The NHX Family of Na+-H+ Exchangers in Caenorhabditis elegans

Na+-H+ exchangers prevent cellular acidification by catalyzing the electroneutral exchange of extracellular sodium for an intracellular proton. To date, seven Na+-H+ exchangers have been identified in mammals, and although several members of this family have been extensively studied and characterized, it is clear that there are major gaps in our understanding with respect to the remaining family members. To initiate the study of Na+-H+ exchangers in a genomically defined and genetically tractable model system, we have cloned the complete cDNAs and analyzed splice site variation for nine putative homologs from the nematode Caenorhabditis elegans, which we have called NHX-1 through -9. The expression patterns and cellular distributions of the NHX proteins were determined using transcriptional and translational promoter-transgene fusion constructs to green fluorescent protein. Four of the putative exchangers were expressed at the cell surface, whereas five of the exchangers were associated with the membranes of intracellular organelles. Individual isoforms were expressed exclusively in the intestine, seam cells, hypodermal cells of the main body syncytium, and the excretory cell, all of which are polarized epithelial cells, suggesting a role for these proteins in epithelial membrane transport processes in the nematode. Other isoforms were found to express either ubiquitously or in a pan-neural pattern, suggesting a more conserved role in cell pH regulation or neuronal function. Finally, we show that recombinant NHX-4, the ubiquitous nematode Na+-H+ exchanger, mediates Na+-dependent pH recovery after intracellular acidification. NHX-4 has a K a for Na+of ∼32 mm, is not Cl−-dependent, and is relatively insensitive to the amiloride analog EIPA. Na+-H+ exchangers prevent cellular acidification by catalyzing the electroneutral exchange of extracellular sodium for an intracellular proton. To date, seven Na+-H+ exchangers have been identified in mammals, and although several members of this family have been extensively studied and characterized, it is clear that there are major gaps in our understanding with respect to the remaining family members. To initiate the study of Na+-H+ exchangers in a genomically defined and genetically tractable model system, we have cloned the complete cDNAs and analyzed splice site variation for nine putative homologs from the nematode Caenorhabditis elegans, which we have called NHX-1 through -9. The expression patterns and cellular distributions of the NHX proteins were determined using transcriptional and translational promoter-transgene fusion constructs to green fluorescent protein. Four of the putative exchangers were expressed at the cell surface, whereas five of the exchangers were associated with the membranes of intracellular organelles. Individual isoforms were expressed exclusively in the intestine, seam cells, hypodermal cells of the main body syncytium, and the excretory cell, all of which are polarized epithelial cells, suggesting a role for these proteins in epithelial membrane transport processes in the nematode. Other isoforms were found to express either ubiquitously or in a pan-neural pattern, suggesting a more conserved role in cell pH regulation or neuronal function. Finally, we show that recombinant NHX-4, the ubiquitous nematode Na+-H+ exchanger, mediates Na+-dependent pH recovery after intracellular acidification. NHX-4 has a K a for Na+of ∼32 mm, is not Cl−-dependent, and is relatively insensitive to the amiloride analog EIPA. electroneutral Na+-H+ exchanger kilobase(s) green fluorescent protein 2′,7′-bis(2-carboxyethyl)-4(6)-carboxyfluorescein 5-(N-ethyl-N-isopropyl)amiloride Na+-H+ exchangers are a family of integral membrane phosphoglycoproteins that play an important role in the regulation of intracellular pH and sodium homeostasis by mediating the countertransport of extracellular sodium and intracellular protons (for review, see Refs. 1Wakabayashi S. Shigekawa M. Pouyssegur J. Physiol. Rev. 1997; 77: 51-74Crossref PubMed Scopus (563) Google Scholar and 2Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar). Although Na+-H+exchange activity is present in organisms ranging from bacteria, yeast, and plants to animals, the functional basis for these activities is in some cases quite different. Bacteria exhibit electrogenic Na+-H+ exchange, with a stoichiometry of 1Na/2H; as a result of the net uptake of protons for net loss of sodium, the organism is able to live in extreme hypersaline or hyperalkaline environments (3Taglicht D. Padan E. Schuldiner S. J. Biol. Chem. 1993; 268: 5382-5387Abstract Full Text PDF PubMed Google Scholar). The bacterial Na+-H+ exchange proteins are generally around 332 amino acids in length and contain 10 transmembrane domains (4Waser M. Hess-Bienz D. Davies K. Solioz M. J. Biol. Chem. 1992; 267: 5396-5400Abstract Full Text PDF PubMed Google Scholar, 5Karpel R. Olami Y. Taglicht D. Schuldiner S. Padan E. J. Biol. Chem. 1988; 263: 10408-10414Abstract Full Text PDF PubMed Google Scholar, 6Goldberg E.B. Arbel T. Chen J. Karpel R. Mackie G.A. Schuldiner S. Padan E. