. Albus U. Lang H.J. Linz W. Martorana P.A. Englert H.C. Schölkens B.A. Br. J. Pharmacol. 1993; 109: 562-568Crossref PubMed Scopus (187) Google Scholar, 20Harper I.S. Bond J.M. Chacon E. Reece J.M. Herman B. Lemasters J.J. Basic Res. Cardiol. 1993; 88: 430-442Crossref PubMed Scopus (60) Google Scholar, 21Humphreys R.A. Haist J.V. Chakrabarti S. Feng Q.P. Malcolm J. Arnold O. Karmazyn M. Am. J. Physiol. Heart Circ. Physiol. 1999; 276: 749-757Crossref PubMed Google Scholar, 22Rupprecht H.J. Vom D. Terres W. Seyfarth K.M. Richardt M. F.H. H. 2000; PubMed Scopus Google Scholar) and in the of K. K.M. Circ. Res. 2000; PubMed Scopus Google Scholar). a of NHE3 has been that also facilitate studies of this isoform in and Lang H.-J. W. M. J. Physiol. 1998; PubMed Scopus Google Scholar, K. M. Am. J. Physiol. 2000; PubMed Google Scholar). that inhibition by amiloride compounds J. Biol. Chem. Full Text PDF PubMed Google Scholar) and (15Counillon L. Scholz W. Lang H.J. Pouysségur J. Mol. Pharmacol. 1993; 44: 1041-1045PubMed Google Scholar) is reduced by inhibition they the and also share a under amiloride and its also inhibit transport that the Nao+ and amiloride binding sites not be Warnock D.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, D.G. Cragoe Jr., E.J. J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar). the and sites can be of other A. Cragoe Jr., E.J. Pouysségur J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). these data that amiloride and other with sites on the Consistent with the above have identified residues in the predicted fourth membrane-spanning helix (M4) of NHE1 and of that sensitivity to amiloride and its analogues but not affect Nao+ affinity L. A. Pouysségur J. U. S. A. 1993; PubMed Scopus Google Scholar, L. J. R.A. Pouysségur J. 1997; PubMed Scopus Google Scholar). of a found to decrease affinities for both Nao+ and HOE642 N. P. Counillon L. 2001; PubMed Scopus Google Scholar). In addition, pharmacological of of NHE1 and NHE3 a 66-amino acid segment encompassing M9 and its of as a major of the drug sensitivity between these two isoforms J. Kandasamy R.A. J. Biol. Chem. 1996; 271: 19922-19927Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). substitution of this region between NHE1 and NHE3 a in their drug by orders of magnitude, with the for the more and A for this region in drug recognition is by of D. Warnock D.G. Am. J. Physiol. 1995; 269: PubMed Google found that of in the M9 of NHE1 a or or decrease or in amiloride sensitivity, other amino acid this is to for the large for the the of other sites as critical determinants of drug sensitivity. The of the present to specific residues that contribute to the large in drug sensitivity between NHE1 and NHE3. The show that sites within M9 as as between M3 and M4 are determinants of drug recognition. These sites were also found to be for cation translocation. These data in NHE drug and in the of more with from Amiloride and were from and the amiloride from by J. Lang and were from to NHE1 were by with a containing to the C-terminal amino of the exchanger and as S. M. Orlowski J. Pouysségur J. J. 1993; PubMed Scopus Google Scholar). The from Cell and were from other and in these were from or and were of the The NHE1 and NHE3 to a of sites to for were a mammalian expression under the of the region from the of and as J. Kandasamy R.A. J. Biol. Chem. 1996; 271: 19922-19927Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). These the amino acid in a in substitution of an amino acid present in one isoform with the of the other isoform and on their respective of various amino by the PubMed Scopus Google Scholar). The were to the of the mutations and to that other mutations were not cells of NHE activity D. Grinstein S. Am. J. Physiol. PubMed Google Scholar) were transfected with containing the various NHE by the of and C. H. Mol. Biol. PubMed Scopus Google Scholar). the cells were for in response to over a acid (i.e. (10Orlowski J. J. Biol. Chem. 1993; 268: 16369-16377Abstract Full Text PDF PubMed Google Scholar, A. D. Pouysségur J. U. S. A. PubMed Scopus Google Scholar) to between and The for were and for The cells were to in NHE activity by the cells with the P. J. Physiol. PubMed Scopus Google the as (10Orlowski J. J. Biol. Chem. 1993; 268: 16369-16377Abstract Full Text PDF PubMed Google Scholar). the cell and by 