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The Expanding Family of Eucaryotic Na+/H+Exchangers

2000; Elsevier BV; Volume: 275; Issue: 1 Linguagem: Inglês

10.1074/jbc.275.1.1

1083-351X

Laurent Counillon, Jacques Pouysségur,

Plant Molecular Biology Research

Na+/H+ exchanger reverse transcription-polymerase chain reaction mitogen-activated protein kinase NHE regulatory factor Maintaining intracellular pH values close to neutrality is a crucial task for a wide variety of cells. Hence, various mechanisms for pH regulation have been selected early in evolution and are ubiquitously distributed. Among the actors in this scene, the members of the Na+/H+ exchanger gene family (NHE1 isoforms) are widely expressed and constitute extremely efficient systems for protecting cells against internal acidification. To date, at least six genes have been identified in mammalian cells, and to various extents, the corresponding proteins have been molecularly and functionally characterized. In this short review, we will update our current knowledge on these NHE family members and highlight the most important aspects of the basic function of these transporters. Then, in a broader physiological context, we will present what we think are the most prominent specific features of the different NHE isoforms.Structural and Functional Domains of NHEsThe first cDNA encoding the NHE-1 isoform was cloned using an expression strategy based on the ability of Na+/H+ exchangers to protect antiporter-deficient cells (1Pouysségur J. Sardet C. Franchi A. L'Allemain G. Paris S. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4833-4837Crossref PubMed Scopus (438) Google Scholar) against otherwise lethal intracellular acidification (2Sardet C. Franchi A. Pouysségur J. Cell. 1989; 56: 271-280Abstract Full Text PDF PubMed Scopus (668) Google Scholar). Variations in the hormonal regulation and pharmacological features of Na+/H+ exchange were the first indications that a large family of Na+/H+ exchange molecules existed (3Clark J.D. Limbird L.E. Am. J. Physiol. 1991; 261: C945-C953Crossref PubMed Google Scholar). Therefore, using the NHE-1 cDNA as a probe led to the molecular identification of the NHE-2, -3, and -4 (4Tse C.M. Brant S.R. Walker M.S. Pouysségur J. Donowitz M. J. Biol. Chem. 1992; 267: 9340-9346Abstract Full Text PDF PubMed Google Scholar, 5Tse C.M. Levine S.A. Yun C.H. Montrose M.H. Little P.J. Pouysségur J. Donowitz M. J. Biol. Chem. 1993; 268: 11917-11924Abstract Full Text PDF PubMed Google Scholar, 6Wang Z. Orlowski J. Shull G.E. J. Biol. Chem. 1993; 268: 11925-11928Abstract Full Text PDF PubMed Google Scholar, 7Orlowski J. Kandasamy R.A. Shull G.E. J. Biol. Chem. 1992; 267: 9331-9339Abstract Full Text PDF PubMed Google Scholar) isoforms. In addition, non-epithelial isoforms such as NHE-5 (8Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. et al.Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar, 9Baird N.R. Orlowski J. Szabo E.Z. Zaun H.C. Schultheis P.J. Menon A.G. Shull G.E. J. Biol. Chem. 1999; 274: 4377-4382Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 10Sun A.M. Liu Y. Centracchio J. Dworkin L.D. J. Membr. Biol. 1998; 164: 293-300Crossref PubMed Scopus (24) Google Scholar) and NHE-6 have been cloned recently. By contrast to the other Na+/H+ exchangers, NHE-6 is not expressed at the plasma membrane but in the mitochondria (11Numata M. Petrecca K. Lake N. Orlowski J. J. Biol. Chem. 1998; 273: 6951-6959Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar).Topological Features and Sequence ConservationThe highly hydrophobic N-terminal region of the protein is predicted to span the membrane 10–12 times depending on the algorithm used to calculate the hydropathy plot of the protein. In particular, the region situated in the central part of the transmembrane domain (residues 226–281 in the human NHE-1) is quite hydrophobic but contains several negatively charged residues. By contrast the C-terminal region of the Na+/H+ exchangers is hydrophilic and has been shown to be located in the cell cytoplasm, at least for the NHE-1 isoform (12Shrode L.D. Gan B.S. D'Souza S.J. Orlowski J. Grinstein S. Am. J. Physiol. 1998; 275: C431-C439Crossref PubMed Google Scholar, 13Sardet C. Counillon L. Franchi A. Pouysségur J. Science. 