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2615-2619Crossref PubMed Scopus (183) Google Scholar). In yeast, the plasma membrane antiporters are variable in size from 450 to more than 900 amino acids, depending upon the species, and can mediate potassium as well as sodium efflux (7Jia Z.P. McCullough N. Martel R. Hemmingsen S. Young P.G. EMBO J. 1992; 11: 1631-1640Crossref PubMed Scopus (158) Google Scholar, 8Prior C. Potier S. Souciet J.L. Sychrova H. FEBS Lett. 1996; 387: 89-93Crossref PubMed Scopus (149) Google Scholar, 9Banuelos M.A. Sychrova H. Bleykasten-Grosshans C. Souciet J.L. Potier S. Microbiology. 1998; 144: 2749-2758Crossref PubMed Scopus (200) Google Scholar); in addition, an intracellular Na+-H+ exchanger in yeast is associated with a late endosomal compartment and functions in protein trafficking and osmotolerance (10Nass R. Cunningham K.W. Rao R. J. Biol. Chem. 1997; 272: 26145-26152Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 11Nass R. Rao R. J. Biol. Chem. 1998; 273: 21054-21060Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 12Bowers K. Levi B.P. Patel F.I. Stevens T.H. Mol. Biol. Cell. 2000; 11: 4277-4294Crossref PubMed Scopus (151) Google Scholar). In plants, salt tolerance can be conferred by overexpression of a vacuolar Na+-H+ antiporter with great similarity to that found in yeast (13Apse M.P. Aharon G.S. Snedden W.A. Blumwald E. Science. 1999; 285: 1256-1258Crossref PubMed Scopus (1584) Google Scholar). Finally, the single Na+-H+exchanger that has been characterized from invertebrates shows remarkable similarity to the mammalian Na+-H+exchangers; however, the stoichiometry of exchange is 2Na/1H (for review, see Ref. 14Ahearn G.A. Mandal P.K. Mandal A. J. Exp. Zool. 2001; 289: 232-244Crossref PubMed Scopus (40) Google Scholar). In mammals, seven members of the electroneutral Na+-H+ exchanger (NHE)1 family have been identified to date (15Numata M. Orlowski J. J. Biol. 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These exchangers are typically from 600 to 700 amino acids in length, contain 10–12 predicted transmembrane domains, and have a large, exposed cytoplasmic carboxyl terminus. NHE1 is expressed ubiquitously and is believed to play a housekeeping role in establishing cytosolic pH (22Noel J. Pouyssegur J. Am. J. Physiol. Cell Physiol. 1995; 268: 283-296Crossref PubMed Google Scholar), whereas NHE2, -3, -4, and -5 display more restricted tissue distributions, perhaps reflecting more specialized functions (19Wang Z. Orlowski J. Shull G.E. J. Biol. Chem. 1993; 268: 11925-11928Abstract Full Text PDF PubMed Google Scholar, 23Tse C.M. Levine S.A. Yun C.H. Montrose M.H. Little P.J. Pouyssegur J. Donowitz M. J. Biol. Chem. 1993; 268: 11917-11924Abstract Full Text PDF PubMed Google Scholar). NHE6 and NHE7 are expressed ubiquitously and appear to be intracellular exchangers (15Numata M. Orlowski J. J. Biol. Chem. 2001; 276: 17387-17394Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar,18Numata M. Petrecca K. Lake N. Orlowski J. J. Biol. Chem. 1998; 273: 6951-6959Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Insights into the function of the NHE exchanger family have come lately from gene ablation studies on mouse isoforms 1, 2, and 3. Targeted disruption of the ubiquitously expressed NHE1 was expected to be lethal; however, null mutant mice were not only viable but exhibited only a slightly retarded postnatal growth rate (24Bell 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. Cell Physiol. 1999; 276: 788-795Crossref PubMed Google Scholar). Because NHE1 is generally expressed at low levels in the brain, it was surprising to find that NHE1-null mice exhibited ataxia and epileptic-like seizures. Consistent with NHE1 playing a “housekeeping” role in maintaining intracellular and and cells from mice appear to the to from an intracellular (24Bell 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. Cell Physiol. 1999; 276: 788-795Crossref PubMed Google Scholar, Shull G.E. Melvin J.E. J. Physiol. 2000; PubMed Scopus Google Scholar, G.A. C.M. D. A. J.L. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, R.L. S.M. Schultheis P.J. Shull G.E. Melvin J.E. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). of NHE1 in a in with a role in exchangers by the K. Evans R.L. G.E. K. S.M. Schultheis P.J. Shull G.E. Melvin J.E. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). of to in the of the as well as a in net in P.J. M. T. Miller M.L. Shull G.E. J. 1998; PubMed Scopus Google Scholar). is that the of loss are to of cells than the to of to and as a and as well as a to and in the P.J. Miller M.L. M. T. T. Shull G.E. 1998; PubMed Scopus Google Scholar). in by of the and levels of were to for loss of of was in that the levels of exchanger and sodium as well as were in null These that the proteins and processes in in the and are and that a major role in sodium homeostasis in these although a great is the function of several Na+-H+ exchanger isoforms in mammals, there are major gaps in our understanding of the remaining family members. In addition, the of after mammalian gene ablation some in membrane transport processes with the null To our understanding