2 pH were in this for at in a acid the were with 2 pH were by the cells in containing and of The and to the transport by the and the by the cell with of 2 pH The cell were in of and the were with of the cell and were to and a the conditions of in this with for low Na+ were the of NHE activity as a of the Na+ found with for only Nao+ to an of for this of the from to specific to the Na+/H+ exchanger were as the between the of in the and of 2 amiloride or to inhibit NHE1 or NHE3 under these experimental the To NHE activity as a of the intracellular the at over the range of by the cells in of varying containing the exchange as O. H.C. T. Orlowski J. Grinstein S. J. Cell Biol. 2000; PubMed Scopus Google Scholar). transfected cells were to in and with cellular were by and The were with with in with and with a with the were with with were by on of the of in and the were to that the were within the range of the Earlier studies have identified sites within M4 (Phe165, Phe166, Leu167, and to the NHE1 and a 66-amino acid segment encompassing M9 as of NHE drug recognition. The of these sites are in a of NHE1 as by studies S. T. Shigekawa M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The region M9 is of to for large differences orders of magnitude) in the drug sensitivity of NHE1 and NHE3 J. Kandasamy R.A. J. Biol. Chem. 1996; 271: 19922-19927Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) and is to of the region with M4 S. T. Shigekawa M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). These two isoforms share amino acid identity in this but the amino that this drug sensitivity are To identity these amino this region of NHE1 were with the corresponding residues present in NHE3 by sites are in and The were transfected in cells that NHE activity and for NHE1 the with varying levels of NHE1 as by of in of non-conserved sites encompassing helix M9 that differences in drug sensitivity between NHE1 and NHE3. of amino acid of NHE1 and NHE3 in the region containing helix The sites in that were to the residues in The and sites or significantly drug sensitivity, cells wild type wild type or were to in the cells were with the were with and in containing and of amiloride or were as a of the of in the of the of two or in of of and of wild wild data for of of or transfected cells wild type or intracellular with a of of in a data for of of or transfected cells wild type or intracellular with a of of To their drug sensitivity, were with amiloride and the more selective NHE antagonists, an mutations that did not the apparent inhibition for by more than were wild type in and not these only the substitution significantly the drug sensitivity of its apparent inhibition by and by and and To the molecular of this other and and amino were at this were for but only the and with activity to their drug inhibition the both and significantly sensitivity by and and These data suggest that in than are to drug recognition. The of the however, did not with the of also that the a but response to with transport activity in the of low drug To the of this the substitution in shown in of with at the of (i.e. a in its sensitivity to inhibition by these data that this in M9 is an of drug recognition of the with the for the more of drug inhibition of wild type and Na+/H+ for inhibition were from the of the inhibition data in The the the of as a of the The is the concentration the of two to in not in a of of on the drug sensitivity of NHE3. cells wild type and exchangers were to in and their inhibition by varying the of activity as in the of the of in for inhibition were from the of the inhibition data in The the the of as a of the The is the concentration the of two to in not the above a amino acid that for the drug sensitivity between NHE1 and is that amino to both isoforms in this region are also in drug recognition NHE3 can be by drug To we the of the NHE isoforms (NHE1 to and identified highly conserved 12 residues within M9 and the predicted loop of were for sites are in and the were of cells from acid NHE activity only mutations at a in drug sensitivity. of with the reduced sensitivity to amiloride and by and and for inhibition are in and that reduced the also in recognition these in their of transport in the of low of also that the of the not be by amiloride or only inhibition at the wild type and were mutations of