1990; 247: 723-726Crossref PubMed Scopus (375) Google Scholar). Interestingly, recent experiments on the NHE-3 isoform seem to indicate that epitopes within the C-terminal region of this protein are extracellularly exposed (14Biemesderfer D. DeGray B. Aronson P.S. J. Biol. Chem. 1998; 273: 12391-12396Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). For NHE-1 and -2, the loop between the putative transmembrane segments 1 and 2 is glycosylated and therefore extracellular (15Counillon L. Pouysségur J. Reithmeier R.A. Biochemistry. 1994; 33: 10463-10469Crossref PubMed Scopus (123) Google Scholar, 16Tse C.M. Levine S.A. Yun C.H. Khurana S. Donowitz M. Biochemistry. 1994; 33: 12954-12961Crossref PubMed Scopus (75) Google Scholar). Methods such as scanning N-glycosylation mutagenesis (17Popov M. Tam L.Y. Li J. Reithmeier R.A. J. Biol. Chem. 1997; 272: 18325-18332Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) will be necessary to gain further topological information.Sequence Comparison between NHE IsoformsThe transmembrane domain exhibits from 45 to 65% amino acid identity, although this score drops to about 25–35% for the cytoplasmic domain. A more detailed analysis of the sequence homology clusters reveals the presence of two subfamilies of isoforms that have probably diverged later in evolution: NHE-2 and -4 as well as NHE-3 and -5. The central part of the domain (putative transmembrane segments 5a and 5b between residues 226 and 281 in human NHE-1) is nearly identical between all the NHE isoforms. This part of the polypeptide possesses negatively charged residues (aspartates 226, 238, and 267 and glutamates 247, 248, 253, and 262 in the human NHE-1) included in a highly hydrophobic stretch of sequence, and the substitution of Glu-262 in the NHE-1 isoform results in the inactivation of the transporter (18Fafournoux P. Noel J. Pouysségur J. J. Biol. Chem. 1994; 269: 2589-2596Abstract Full Text PDF PubMed Google Scholar). Although it is not possible to rule out an indirect effect of this mutation, this result in association with the extreme sequence conservation of this region among the NHE members strongly suggests that these two transmembrane segments of the exchangers constitute the catalytic core of the Na+/H+ exchangers.By contrast, the first putative transmembrane segment of the Na+/H+ exchangers and the first extracellular loop are not well conserved in the NHE family. These N-terminal sequences are divergent even in the same isoform cloned from various mammalian species (19Counillon L. Pouysségur J. Biochim. Biophys. Acta. 1993; 1172: 343-345Crossref PubMed Scopus (23) Google Scholar), indicating that sequence conservation in the first extracellular loop is not crucial for the function of the protein. A closer analysis of the first stretch of hydrophobic residues using the von Heijne rules (20von Heijne G. Nucleic Acids Res. 1986; 14: 4683-4690Crossref PubMed Scopus (3686) Google Scholar, 21von Heijne G. J. Membr. Biol. 1990; 115: 195-201Crossref PubMed Scopus (854) Google Scholar) reveals that this first putative transmembrane segment has the features of a signal peptide, including a positively charged N-terminal end and a relatively short hydrophobic stretch.Although the cytosolic domain sequence seems to be more poorly conserved, alignment methods based on the presence of hydrophobic secondary structures (hydrophobic score analysis) (22Gaboriaud C. Bissery V. Benchetrit T. Mornon J.P. FEBS Lett. 1987; 224: 149-155Crossref PubMed Scopus (541) Google Scholar, 23Callebaut I. Labesse G. Durand P. Poupon A. Canard L. Chomilier J. Henrissat B. Mornon J.P. Cell. Mol. Life Sci. 1997; 53: 621-645Crossref PubMed Scopus (430) Google Scholar) show that these C-terminal domains clearly exhibit structural similarities. 