Portal de Periódicos da CAPES

Molecular Cloning, Genomic Organization, and Functional Expression of Na+/H+ Exchanger Isoform 5 (NHE5) from Human Brain

1999; Elsevier BV; Volume: 274; Issue: 7 Linguagem: Inglês

10.1074/jbc.274.7.4377

1083-351X

Nancy R. Baird, John Orlowski, Elöd Z. Szabó, Hans C. Zaun, Patrick J. Schultheis, Anil G. Menon, Gary E. Shull,

Receptor Mechanisms and Signaling

To isolate a cDNA encoding Na+/H+ exchanger isoform 5 (NHE5), we screened a human spleen library using exon sequences of theNHE5 gene. Clones spanning 2.9 kilobase pairs were isolated; however, they contained several introns and were missing coding sequences at both the 5′ and 3′ ends. The missing 5′ sequences were obtained by 5′-rapid amplification of cDNA ends and by analysis of an NHE5 genomic clone, and the missing 3′ sequences were obtained by 3′-rapid amplification of cDNA ends. Polymerase chain reaction amplification of brain cDNA yielded products in which each of the introns had been correctly excised, whereas the introns were retained in products from spleen and testis, suggesting that the NHE5 transcripts expressed in these organs do not encode a functional transporter. The intron/exon organization of theNHE5 gene was analyzed and found to be very similar to that of the NHE3 gene. The NHE5 cDNA, which encodes an 896-amino acid protein that is most closely related to NHE3, was expressed in Na+/H+ exchanger-deficient fibroblasts and shown to mediate Na+/H+exchange activity. Northern blot analysis demonstrated that the mRNA encoding NHE5 is expressed in multiple regions of the brain, including hippocampus, consistent with the possibility that it regulates intracellular pH in hippocampal and other neurons. To isolate a cDNA encoding Na+/H+ exchanger isoform 5 (NHE5), we screened a human spleen library using exon sequences of theNHE5 gene. Clones spanning 2.9 kilobase pairs were isolated; however, they contained several introns and were missing coding sequences at both the 5′ and 3′ ends. The missing 5′ sequences were obtained by 5′-rapid amplification of cDNA ends and by analysis of an NHE5 genomic clone, and the missing 3′ sequences were obtained by 3′-rapid amplification of cDNA ends. Polymerase chain reaction amplification of brain cDNA yielded products in which each of the introns had been correctly excised, whereas the introns were retained in products from spleen and testis, suggesting that the NHE5 transcripts expressed in these organs do not encode a functional transporter. The intron/exon organization of theNHE5 gene was analyzed and found to be very similar to that of the NHE3 gene. The NHE5 cDNA, which encodes an 896-amino acid protein that is most closely related to NHE3, was expressed in Na+/H+ exchanger-deficient fibroblasts and shown to mediate Na+/H+exchange activity. Northern blot analysis demonstrated that the mRNA encoding NHE5 is expressed in multiple regions of the brain, including hippocampus, consistent with the possibility that it regulates intracellular pH in hippocampal and other neurons. Six members of the mammalian Na+/H+exchanger (NHE) 1The abbreviations used are: NHE, Na+/H+ exchanger; NHE1–6, Na+/H+ exchanger isoforms 1–6; nt(s), nucleotides; kb, kilobase; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction.1The abbreviations used are: NHE, Na+/H+ exchanger; NHE1–6, Na+/H+ exchanger isoforms 1–6; nt(s), nucleotides; kb, kilobase; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction. family, including five plasma membrane (NHE1–5) and one mitochondrial (NHE6) exchanger, have been identified by molecular cloning studies (1Sardet C. Franchi A. Pouyssegur J. Cell. 1989; 86: 271-280Abstract Full Text PDF Scopus (668) Google Scholar, 2Orlowski J. Kandasamy R.A. Shull G.E. J. Biol. Chem. 1992; 267: 9331-9339Abstract Full Text PDF PubMed Google Scholar, 3Tse C.-M. Brant S.R. Walker M.S. Pouyssegur J. Donowitz M. J. Biol. Chem. 1992; 267: 9340-9346Abstract Full Text PDF PubMed Google Scholar, 4Wang Z. Orlowski J. Shull G.E. J. Biol. Chem. 1993; 268: 11925-11928Abstract Full Text PDF PubMed Google Scholar, 5Tse C.-M. Levine S.A. Yun C.H.C. Montrose M.H. Little P.J. Pouyssegur J. Donowitz M. J. Biol. Chem. 1993; 268: 11917-11924Abstract Full Text PDF PubMed Google Scholar, 6Numata M. Petrecca K. Lake N. Orlowski J. J. Biol. Chem. 1998; 273: 6951-6959Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 7Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. Shull G.E. Menon A.G. Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar). Full-length cDNAs encoding NHE1–4 (1Sardet C. Franchi A. Pouyssegur J. Cell. 1989; 86: 271-280Abstract Full Text PDF Scopus (668) Google Scholar, 2Orlowski J. Kandasamy R.A. Shull G.E. J. Biol. Chem. 1992; 267: 9331-9339Abstract Full Text PDF PubMed Google Scholar, 3Tse C.-M. Brant S.R. Walker M.S. Pouyssegur J. Donowitz M. J. Biol. Chem. 1992; 267: 9340-9346Abstract Full Text PDF PubMed Google Scholar, 4Wang Z. Orlowski J. Shull G.E. J. Biol. Chem. 1993; 268: 11925-11928Abstract Full Text PDF PubMed Google Scholar, 5Tse C.