residues within M4 of NHE1 Phe166, Leu167, and Gly178) have been shown to influence drug sensitivity. only the at is between NHE1 and NHE3. To residues in this region contribute to drug we the amino acid in the predicted loop between M3 and M4 of NHE isoforms that broad differences in drug sensitivity. shown in residues in this region (Gly152, to the NHE1 are the more drug-sensitive isoforms (e.g. NHE1 and but in the more drug-resistant isoforms (e.g. NHE3 and and residues are known to significantly influence the of of α-helices in the of also they contribute to the drug sensitivity of the NHE isoforms. To this these sites were in to the corresponding residues present in (i.e. and double a within M4 that is known to drug sensitivity also L. A. Pouysségur J. U. S. A. 1993; PubMed Scopus Google Scholar). shown in and in both the and modestly reduced sensitivity to by and Consistent with the significantly reduced the of to These data the loop as an with M4 in drug recognition. The above suggest that M4 and M9 be in to other to of the drug binding one that of sites in both this by and a shown in this double a affinity for with wild type and the affinity of the drug for than the substitution a in drug sensitivity. These data the that these two sites are critical determinants of the drug sensitivity between NHE1 and NHE3. To the above mutations other than drug sensitivity, we a of their kinetic properties affinities and catalytic Earlier kinetic that NHE can as J. Biol. Chem. Full Text PDF PubMed Google Scholar) under Warnock D.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, D.G. Cragoe Jr., E.J. J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar) of Na+ Consistent with these of certain sites that drug sensitivity and were found to have affect on Nao+ affinity L. A. Pouysségur J. U. S. A. 1993; PubMed Scopus Google Scholar, L. J. R.A. Pouysségur J. 1997; PubMed Scopus Google Scholar, D. Warnock D.G. Am. J. Physiol. 1995; 269: PubMed Google of a both Na+ and drug affinities N. P. Counillon L. 2001; PubMed Scopus Google Scholar). of to the or double L167F/G356A) in this also Nao+ the in the NHE as the of as a of the Nao+ concentration in the wild type and in the with Nao+ for wild type and with of the data a apparent Nao+ affinity for the that were not appreciably from wild type and for the drug-sensitive sites identified not contribute to Na+ binding are more in drug of the apparent affinity of the wild type and Na+/H+ exchangers for the of two in in a the of two in The transport of the M9 drug-resistant (i.e. and were also as a of to The residues in the loop between M3 and M4 were not they are to affect sensitivity. shown in both mutations on the of the exchanger over the range of other at these sites (i.e. and did not affinity not we the catalytic turnover of the To this to the of NHE1 at the cell surface from the that within intracellular of the and these in to the cellular of under by an to the of the exchanger by of the on To a of cellular NHE1 the of cellular by in and were to that the were within the range of the are shown in A. The two major a with an apparent molecular of that has been by and to at the L. Pouysségur J. PubMed Scopus Google Scholar, L.D. Orlowski J. Grinstein S. Am. J. Physiol. Cell Physiol. 1998; 275: PubMed Google Scholar) and an of the of that within the A of in cell and as a of on the the various that the for more of A more for the that a in the of the and a corresponding in the is that this substitution the in and in of the and of the data to that of the wild type exchanger is shown in B. In the of the were expressed at or higher levels relative to the wild type in transfected cells but or of on a To the relative activity or turnover of the the cellular of were expressed as a of their respective levels the various only the a of activity that with wild the a in that these sites not affect Nao+ or they influence the of the be in cation translocation. The of this to the in drug recognition by mammalian Na+/H+ of of the drug-sensitive NHE1 and drug-resistant NHE3 isoforms a 66-amino acid containing helix M9 as a major segment for drug recognition J. Kandasamy R.A. J. Biol. Chem. 1996; 271: 19922-19927Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The present this region by that of is to within M9, is a crucial of NHE drug sensitivity. of this amino acid to the