2L. Counillon, unpublished results. Recently, circular dichroism measurements performed on the Escherichia coli-expressed NHE-1 C-terminal region confirmed that this part of the protein possesses a high degree of structural organization (24Gebreselassie D. Rajarathnam K. Fliegel L. Biochem. Cell Biol. 1998; 76: 837-842Crossref PubMed Scopus (20) Google Scholar).Physiological Roles and Regulation of NHE IsoformsThe NHE-1 isoform is expressed in virtually all cells and tissues, although the expression pattern of the other NHE isoforms exhibits striking variation among different tissues. The least ambiguous expression pattern is that of NHE-3, which is highly expressed in the kidney (proximal tubule, thin and thick limbs of the loop of Henle) (59Soleimani M. Singh G. Bizal G.L. Gullans S.R. McAteer J.A. J. Biol. Chem. 1994; 269: 27973-27978Abstract Full Text PDF PubMed Google Scholar) and intestine (jejunum, ileum, ascending and descending colon, and rectum) (60Dujeda P.K. Rao D.D. Syed I. Joshi V. Dahdal R.Y. Gardner C. Risk M.C. Schmidt L. Bavishi D. Kim K.E. Harig J.M. Goldstein J.L. Layden T.J. Ramaswamy K. Am. J. Physiol. 1996; 271: G438-G493PubMed Google Scholar). Whereas NHE-1 is found mostly on the basolateral membrane of epithelial cells (61Coupaye-Gerard B. Bookstein C. Duncan P. Chen X.Y. Smith P.R. Musch M. Ernst S.A. Chang E.B. Kleyman T.R. Am. J. Physiol. 1996; 271: C1639-C1645Crossref PubMed Google Scholar) or both in the basolateral and apical membranes in epithelial cell lines such as opossum kidney or Madin-Darby canine kidney cells (62Noel J. Roux D. Pouysségur J. J. Cell Sci. 1996; 109: 929-939PubMed Google Scholar), NHE-3 is specifically targeted to the apical membrane (62Noel J. Roux D. Pouysségur J. J. Cell Sci. 1996; 109: 929-939PubMed Google Scholar, 63Hoogerwerf W.A. Tsao S.C. Devuyst O. Levine S.A. Yun C.H. Yip J.W. Cohen M.E. Wilson P.D. Lazenby A.J. Tse C.M. Donowitz M. Am. J. Physiol. 1996; 270: G29-G41PubMed Google Scholar).NHE-2, like NHE-3, has been detected both in intestine and kidney and is also targeted to the apical membrane of epithelial cells. However, whereas the presence of NHE-2 in the intestine has been confirmed by independent investigations (see for example Ref. 60Dujeda P.K. Rao D.D. Syed I. Joshi V. Dahdal R.Y. Gardner C. Risk M.C. Schmidt L. Bavishi D. Kim K.E. Harig J.M. Goldstein J.L. Layden T.J. Ramaswamy K. Am. J. Physiol. 1996; 271: G438-G493PubMed Google Scholar), the expression of this protein in the kidney is somewhat controversial (59Soleimani M. Singh G. Bizal G.L. Gullans S.R. McAteer J.A. J. Biol. Chem. 1994; 269: 27973-27978Abstract Full Text PDF PubMed Google Scholar, 64Sun A.M. Liu Y. Dworkin L.D. Tse C.M. Donowitz M. Yip K.P. J. Membr. Biol. 1997; 160: 85-90Crossref PubMed Scopus (60) Google Scholar, 65Bookstein C. Xie Y. Rabenau K. Musch M.W. McSwine R.L. Rao M.C. Chang E.B. Am. J. Physiol. 1997; 273: C1496-C1505Crossref PubMed Google Scholar).NHE-4 mRNA can be found in the stomach, intestine, kidney, and in the cavi ammoni fields of the hippocampus. In the kidney, NHE-4 is mostly present in the inner medulla collecting duct and has also been found heterogeneously distributed on the basolateral membrane of cortical tubule cells (65Bookstein C. Xie Y. Rabenau K. Musch M.W. McSwine R.L. Rao M.C. Chang E.B. Am. J. Physiol. 1997; 273: C1496-C1505Crossref PubMed Google Scholar).The recently cloned NHE-5 isoform has been detected predominantly in brain but also in testis, spleen, and skeletal muscle by Northern blot (8Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. et al.Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar), whereas NHE-6, which is expressed in mitochondria, has a wide tissue distribution.The common mechanism by which intracellular signaling pathways modulate the Na+/H+ exchangers involves the C-terminal region of these proteins, as shown in a series of key experiments. For example, the expression of a chimera consisting of the transmembrane region of the cAMP-insensitive human NHE-1 and the cytosolic region of the cAMP-activable β-NHE-1 of trout red cells results in a protein which is activated by cAMP, identical to the way the complete β-NHE-1 isoform behaves (66Borgese F. Sardet C. Cappadoro M. Pouysségur J. Motais R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6765-6769Crossref PubMed Scopus (124) Google Scholar, 67Borgese F. Malapert M. Fievet B. Pouysségur J. Motais R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5431-5435Crossref PubMed Scopus (37) Google Scholar). Conversely, NHE-3 is inhibited by cAMP in epithelial cells, and a chimeric construct between the transmembrane region of NHE-1 and the cytosolic region of NHE-3 becomes