-M. Levine S.A. Yun C.H.C. Montrose M.H. Little P.J. Pouyssegur J. Donowitz M. J. Biol. Chem. 1993; 268: 11917-11924Abstract Full Text PDF PubMed Google Scholar) and NHE6 (6Numata M. Petrecca K. Lake N. Orlowski J. J. Biol. Chem. 1998; 273: 6951-6959Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar) have been characterized, thereby providing their amino acid sequences and allowing the development of expression constructs and isoform-specific nucleic acid and antibody probes. The use of these reagents has led to a substantial body of information about the expression patterns of these isoforms and their functional characteristics (reviewed in Refs.8Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Crossref PubMed Scopus (517) Google Scholar and 9Wakabayashi S. Shigekawa M. Pouyssegur J. Physiol. Rev. 1997; 77: 51-74Crossref PubMed Scopus (562) Google Scholar). In recent studies, mice carrying mutations in the genes encoding NHE1, -2, and -3 have been developed, and their phenotypes have been analyzed to determine their specific physiological functionsin vivo (10Cox 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 (236) Google Scholar, 11Schultheis 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, 12Schultheis 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). In contrast, the complete primary structure of NHE5 has not been determined, and nothing is known about its functional characteristics and physiological roles.The human NHE5 gene was identified several years ago by low stringency screening of a cosmid library using an NHE2 cDNA probe and mapped to chromosome 16q22.1 (7Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. Shull G.E. Menon A.G. Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar). Northern blot analysis demonstrated that NHE5 transcripts were expressed at easily detected levels in brain, spleen, and testis and at trace levels in skeletal muscle and revealed differences in the sizes of the transcripts in each tissue. Unlike the other isoforms, expression of NHE5 was not observed in epithelial tissues. Based on the partial coding sequences obtained and limited analysis of the structure of the gene, NHE5 appeared to be more closely related to the amiloride-resistant NHE3 isoform than to other members of the family. Its abundant expression in brain and apparent similarity to NHE3 raised the possibility that NHE5 might be the amiloride-resistant Na+/H+ exchanger that was described in hippocampal neurons (13Raley-Susman K.M. Cragoe Jr., E.J. Sapolsky R.M. Kopito R.R. J. Biol. Chem. 1991; 266: 2739-2745Abstract Full Text PDF PubMed Google Scholar), particularly since NHE3 expression is negligible in brain (2Orlowski J. Kandasamy R.A. Shull G.E. J. Biol. Chem. 1992; 267: 9331-9339Abstract Full Text PDF PubMed Google Scholar). To begin a systematic analysis of the functional characteristics and physiological roles of NHE5, we have cloned and functionally expressed a cDNA encoding the complete amino acid sequence of human NHE5, determined the intron-exon organization of its gene, and demonstrated that its mRNA is expressed in hippocampus and other structures of the brain.DISCUSSIONThe major objectives of this study were to determine the primary structure of human NHE5 and to explore the possibility that it corresponds to an amiloride-resistant Na+/H+exchanger that was identified previously in hippocampal neurons (13Raley-Susman K.M. Cragoe Jr., E.J. Sapolsky R.M. Kopito R.R. J. Biol. Chem. 1991; 266: 2739-2745Abstract Full Text PDF PubMed Google Scholar). The human NHE5 gene was identified several years ago and was shown to be expressed in brain, spleen, and testis (7Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. Shull G.E. Menon A.G. Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar). In the present study we were unable to isolate NHE5 cDNAs corresponding to fully processed transcripts in human spleen or testis; however, PCR analysis of brain cDNA did yield products that were correctly processed. These cloning experiments and the previous observation that NHE5 transcripts in human spleen and testis are larger than those in brain suggest that intron sequences, which disrupt the open reading frame, are retained in the spleen and testis mRNAs. While this study was in progress we had frequent discussions with Attaphitaya et al. (21Attaphitaya S. Park K. Melvin J.E. J. Biol. Chem. 1999; 274: 4383-4388Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), who were conducting similar studies of rat NHE5. When cloning rat NHE5 from brain, these investigators did not encounter processing intermediates or aberrantly spliced transcripts, and their Northern blot analyses revealed high levels of NHE5 mRNA in brain and only trace levels in spleen, testis, and other tissues. Thus, the results from both laboratories suggest that expression of functional NHE5 mRNA may be restricted to brain, although they do not rule out the possibility of expression in tissues and developmental stages that were not examined in these experiments.On the basis of the current and previous studies (7Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. Shull G.E. Menon A.G. Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar, 14Kandasamy R.A. Orlowski J. J. Biol. Chem. 1996; 271: 10551-10559Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), it is apparent that NHE5 and NHE3 are more closely related to each other, in both genomic organization and amino acid sequence, than to the other isoforms. As shown in Fig. 2, the location of exon boundaries relative to the coding sequences are almost identical in the NHE5 andnhe3 genes, and only the last intron of the nhe3gene lacks a counterpart in the NHE5 gene. In contrast, introns 2, 4, and 10–15 of the NHE5 and nhe3genes have no counterparts in the NHE1 gene (14Kandasamy R.A. Orlowski J. J. Biol. Chem. 1996; 271: 10551-10559Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 22Miller R.T. Counillon L. Pages G. Lifton R.P. Sardet C. Pouyssegur J. J. Biol. Chem. 1991; 266: 10813-10819Abstract Full Text PDF PubMed Google Scholar), and introns 8–11 of the NHE1 gene have no counterparts in theNHE5 and nhe3 genes. The complete genomic organization of the nhe2 and nhe4 genes, which are tightly linked in the mouse genome (23Pathak B.P. Shull G.E. Jenkins N.A. Copeland N.G. Genomics. 1996; 31: 261-263Crossref PubMed Scopus (17) Google Scholar), have not been determined. However, limited analyses of the mouse nhe2 andnhe4 genes (7Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. Shull G.E. Menon A.G. Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar) indicate that they are more closely related to NHE1 than to NHE5 and nhe3, a conclusion that is supported by comparisons of amino acid sequences (Table II and see Refs. 4Wang Z. Orlowski J. Shull G.E. J. Biol. Chem. 1993; 268: 11925-11928Abstract Full Text PDF PubMed Google Scholar, 5Tse C.-M. Levine S.A. Yun C.H.C. Montrose M.H. Little P.J. Pouyssegur J. Donowitz M. J. Biol. Chem. 1993; 268: 11917-11924Abstract Full Text PDF PubMed Google Scholar, and 7Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. Shull G.E. Menon A.G. Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar).An unusual feature of the NHE5 gene is the presence of GC dinucleotides, rather than GT dinucleotides, at the beginning of introns 13 and 14 (Table I). This departure from the consensus sequence is uncommon, but several dozen examples of GC dinucleotides at the beginning of an intron have been documented (24Jackson I.J. Nucleic Acids Res. 1991; 14: 3795-3798Crossref Scopus (278) Google Scholar). For example, the sequences, AAG/GCATGT and CAG/GCAAGC, at the donor sites of exons 13 and 14, respectively, are identical to donor sites observed in the RNA polymerase II (25Ahearn Jr., J.M. Bartolomei M.S. West M.L. Cisek L.J. Corden J.L. J. Biol. Chem. 1987; 262: 10695-10705Abstract Full Text PDF PubMed Google Scholar) and heme oxygenase (26Muller R.M. Taguchi H. Shibahara S. J. Biol. Chem. 1987; 262: 6795-6802Abstract Full Text PDF PubMed Google Scholar) genes, respectively.The data shown in Fig. 3 demonstrate that human NHE5 mediates H+-dependent Na+ influx and are consistent with the observation that rat NHE5 mediates Na+-dependent pH recovery from an acid load (21Attaphitaya S. Park K. Melvin J.E. J. Biol. Chem. 1999; 274: 4383-4388Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). 22Na+ influx in the clonal isolate shown in Fig. 3 was ∼0.2 pmol/min/mg protein. Under comparable assay conditions the average 22Na+ influx values for AP-1 cell isolates expressing NHE1, NHE2, and NHE3 was between 0.25 and 0.60 pmol/min/mg protein (17Orlowski J. J. Biol. Chem. 1993; 268: 16369-16377Abstract Full Text PDF PubMed Google Scholar, 20Yu F.H. Shull G.E. Orlowski J. J. Biol. Chem. 1993; 268: 25536-25541Abstract Full Text PDF PubMed Google Scholar), with activity in the order NHE1 > NHE2 > NHE3. Because the amount of NHE protein expressed in each of the clonal isolates that have been examined has not been determined, the relative activities of the various isoforms cannot be directly compared; however, the activity of the NHE5 isolate examined here is within the range of values observed for clonal isolates expressing NHE3. Rat NHE5 has been shown to be more resistant than NHE1 to 5-(N-ethyl-N-isopropyl)amiloride (21Attaphitaya S. Park K. Melvin J.E. J. Biol. Chem. 1999; 274: 4383-4388Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), and detailed pharmacological and biochemical analyses of human NHE5 that are currently in progress 2E. Szabo and J. Orlowski, unpublished observations. indicate that human NHE5 is also quite resistant to pharmacological antagonists, although not to as great an extent as NHE3. Thus, both human and rat NHE5 are, in fact, Na+/H+exchangers and have pharmacological characteristics that more closely resemble NHE3 than NHE1.When we began this study we were interested in determining whether NHE5 might be the transporter responsible for the amiloride-resistant Na+/H+ exchange activity identified in rat hippocampal neurons by Raley-Susman et al. (13Raley-Susman K.M. Cragoe Jr., E.J. Sapolsky R.M. Kopito R.R. J. Biol. Chem. 1991; 266: 2739-2745Abstract Full Text PDF PubMed Google Scholar). Our Northern blot data showing NHE5 expression in hippocampus of human brain and in situ hybridization analysis showing NHE5 expression in rat hippocampus (21Attaphitaya S. Park K. Melvin J.E. J. Biol. Chem. 1999; 274: 4383-4388Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) are consistent with this possibility. However, the Na+/H+ exchange activity observed in hippocampal neurons was completely insensitive