corresponding present in NHE3 reduced drug recognition. the were for the more and HOE694, than is with of the J. Kandasamy R.A. J. Biol. Chem. 1996; 271: 19922-19927Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The more for with amiloride suggest that or the of the of with this the sensitivity to HOE694, is similar to and also has a large group at an of its benzoyl the in NHE3 a in drug sensitivity. the of this for is than the of drug sensitivity for the is a is more of a that to drug recognition than a in The of this in drug sensitivity is by the of an of mutations at amino that between NHE1 and NHE3. is also that drug affinities that are to NHE1 and NHE3 E.Z. Numata M. Shull G.E. Orlowski J. J. Biol. Chem. 2000; 275: 6302-6307Abstract Full Text Full Text PDF PubMed Scopus (61) Google also has an at the these data the critical of this in drug recognition and In to residues that are between NHE1 and NHE3 in the exomembrane loop between M3 and M4 were also found to influence drug sensitivity. of and in NHE1 with the corresponding residues present in and reduced drug sensitivity. also that of with at the of in M4 also significantly reduced drug sensitivity L. A. Pouysségur J. U. S. A. 1993; PubMed Scopus Google Scholar). this to be for drug recognition by the of the in NHE2 also reduced its sensitivity to amiloride compounds C.H.C. Nath S.K. Levine S.A. Pouysségur J. Tse Donowitz M. Biophys. Res. 1993; PubMed Scopus Google Scholar). To the of M4 and M9 in drug we the and this substitution a in drug sensitivity of NHE1 to levels for wild type NHE3. these two sites are to for of the in drug sensitivity between NHE1 and NHE3 and other isoforms as these data the in drug recognition to not only sites within M4 but also that on the surface between and within from these we also identified a highly conserved (Glu350) in M9 that a in drug sensitivity. that or and were that be to of other conserved residues this on drug sensitivity, an for this in drug recognition and one that be mammalian including as as the more organellar isoforms. To these sites were in other of exchanger we a kinetic of the various studies show that the compounds J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. Pouysségur J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. Cragoe Jr., E.J. Pouysségur J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) or Warnock D.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, D.G. Cragoe Jr., E.J. J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar) inhibition at the that the Na+ and amiloride binding sites not be certain guanidinium that by with Nao+ were not effective of amiloride binding to the exchanger C. P. P. M. J. PubMed Scopus Google the of at two sites in drug In this kinetic of mutations of NHE1 at L. A. Pouysségur J. U. S. A. 1993; PubMed Scopus Google Scholar) and L. J. R.A. Pouysségur J. 1997; PubMed Scopus Google Scholar) in a did a in Na+ affinity to L. J. R.A. Pouysségur J. 1997; PubMed Scopus Google Scholar). of a to in found to decrease both and sensitivity to HOE642 affinities for other such as and or the guanidinium were N. P. Counillon L. 2001; PubMed Scopus Google Scholar). In the present report, mutations that drug sensitivity and L167F/G356A) did not affect Nao+ In the of and intracellular affinity also The of an on cation affinities for the a for cation binding its within a and its mammalian more comparisons of the that many (i.e. and such as can amino at this including and not that a at this is not for the of an on cation the of mutations that drug binding also reduced the or turnover of the relative to wild with the of that these sites be for the that cation binding and translocation. In a mutations at these sites by translocation. In we have several distinct sites between (Gly152, Pro158) and within M9 that for the drug between NHE1 and NHE3 and to other isoforms as mutations of appear to have a on binding of the more such as with In addition, we have identified a highly conserved (Glu350) in the helix that is also a critical of drug recognition by mammalian these sites are not in the between Nao+ and drug binding appear to contribute significantly to cation translocation. molecular of the drug binding region facilitate the of more and that be therapeutically in the treatment of certain

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