inhibited by cAMP (68Cabado A.G. Yu F.H. Kapus A. Lukacs G. Grinstein S. Orlowski J. J. Biol. Chem. 1996; 271: 3590-3599Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). These key findings indicate that the C-terminal domain dictates the type of hormonal regulation in a given cell.Molecular Dissection of NHE-1 ActivationNHE-1 activation by an extreme variety of extracellular stimuli, including hormones, integrins, and virtually all growth factors results from an increase in affinity of the transporter for intracellular protons. The simplest model that has been proposed is that the cytoplasmic tail cooperates with the central pH i sensor to decrease the pH i threshold value of NHE-1. In this regard, the cytoplasmic tail is seen as a signal integrator capable of transmitting hormonal signals to the transmembrane built-in pH i sensor (69Wakabayashi S. Fafournoux P. Sardet C. Pouysségur J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2424-2428Crossref PubMed Scopus (236) Google Scholar). Therefore, as for a promoter region of a regulated gene, it is not surprising to see that the NHE-1 cytoplasmic tail has “collected” regulatory boxes that convey specific extracellular signals. For example, all growth factors have been shown to induce a very rapid and transient rise in cytoplasmic calcium as well as a more or less sustained activation of the p42/p44 MAPK cascade. Interestingly and as presented below, the NHE-1 cytoplasmic domain intercepts these distinct signals for transmission into a cytoplasmic alkalinization. Bertrand et al. (70Bertrand B. Wakabayashi S. Ikeda T. Pouysségur J. Shigekawa M. J. Biol. Chem. 1994; 269: 13703-13709Abstract Full Text PDF PubMed Google Scholar) demonstrated that calmodulin physically interacts with a particular subdomain of the NHE-1 cytosolic region (71Wakabayashi S. Bertrand B. Ikeda T. Pouysségur J. Shigekawa M. J. Biol. Chem. 1994; 269: 13710-13715Abstract Full Text PDF PubMed Google Scholar) releasing a negative constraint, thus resulting in the activation of NHE-1 by increases in intracellular Ca2+. Therefore, this calmodulin-binding regulatory box is sufficient to account for the rapid and transient activation of NHE-1 in response to growth factors and other Ca2+-mobilizing agonists. By contrast, a similar sequence is not found in NHE-3, which is also regulated by calmodulin, both in a calmodulin kinase-dependent and -independent manner (72Levine S.A. Nath S.K. Yun C.H. Yip J.W. Montrose M. Donowitz M. Tse C.M. J. Biol. Chem. 1995; 270: 13716-13725Crossref PubMed Scopus (103) Google Scholar).Direct phosphorylation of NHE1 and/or phosphorylation of ancillary proteins could account for more robust and sustained activation of NHE-1. Both mechanisms have been well documented. First, it was demonstrated that NHE-1 is a phosphoprotein and that its level of phosphorylation is increased in mitogen-stimulated cells when compared with unstimulated controls (13Sardet C. Counillon L. Franchi A. Pouysségur J. Science. 1990; 247: 723-726Crossref PubMed Scopus (375) Google Scholar, 73Livne A.A. Sardet C. Pouysségur J. FEBS Lett. 1991; 284: 219-222Crossref PubMed Scopus (43) Google Scholar, 74Wang H. Silva N.L. Lucchesi P.A. Haworth R. Wang K. Michalak M. Pelech S. Fliegel L. Biochemistry. 1997; 36: 9151-9158Crossref PubMed Scopus (85) Google Scholar, 75Sardet C. Fafournoux P. Pouysségur J. J. Biol. Chem. 1991; 266: 19166-19171Abstract Full Text PDF PubMed Google Scholar). Phosphopeptide mapping carried out on wild-type and deletion mutants of the cytoplasmic region (76Wakabayashi S. Bertrand B. Shigekawa M. Fafournoux P. Pouysségur J. J. Biol. Chem. 1994; 269: 5583-5588Abstract Full Text PDF PubMed Google Scholar) revealed that the phosphorylation sites are located in the C-terminal cytoplasmic region of the protein. Ser-703 was recently demonstrated to be phosphorylated in vivo by the p42/p44 MAPK-activated target, p90RSK (77Takahashi E. Abe J. Gallis B. Aebersold R. Spring D.J. Krebs E.G. Berk B.C. J. Biol. Chem. 1999; 274: 20206-20214Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), and to represent a major site for serum activation. This result is in agreement with our demonstration, using a Raf-activable construct, that p42/p44 MAPK plays a key role