to 1 mm amiloride, whereas human NHE5 expressed in AP-1 cells is fully inhibited by this concentration of amiloride (Fig. 3). This apparent difference in drug sensitivity could be due to the different conditions under which Na+/H+ exchange activity was measured in the two studies. For example, in the study of Na+/H+ exchange in hippocampal neurons (13Raley-Susman K.M. Cragoe Jr., E.J. Sapolsky R.M. Kopito R.R. J. Biol. Chem. 1991; 266: 2739-2745Abstract Full Text PDF PubMed Google Scholar), the concentration of extracellular Na+, which is a competitive inhibitor of amiloride binding, was 135 mm, whereas in our own studies only trace levels of22Na+ were present. NHE4 has been localized to cavi amnoni neurons in rat hippocampus (27Bookstein C. Musch M.W. DePaoli A. Xie Y. Rabenau K. Villereal M. Rao M.C. Chang E.B. Am. J. Physiol. 1996; 271: C1629-C1638Crossref PubMed Google Scholar), but this isoform also has been shown to exhibit some sensitivity to 1 mm amiloride. Additional studies will be needed to resolve the questions of which isoform(s) mediate control of intracellular pH in hippocampal neurons.In summary, we have cloned and expressed a fifth member of the plasma membrane Na+/H+ exchanger family and have obtained data indicating that functional expression of this isoform may be restricted to brain. Northern blot analysis showed that it is widely distributed in brain, with significant mRNA expression detected in hippocampus, amygdala, caudate nucleus, hypothalamus, subthalamic nucleus, and thalamus. The detection of NHE5 mRNA in multiple structures of the brain, but the absence of a significant hybridization signal in corpus callosum, which consists primarily of axons and glial cells and has few neuronal cell bodies, raises the possibility that NHE5 may be a neuron-specific isoform. If this proves to be the case, then this isoform would be the most highly cell type-specific member of the Na+/H+ exchanger family. Six members of the mammalian Na+/H+exchanger (NHE) 1The abbreviations used are: NHE, Na+/H+ exchanger; NHE1–6, Na+/H+ exchanger isoforms 1–6; nt(s), nucleotides; kb, kilobase; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction.1The abbreviations used are: NHE, Na+/H+ exchanger; NHE1–6, Na+/H+ exchanger isoforms 1–6; nt(s), nucleotides; kb, kilobase; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction. family, including five plasma membrane (NHE1–5) and one mitochondrial (NHE6) exchanger, have been identified by molecular cloning studies (1Sardet C. Franchi A. Pouyssegur J. Cell. 1989; 86: 271-280Abstract Full Text PDF Scopus (668) Google Scholar, 2Orlowski J. Kandasamy R.A. Shull G.E. J. Biol. Chem. 1992; 267: 9331-9339Abstract Full Text PDF PubMed Google Scholar, 3Tse C.-M. Brant S.R. Walker M.S. Pouyssegur J. Donowitz M. J. Biol. Chem. 1992; 267: 9340-9346Abstract Full Text PDF PubMed Google Scholar, 4Wang Z. Orlowski J. Shull G.E. J. Biol. Chem. 1993; 268: 11925-11928Abstract Full Text PDF PubMed Google Scholar, 5Tse C.-M. Levine S.A. Yun C.H.C. Montrose M.H. Little P.J. Pouyssegur J. Donowitz M. J. Biol. Chem. 1993; 268: 11917-11924Abstract Full Text PDF PubMed Google Scholar, 6Numata M. Petrecca K. Lake N. Orlowski J. J. Biol. Chem. 1998; 273: 6951-6959Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 7Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. Shull G.E. Menon A.G. Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar). Full-length cDNAs encoding NHE1–4 (1Sardet C. Franchi A. Pouyssegur J. Cell. 1989; 86: 271-280Abstract Full Text PDF Scopus (668) Google Scholar, 2Orlowski J. Kandasamy R.A. Shull G.E. J. Biol. Chem. 1992; 267: 9331-9339Abstract Full Text PDF PubMed Google Scholar, 3Tse C.-M. Brant S.R. Walker M.S. Pouyssegur J. Donowitz M. J. Biol. Chem. 1992; 267: 9340-9346Abstract Full Text PDF PubMed Google Scholar, 4Wang Z. Orlowski J. Shull G.E. J. Biol. Chem. 1993; 268: 11925-11928Abstract Full Text PDF PubMed Google Scholar, 5Tse C.-M. Levine S.A. Yun C.H.C. Montrose M.H. Little P.J. Pouyssegur J. Donowitz M. J. Biol. Chem. 1993; 268: 11917-11924Abstract Full Text PDF PubMed Google Scholar) and NHE6 (6Numata M. Petrecca K. Lake N. Orlowski J. J. Biol. Chem. 1998; 273: 6951-6959Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar) have been characterized, thereby providing their amino acid sequences and allowing the development of expression constructs and isoform-specific nucleic acid and antibody probes. The use of these reagents has led to a substantial body of information about the expression patterns of these isoforms and their functional characteristics (reviewed in Refs.8Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Crossref PubMed Scopus (517) Google Scholar and 9Wakabayashi S. Shigekawa M. Pouyssegur J. Physiol. Rev. 1997; 77: 51-74Crossref PubMed Scopus (562) Google Scholar). In recent studies, mice carrying mutations in the genes encoding NHE1, -2, and -3 have been developed, and their phenotypes have been analyzed to determine their specific physiological functionsin vivo (10Cox 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 (236) Google Scholar, 11Schultheis 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, 12Schultheis 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). In contrast, the complete primary structure of NHE5 has not been determined, and nothing is known about its functional characteristics and physiological roles. The human NHE5 gene was identified several years ago by low stringency screening of a cosmid library using an NHE2 cDNA probe and mapped to chromosome 16q22.1 (7Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. Shull G.E. Menon A.G. Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar). Northern blot analysis demonstrated that NHE5 transcripts were expressed at easily detected levels in brain, spleen, and testis and at trace levels in skeletal muscle and revealed differences in the sizes of the transcripts in each tissue. Unlike the other isoforms, expression of NHE5 was not observed in epithelial tissues. Based on the partial coding sequences obtained and limited analysis of the structure of the gene, NHE5 appeared to be more closely related to the amiloride-resistant NHE3 isoform than to other members of the family. Its abundant expression in brain and apparent similarity to NHE3 raised the possibility that NHE5 might be the amiloride-resistant Na+/H+ exchanger that was described in hippocampal neurons (13Raley-Susman K.M. Cragoe Jr., E.J. Sapolsky R.M. Kopito R.R. J. Biol. Chem. 1991; 266: 2739-2745Abstract Full Text PDF PubMed Google Scholar), particularly since NHE3 expression is negligible in brain (2Orlowski J. Kandasamy R.A. Shull G.E. J. Biol. Chem. 1992; 267: 9331-9339Abstract Full Text PDF PubMed Google Scholar). To begin a systematic analysis of the functional characteristics and physiological roles of NHE5, we have cloned and functionally expressed a cDNA encoding the complete amino acid sequence of human NHE5, determined the intron-exon organization of its gene, and demonstrated that its mRNA is expressed in hippocampus and other structures of the brain. DISCUSSIONThe major objectives of this study were to determine the primary structure of human NHE5 and to explore the possibility that it corresponds to an amiloride-resistant Na+/H+exchanger that was identified previously in hippocampal neurons (13Raley-Susman K.M. Cragoe Jr., E.J. Sapolsky R.M. Kopito R.R. J. Biol. Chem. 1991; 266: 2739-2745Abstract Full Text PDF PubMed Google Scholar). The human NHE5 gene was identified several years ago and was shown to be expressed in brain, spleen, and testis (7Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. Shull G.E. Menon A.G. Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar). In the present study we were unable to isolate NHE5 cDNAs corresponding to fully processed transcripts in human spleen or testis; however, PCR analysis of brain cDNA did yield products that were correctly processed. These cloning experiments and the previous observation that NHE5 transcripts in human spleen and testis are larger than those in brain suggest that intron sequences, which disrupt the open reading frame, are retained in the spleen and testis mRNAs. While this study was in progress we had frequent discussions with Attaphitaya et al. (21Attaphitaya S. Park K. Melvin J.E. J. Biol. Chem. 1999; 274: 4383-4388Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), who were conducting similar studies of rat NHE5. When cloning rat NHE5 from brain, these investigators did not encounter processing intermediates or aberrantly spliced transcripts, and their Northern blot analyses revealed high levels of NHE5 mRNA in brain and only trace levels in spleen, testis, and other tissues. Thus, the results from both laboratories suggest that expression of functional NHE5 mRNA may be restricted to brain, although they do not rule out the possibility of expression in tissues and developmental stages that were not examined in these experiments.On the basis of the current and previous studies (7Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. Shull G.E. Menon A.G. Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar, 14Kandasamy R.A. Orlowski J. J. Biol. Chem. 1996; 271: 10551-10559Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), it is apparent that NHE5 and NHE3 are more closely related to each other, in both genomic organization and amino acid sequence, than to the other isoforms. As shown in Fig. 2, the location of exon boundaries relative to the coding sequences are almost identical in the NHE5 andnhe3 genes, and only the last intron of the nhe3gene lacks a counterpart in the NHE5 gene. In contrast, introns 2, 4, and 10–15 of the NHE5 and nhe3genes have no counterparts in the NHE1 gene (14Kandasamy R.A. Orlowski J. J. Biol. Chem. 1996; 271: 10551-10559Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 22Miller R.T. Counillon L. Pages G. Lifton R.P. Sardet C. Pouyssegur J. J. Biol. Chem. 1991; 266: 10813-10819Abstract Full Text PDF PubMed Google Scholar), and introns 8–11 of the NHE1 gene have no counterparts in theNHE5 and nhe3 genes. The complete genomic organization of the nhe2 and nhe4 genes, which are tightly linked in the mouse genome (23Pathak B.P. Shull G.E. Jenkins N.A. Copeland N.G. Genomics. 1996; 31: 261-263Crossref PubMed Scopus (17) Google Scholar), have not been determined. However, limited analyses of the mouse nhe2 andnhe4 genes (7Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. Shull G.E. Menon A.G. Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar) indicate that they are more closely related to NHE1 than to NHE5 and nhe3, a conclusion that is supported by comparisons of amino acid sequences (Table II and see Refs. 4Wang Z. Orlowski J. Shull G.E. J. Biol. Chem. 1993; 268: 11925-11928Abstract Full Text PDF PubMed Google Scholar, 5Tse C.-M. Levine S.A. Yun C.H.C. Montrose M.H. Little P.J. Pouyssegur J. Donowitz M. J. Biol. Chem. 1993; 268: 11917-11924Abstract Full Text PDF PubMed Google Scholar, and 7Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. Shull G.E. Menon A.G. Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar).An unusual feature of the NHE5 gene is the presence of GC dinucleotides, rather than GT dinucleotides, at the beginning of introns 13 and 14 (Table I). This departure from the consensus sequence is uncommon, but several dozen examples of GC dinucleotides at the beginning of an intron have been documented (24Jackson I.J. Nucleic Acids Res. 1991; 14: 3795-3798Crossref Scopus (278) Google Scholar). For example, the sequences, AAG/GCATGT and CAG/GCAAGC, at the donor sites of exons 13 and 14, respectively, are identical to donor sites observed in the RNA polymerase II (25Ahearn Jr., J.M. Bartolomei M.S. West M.L. Cisek L.J. Corden J.L. J. Biol. Chem. 1987; 262: 10695-10705Abstract Full Text PDF PubMed Google Scholar) and heme oxygenase (26Muller R.M. Taguchi H. Shibahara S. J. Biol. Chem. 1987; 262: 6795-6802Abstract Full Text PDF PubMed Google Scholar) genes, respectively.The data shown in Fig. 3 demonstrate that human NHE5 mediates H+-dependent Na+ influx and are consistent with the observation that rat NHE5 mediates Na+-dependent pH recovery from an acid load (21Attaphitaya S. Park K. Melvin J.E. J. Biol. Chem. 1999; 274: 4383-4388Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). 22Na+ influx in the clonal isolate shown in Fig. 3 was ∼0.2 pmol/min/mg protein. Under comparable assay conditions the average 22Na+ influx values for AP-1 cell isolates expressing NHE1, NHE2, and NHE3 was between 0.25 and 0.60 pmol/min/mg protein (17Orlowski J. J. Biol. Chem. 1993; 268: 16369-16377Abstract Full Text PDF PubMed Google Scholar, 20Yu F.H. Shull G.E. Orlowski J. J. Biol. Chem. 1993; 268: 25536-25541Abstract Full Text PDF PubMed Google Scholar), with activity in the order NHE1 > NHE2 > NHE3. Because the amount of NHE protein expressed in each of the clonal isolates that have been examined has not been determined, the relative activities of the various isoforms cannot be directly compared; however, the activity of the NHE5 isolate examined here is within the range of values observed for clonal isolates expressing NHE3. Rat NHE5 has been shown to be more resistant than NHE1 to 5-(N-ethyl-N-isopropyl)amiloride (21Attaphitaya S. Park K. Melvin J.E. J. Biol. Chem. 1999; 274: 4383-4388Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), and detailed pharmacological and biochemical analyses of human NHE5 that are currently in progress 2E. Szabo and J. Orlowski, unpublished observations. indicate that human NHE5 is also quite resistant to pharmacological antagonists, although not to as great an extent as NHE3. Thus, both human and rat NHE5 are, in fact, Na+/H+exchangers and have pharmacological characteristics that more closely resemble NHE3 than NHE1.When we began this study we were interested in determining whether NHE5 might be the transporter responsible for the amiloride-resistant Na+/H+ exchange activity identified in rat hippocampal neurons by Raley-Susman et al. (13Raley-Susman K.M. Cragoe Jr., E.J. Sapolsky R.M. Kopito R.R. J. Biol. Chem. 1991; 266: 2739-2745Abstract Full Text PDF PubMed Google Scholar). Our Northern blot data showing NHE5 expression in hippocampus of human brain and in situ hybridization analysis showing NHE5 expression in rat hippocampus (21Attaphitaya S. Park K. Melvin J.E. J. Biol. Chem. 1999; 274: 4383-4388Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) are consistent with this possibility. However, the Na+/H+ exchange activity observed in hippocampal neurons was completely insensitive to 1 mm amiloride, whereas human NHE5 expressed in AP-1 cells is fully inhibited by this concentration of amiloride (Fig. 3). This apparent difference in drug sensitivity could be due to the different conditions under which Na+/H+ exchange activity was measured in the two studies. For example, in the study of Na+/H+ exchange in hippocampal neurons (13Raley-Susman K.M. Cragoe Jr., E.J. Sapolsky R.M. Kopito R.R. J. Biol. Chem. 1991; 266: 2739-2745Abstract Full Text PDF PubMed Google Scholar), the concentration of extracellular Na+, which is a competitive inhibitor of amiloride binding, was 135 mm, whereas in our own studies only trace levels of22Na+ were present. NHE4 has been localized to cavi amnoni neurons in rat hippocampus (27Bookstein C. Musch M.W. DePaoli A. Xie Y. Rabenau K. Villereal M. Rao M.C. Chang E.B. Am. J. Physiol. 