in NHE1 activation (78Bianchini L. L'Allemain G. Pouysségur J. J. Biol. Chem. 1997; 272: 271-279Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Besides the MAPK pathway, NHE-1 has been shown to be phosphorylated by p160 ROCK (79Sahai E. Alberts A.S. Treisman R. EMBO J. 1998; 17: 1350-1361Crossref PubMed Scopus (229) Google Scholar), a Rho effector associated with the assembly of stress fibers and focal adhesions. However, p42/p44 MAPK-mediated NHE-1 activation cannot be entirely explained by the direct phosphorylation of NHE-1. First, deletion of the distal cytoplasmic tail containing Ser-703 and other major phosphorylation sites attenuates but does not abolish growth factor activation. The residual activation (about 50%) remains sensitive to the MEK inhibitor PD98059 (78Bianchini L. L'Allemain G. Pouysségur J. J. Biol. Chem. 1997; 272: 271-279Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The simplest working model taking into account this set of results is that additional regulatory proteins, which may themselves be phosphorylated, interact with various domains of the cytosolic region of the exchanger. Candidate proteins have been identified, such as p24 NHE-1 (80Goss G. Orlowski J. Grinstein S. Am. J. Physiol. 1996; 270: C1493-C1502Crossref PubMed Google Scholar), HSP70 (81Silva N.L. Haworth R.S. Singh D. Fliegel L. Biochemistry. 1995; 34: 10412-10420Crossref PubMed Scopus (84) Google Scholar), CHP (82Lin X. Barber D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12631-12636Crossref PubMed Scopus (151) Google Scholar), and other proteins obtained by double hybrid screening, such as myosin light chain phosphatase. 4P. Fafournoux and J. Pouysségur, unpublished results. Additionally, NHE-1 can be activated by different mechanical stimuli such as osmotic stress or cell spreading. Grinstein et al. (83Grinstein S. Woodside M. Sardet C. Pouysségur J. Rotin D. J. Biol. Chem. 1992; 267: 23823-23828Abstract Full Text PDF PubMed Google Scholar) have demonstrated that the mechanism of this activation is phosphorylation-independent. In view of their finding that NHE-1 is associated with the actin cytoskeleton in focal adhesion plaques (84Grinstein S. Woodside M. Waddell T.K. Downey G.P. Orlowski J. Pouysségur J. Wong D.C. Foskett J.K. EMBO J. 1993; 12: 5209-5218Crossref PubMed Scopus (169) Google Scholar), these results suggest that this activation might be mediated by direct contact with cytoskeletal proteins. This hypothesis is reinforced by the fact that the presence of relatively high concentrations of ATP is required for optimal NHE-1 activation (85Demaurex N. Romanek R.R. Orlowski J. Grinstein S. J. Gen. Physiol. 1997; 109: 117-128Crossref PubMed Scopus (54) Google Scholar) and that ATP depletion results in a more homogeneous plasma membrane distribution of NHE-1. As the NHE-1 phosphorylation level is not changed upon cellular ATP depletion, NHE-1 might interact in an ATP-dependent manner with an ancillary protein mediating NHE-1 interaction with cytoskeletal elements (86Goss G.G. Woodside M. Wakabayashi S. Pouysségur J. Waddell T. Downey G.P. Grinstein S. J. Biol. Chem. 1994; 269: 8741-8748Abstract Full Text PDF PubMed Google Scholar). Bianchiniet al. (78Bianchini L. L'Allemain G. Pouysségur J. J. Biol. Chem. 1997; 272: 271-279Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) demonstrated that the p42/44 MAPK is a major pathway for NHE-1 activation by many growth stimuli, whereas the stress-activated kinases and stress kinase pathways (JNK and p38 MAPK) are not involved in the activation of NHE-1 by hypertonicity. Thus the C-terminal domain of NHE-1 can be viewed as a series of regulatory cassettes, where upon phosphorylation or binding of regulatory proteins, the affinity of the transporter for intracellular protons is modulated.NHE-3 RegulationThe NHE-3 regulation mechanism is completely different from the NHE-1 activation because the changes in activity reflect modifications of the V max of the transporter instead of changes in its apparent affinity for intracellular protons. Using immunofluorescence techniques and confocal microscopy, it is possible to detect NHE-3 accumulation in recycling endosomes (87D'Souza S. Garcia-Cabado A. Yu F. Teter K. Lukacs G. Skorecki K. Moore H.P. Orlowski J. Grinstein S. J. Biol. Chem. 1998; 273: 2035-2043Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Weinman and Shenolikar (88Weinman E.J. Shenolikar S. Exp. Nephrol. 