1996; 271: C1629-C1638Crossref PubMed Google Scholar), but this isoform also has been shown to exhibit some sensitivity to 1 mm amiloride. Additional studies will be needed to resolve the questions of which isoform(s) mediate control of intracellular pH in hippocampal neurons.In summary, we have cloned and expressed a fifth member of the plasma membrane Na+/H+ exchanger family and have obtained data indicating that functional expression of this isoform may be restricted to brain. Northern blot analysis showed that it is widely distributed in brain, with significant mRNA expression detected in hippocampus, amygdala, caudate nucleus, hypothalamus, subthalamic nucleus, and thalamus. The detection of NHE5 mRNA in multiple structures of the brain, but the absence of a significant hybridization signal in corpus callosum, which consists primarily of axons and glial cells and has few neuronal cell bodies, raises the possibility that NHE5 may be a neuron-specific isoform. If this proves to be the case, then this isoform would be the most highly cell type-specific member of the Na+/H+ exchanger family. The major objectives of this study were to determine the primary structure of human NHE5 and to explore the possibility that it corresponds to an amiloride-resistant Na+/H+exchanger that was identified previously in hippocampal neurons (13Raley-Susman K.M. Cragoe Jr., E.J. Sapolsky R.M. Kopito R.R. J. Biol. Chem. 1991; 266: 2739-2745Abstract Full Text PDF PubMed Google Scholar). The human NHE5 gene was identified several years ago and was shown to be expressed in brain, spleen, and testis (7Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. Shull G.E. Menon A.G. Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar). In the present study we were unable to isolate NHE5 cDNAs corresponding to fully processed transcripts in human spleen or testis; however, PCR analysis of brain cDNA did yield products that were correctly processed. These cloning experiments and the previous observation that NHE5 transcripts in human spleen and testis are larger than those in brain suggest that intron sequences, which disrupt the open reading frame, are retained in the spleen and testis mRNAs. While this study was in progress we had frequent discussions with Attaphitaya et al. (21Attaphitaya S. Park K. Melvin J.E. J. Biol. Chem. 1999; 274: 4383-4388Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), who were conducting similar studies of rat NHE5. When cloning rat NHE5 from brain, these investigators did not encounter processing intermediates or aberrantly spliced transcripts, and their Northern blot analyses revealed high levels of NHE5 mRNA in brain and only trace levels in spleen, testis, and other tissues. Thus, the results from both laboratories suggest that expression of functional NHE5 mRNA may be restricted to brain, although they do not rule out the possibility of expression in tissues and developmental stages that were not examined in these experiments. On the basis of the current and previous studies (7Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. Shull G.E. Menon A.G. Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar, 14Kandasamy R.A. Orlowski J. J. Biol. Chem. 1996; 271: 10551-10559Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), it is apparent that NHE5 and NHE3 are more closely related to each other, in both genomic organization and amino acid sequence, than to the other isoforms. As shown in Fig. 2, the location of exon boundaries relative to the coding sequences are almost identical in the NHE5 andnhe3 genes, and only the last intron of the nhe3gene lacks a counterpart in the NHE5 gene. In contrast, introns 2, 4, and 10–15 of the NHE5 and nhe3genes have no counterparts in the NHE1 gene (14Kandasamy R.A. Orlowski J. J. Biol. Chem. 1996; 271: 10551-10559Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 22Miller R.T. Counillon L. Pages G. Lifton R.P. Sardet C. Pouyssegur J. J. Biol. Chem. 1991; 266: 10813-10819Abstract Full Text PDF PubMed Google Scholar), and introns 8–11 of the NHE1 gene have no counterparts in theNHE5 and nhe3 genes. The complete genomic organization of the nhe2 and nhe4 genes, which are tightly linked in the mouse genome (23Pathak B.P. Shull G.E. Jenkins N.A. Copeland N.G. Genomics. 1996; 31: 261-263Crossref PubMed Scopus (17) Google Scholar), have not been determined. However, limited analyses of the mouse nhe2 andnhe4 genes (7Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. Shull G.E. Menon A.G. Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar) indicate that they are more closely related to NHE1 than to NHE5 and nhe3, a conclusion that is supported by comparisons of amino acid sequences (Table II and see Refs. 4Wang Z. Orlowski J. Shull G.E. J. Biol. Chem. 1993; 268: 11925-11928Abstract Full Text PDF PubMed Google Scholar, 5Tse C.-M. Levine S.A. Yun C.H.C. Montrose M.H. Little P.J. Pouyssegur J. Donowitz M. J. Biol. Chem. 1993; 268: 11917-11924Abstract Full Text PDF PubMed Google Scholar, and 7Klanke C.A. Su Y.R. Callen D.F. Wang Z. Meneton P. Baird N. Kandasamy R.A. Orlowski J. Otterud B.E. Leppert M. Shull G.E. Menon A.G. Genomics. 1995; 25: 615-622Crossref PubMed Scopus (141) Google Scholar). An unusual feature of the NHE5 gene is the presence of GC dinucleotides, rather than GT dinucleotides, at the beginning of introns 13 and 14 (Table I). This departure from the consensus sequence is uncommon, but several dozen examples of GC dinucleotides at the beginning of an intron have been documented (24Jackson I.J. Nucleic Acids Res. 1991; 14: 3795-3798Crossref Scopus (278) Google Scholar). For example, the sequences, AAG/GCATGT and CAG/GCAAGC, at the donor sites of exons 13 and 14, respectively, are identical to donor sites observed in the RNA polymerase II (25Ahearn Jr., J.M. Bartolomei M.S. West M.L. Cisek L.J. Corden J.L. J. Biol. Chem. 1987; 262: 10695-10705Abstract Full Text PDF PubMed Google Scholar) and heme oxygenase (26Muller R.M. Taguchi H. Shibahara S. J. Biol. Chem. 1987; 262: 6795-6802Abstract Full Text PDF PubMed Google Scholar) genes, respectively. The data shown in Fig. 3 demonstrate that human NHE5 mediates H+-dependent Na+ influx and are consistent with the observation that rat NHE5 mediates Na+-dependent pH recovery from an acid load (21Attaphitaya S. Park K. Melvin J.E. J. Biol. Chem. 1999; 274: 4383-4388Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). 22Na+ influx in the clonal isolate shown in Fig. 3 was ∼0.2 pmol/min/mg protein. Under comparable assay conditions the average 22Na+ influx values for AP-1 cell isolates expressing NHE1, NHE2, and NHE3 was between 0.25 and 0.60 pmol/min/mg protein (17Orlowski J. J. Biol. Chem. 1993; 268: 16369-16377Abstract Full Text PDF PubMed Google Scholar, 20Yu F.H. Shull G.E. Orlowski J. J. Biol. Chem. 1993; 268: 25536-25541Abstract Full Text PDF PubMed Google Scholar), with activity in the order NHE1 > NHE2 > NHE3. Because the amount of NHE protein expressed in each of the clonal isolates that have been examined has not been determined, the relative activities of the various isoforms cannot be directly compared; however, the activity of the NHE5 isolate examined here is within the range of values observed for clonal isolates expressing NHE3. Rat NHE5 has been shown to be more resistant than NHE1 to 5-(N-ethyl-N-isopropyl)amiloride (21Attaphitaya S. Park K. Melvin J.E. J. Biol. Chem. 1999; 274: 4383-4388Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), and detailed pharmacological and biochemical analyses of human NHE5 that are currently in progress 2E. Szabo and J. Orlowski, unpublished observations. indicate that human NHE5 is also quite resistant to pharmacological antagonists, although not to as great an extent as NHE3. Thus, both human and rat NHE5 are, in fact, Na+/H+exchangers and have pharmacological characteristics that more closely resemble NHE3 than NHE1. When we began this study we were interested in determining whether NHE5 might be the transporter responsible for the amiloride-resistant Na+/H+ exchange activity identified in rat hippocampal neurons by Raley-Susman et al. (13Raley-Susman K.M. Cragoe Jr., E.J. Sapolsky R.M. Kopito R.R. J. Biol. Chem. 1991; 266: 2739-2745Abstract Full Text PDF PubMed Google Scholar). Our Northern blot data showing NHE5 expression in hippocampus of human brain and in situ hybridization analysis showing NHE5 expression in rat hippocampus (21Attaphitaya S. Park K. Melvin J.E. J. Biol. Chem. 1999; 274: 4383-4388Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) are consistent with this possibility. However, the Na+/H+ exchange activity observed in hippocampal neurons was completely insensitive to 1 mm amiloride, whereas human NHE5 expressed in AP-1 cells is fully inhibited by this concentration of amiloride (Fig. 3). This apparent difference in drug sensitivity could be due to the different conditions under which Na+/H+ exchange activity was measured in the two studies. For example, in the study of Na+/H+ exchange in hippocampal neurons (13Raley-Susman K.M. Cragoe Jr., E.J. Sapolsky R.M. Kopito R.R. J. Biol. Chem. 1991; 266: 2739-2745Abstract Full Text PDF PubMed Google Scholar), the concentration of extracellular Na+, which is a competitive inhibitor of amiloride binding, was 135 mm, whereas in our own studies only trace levels of22Na+ were present. NHE4 has been localized to cavi amnoni neurons in rat hippocampus (27Bookstein C. Musch M.W. DePaoli A. Xie Y. Rabenau K. Villereal M. Rao M.C. Chang E.B. Am. J. Physiol. 1996; 271: C1629-C1638Crossref PubMed Google Scholar), but this isoform also has been shown to exhibit some sensitivity to 1 mm amiloride. Additional studies will be needed to resolve the questions of which isoform(s) mediate control of intracellular pH in hippocampal neurons. In summary, we have cloned and expressed a fifth member of the plasma membrane Na+/H+ exchanger family and have obtained data indicating that functional expression of this isoform may be restricted to brain. Northern blot analysis showed that it is widely distributed in brain, with significant mRNA expression detected in hippocampus, amygdala, caudate nucleus, hypothalamus, subthalamic nucleus, and thalamus. The detection of NHE5 mRNA in multiple structures of the brain, but the absence of a significant hybridization signal in corpus callosum, which consists primarily of axons and glial cells and has few neuronal cell bodies, raises the possibility that NHE5 may be a neuron-specific isoform. If this proves to be the case, then this isoform would be the most highly cell type-specific member of the Na+/H+ exchanger family. We thank James E. Melvin for critical review of the manuscript and for useful discussions during the course of this work.