1997; 5: 449-452PubMed Google Scholar) have described the cloning and characterization of a 538-amino acid NHE regulatory factor (NHE-RF) (for review, see Ref. 88Weinman E.J. Shenolikar S. Exp. Nephrol. 1997; 5: 449-452PubMed Google Scholar). This protein, which can be either cytoplasmic or membrane-bound, negatively regulates NHE-3 activity by direct binding. When coexpressed in PS120 cells, NHE-RF is able to confer cAMP inhibition to NHE-3. This protein is present in various sections of both the intestine and the kidney tubule. Further, it has been detected in the liver where NHE-3 is not detected, indicating that it could mediate cAMP regulation of proteins other than the NHE-3 isoform. Indeed, NHE-RF-like proteins, such as E3KARP, have been shown to regulate other transmembrane transporters, including the Na-HCO3 or Na-PO4 cotransports. E3KARP can also confer cAMP regulation upon NHE-3, indicating that the NHE regulatory proteins might be multiple (89Yun C.H. Oh S. Zizak M. Steplock D. Tsao S. Tse C.M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (404) Google Scholar). Recently Hall et al. (90Hall R.A. Premont R.T. Chow C.W. Blitzer J.T. Pitcher J.A. Claing A. Stoffel R.H. Barak L.S. Shenolikar S. Weinman E.J. Grinstein S. Lefkowitz R.J. Nature. 1998; 392: 626-630Crossref PubMed Scopus (520) Google Scholar) demonstrated that the β2-adrenergic receptor can directly associate with NHE-RF through its PDZ domain, providing a direct regulation of NHE-3 activity. Similarly, Yun et al. (91Yun C.H. Lamprecht G. Forster D.V. Sidor A. J. Biol. Chem. 1998; 273: 25856-25863Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar) have demonstrated that E3KARP binds directly to an intracellular region within the C-terminal domain of NHE-3 and defined more precisely the protein regions participating in this interaction. Taken together, these results suggest that E3KARP or NHE-RF have a scaffolding function, permitting the co-localization of NHE-3 and kinase A.Similar to NHE-1, the NHE-3 cytosolic region therefore appears to consist of various regulatory cassettes, which seem to integrate activating and inhibitory signals from various signaling pathways (72Levine S.A. Nath S.K. Yun C.H. Yip J.W. Montrose M. Donowitz M. Tse C.M. J. Biol. Chem. 1995; 270: 13716-13725Crossref PubMed Scopus (103) Google Scholar). In an independent work Cabado and co-workers (68Cabado A.G. Yu F.H. Kapus A. Lukacs G. Grinstein S. Orlowski J. J. Biol. Chem. 1996; 271: 3590-3599Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) showed that the region situated between residues 579 and 684 mediates cAMP inhibition. In this region, which contains 6 Ser, only Ser-605 and -634 are crucial for cAMP inhibition (92Kurashima K. Yu F.H. Cabado A.G. Szabo E.Z. Grinstein S. Orlowski J. J. Biol. Chem. 1997; 272: 28672-28679Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Interestingly, although mutation of Ser-634 affects forskolin response, only Ser-605 is indeed phosphorylated.Concluding RemarksGene knockout is an elegant approach to obtain new insights into the physiological role of proteins belonging to multiple gene families. Results from this approach are just emerging for NHE isoforms. Schultheis et al. (93Schultheis P.J. Clarke L.L. Meneton P. Miller M.L. Soleimani M. Gawenis L.R. Riddle T.M. Duffy J.J. Doetschman T. Wang T. Giebisch G. Aronson P.S. Lorenz J.N. Shull G.E. Nat. Genet. 1998; 19: 282-285Crossref PubMed Scopus (696) Google Scholar) have constructed knockout mice for NHE-3, an isoform which was expected to largely contribute intestinal sodium absorption as well as kidney sodium reabsorption coupled to bicarbonate reabsorption. As expected, these homozygous knockout mice exhibit a decrease in blood pressure, they are mildly acidotic, and they present absorptive defects both in kidney and intestine. This important result confirms the predicted physiological role of NHE-3 and shows that this isoform, when compared with NHE-1, -2, and -4, mediates the great majority of the absorptive process in kidney and intestine.By contrast, Cox et al. (94Cox G.A. Lutz C.M. Yang C.L. Biemesderfer D. Bronson R.T. Fu A. Aronson P.S. Noebels J.L. Frankel W.N. Cell. 1997; 91: 139-148Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar) have reported the molecular characterization of the genetic defect present in epileptic and ataxic (SWE) mice. They discovered that these mice possess a point mutation, which introduces a stop codon in the coding sequence of NHE-1, resulting in the production of a truncated, inactive transporter. The homozygous inactivation of NHE-1 gene function reported thereafter (95Bell S.M. Schreiner C.M. Schultheis P.J. Miller M.L. Evans R.L. Vorhees C.V. Shull G.E. Scott W.J. Am. J. Physiol. 1999; 276: C788-C795Crossref PubMed Google Scholar) confirmed the non-lethality of this mutation, a finding which was anticipated in light of the ubiquitous expression of compensatory pH-regulating systems such as sodium-dependent Cl−/HCO3− exchangers. Interestingly, these mice have no apparent defects in their acid-base homeostatic balance or in their kidney or intestine function. Rather, they show defects in brain function; this organ, which is highly sensitive to pH, was not initially hypothesized as the main target of the NHE-1 gene disruption. It is therefore tempting to predict that, as for NHE-1, the disruption of the other isoforms may have surprising physiological consequences. Hence, the recent knockout of the NHE-2 isoform in mouse did not result in detectable modifications in intestinal function but in a modified gastric mucosa, resulting in deficient acid secretion in the stomach (96Schultheis P.J. Clarke L.L. Meneton P. Harline M. Boivin G.P. Stemmermann G. Duffy J.J. Doetschman T. Miller M.L. Shull G.E. J. Clin. Invest. 1998; 101: 1243-1253Crossref PubMed Scopus (220) Google Scholar). Therefore, as has been reported for many gene disruptions, the interpretation of the resulting phenotypes might be confusing because of the possible involvement of the NHE isoforms during critical steps of the embryonic development and the presence of compensatory mechanisms in adult mice. In this case, the recent development of tissue-specific gene disruption and of inducible knockout techniques should prove to be very useful for the future physiological studies of Na+/H+exchange. Maintaining intracellular pH values close to neutrality is a crucial task for a wide variety of cells. Hence, various mechanisms for pH regulation have been selected early in evolution and are ubiquitously distributed. Among the actors in this scene, the members of the Na+/H+ exchanger gene family (NHE1 isoforms) are widely expressed and constitute extremely efficient systems for protecting cells against internal acidification. To date, at least six genes have been identified in mammalian cells, and to various extents, the corresponding proteins have been molecularly and functionally characterized. In this short review, we will update our current knowledge on these NHE family members and highlight the most important aspects of the basic function of these transporters. Then, in a broader physiological context, we will present what we think are the most prominent specific features of the different NHE isoforms. Structural and Functional Domains of NHEsThe first cDNA encoding the NHE-1 isoform was cloned using an expression strategy based on the ability of Na+/H+ exchangers to protect antiporter-deficient cells (1Pouysségur J. Sardet C. Franchi A. L'Allemain G. Paris S. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4833-4837Crossref PubMed Scopus (438) Google Scholar) against otherwise lethal intracellular acidification (2Sardet C. Franchi A. Pouysségur J. Cell. 1989; 56: 271-280Abstract Full Text PDF PubMed Scopus (668) Google Scholar). Variations in the hormonal regulation and pharmacological features of Na+/H+ exchange were the first indications that a large family of Na+/H+ exchange molecules existed (3Clark J.D. Limbird L.E. Am. J. Physiol. 1991; 261: C945-C953Crossref PubMed Google Scholar). Therefore, using the NHE-1 cDNA as a probe led to the molecular identification of the NHE-2, -3, and -4 (4Tse C.M. Brant S.R. Walker M.S. Pouysségur J. Donowitz M. J. Biol. Chem. 1992; 267: 9340-9346Abstract Full Text PDF PubMed Google Scholar, 5Tse C.M.