of Na+-H+ isoforms epithelial membrane transport processes and to maintaining cellular and we have to as a model nematode is to in there only cells in the the that are conserved in on a of the transport in are in with a found family suggesting that the functions of nematode cells are conserved as of the of with C. that it is a genomically defined and genetically tractable for expression is relatively gene a of To an understanding of in the we have cloned nine cDNAs the Na+-H+ exchanger family from C. of these to the seven mammalian NHE exchangers to that conserved functions at the cellular a the role of these putative exchangers in C. elegans, we have the expression patterns of the nine exchangers using translational to have protein to cellular and functional of the ubiquitously expressed NHX-4 protein that members of this family can function as Na+-H+ exchangers and some of the of the transport of and the C. with NHE1 nine putative Na+-H+ exchanger cDNAs several with to E. were not of predicted cDNAs was to upon conserved domains the amino of of the were by using these an and a by R. of were as K. T. J. Melvin J.E. Am. J. Physiol. Cell Physiol. 2000; Google Scholar). for of the NHE homologs were at the of was from of using the as an in with of was to the and was on to splice site and the cDNAs were to and using the gene family as by the C. These are found the to were by of of from of the for using that a to to prevent translational The were cloned into the of the of of which is a of of A. of to is were but a that the than and the was cloned that be with the NHX Because of size and the and translational fusion constructs only of of the were at or on nematode growth with or from an using fusion and which a Park Mol. Cell. Biol. PubMed Scopus Google Scholar), were at in into the of as D. EMBO J. PubMed Scopus Google Scholar). were from at 10 to to for The were on using a with a or and a or as The were using a and analyzed in were using a and a The for was from a and into the The expression and the expression of were at a into the Na+-H+ cell of of using after the cells were and 10 cells were with the fluorescent pH at for in of a low potassium 10 the pH was to with The were to a on the and with Individual cells were identified by with to a with a was to pH The fluorescent were at after at and was by the A. Physiol. Rev. PubMed Scopus Google Scholar). in the was by and after several of the cells were to a in of upon sodium was by using analyzed using The fluorescent was to by in using the In all cells were with cells, which exhibited cells were To in the was with the and to and in the were with potassium and sodium cDNAs nine proteins with to the mammalian NHE family of Na+-H+ which we have called NHX-1 through the gene has been with the Caenorhabditis In predicted NHX protein was from 600 to amino acids in length, with the of which was the of the at amino acids, and protein was predicted to contain from 10 to transmembrane domains with a exposed as is found in all members of the mammalian NHE of the and in the in A. were identified for of the NHX-4 was in that splice were by and of in and exhibited splice variation and was the only nematode Na+-H+ exchanger that a variable and a from an and an with suggesting that it an whereas the an that an to be the result of from was not by an in an the and that the of the putative is in the after the typically not the this upon the seven mammalian NHE proteins can be into The of these the plasma membrane exchangers NHE1 through whereas the the intracellular transport proteins NHE6 and the predicted proteins NHX-1 through with -3, and and Na+-H+exchanger proteins and the to a family members was generally and and from to the conserved the family and with the mammalian Na+-H+ with several of exchangers levels of (for as see and or in the of the mammalian in mammals, the C. appear to be into at with -3, and on and NHX-4, and on the and of the appear to be to the intracellular exchangers NHE6 and is important to however, that is not the of and for the from promoter-transgene fusion constructs by a from of the putative for NHX family In the was by that it not be were identified upon of a the which in a Park Mol. Cell. Biol. PubMed Scopus Google Scholar). in the of these is that the are as and that are for these be an of the gene family cell expression patterns by the of or in In addition, the loss of the cell a as not exhibit the of cells that are to express the the in and of from at of translational fusion the and of gene was to The of fusion protein was determined by either or fluorescent are to the and a a translational fusion protein in a of and hypodermal cells as well as in the The fusion protein an intracellular of and is to the of cell as by The in the is of using a is these and is on the of the is expressed through the intestine, but the of is at the and of the and in the the expression is relatively and is expressed ubiquitously and is to the plasma membrane of K is expressed in the hypodermal cells of the main body as well as the cells of the and the cell of of the protein to in a that is the hypodermal cells and and is a pan-neural Na+-H+ exchanger that as in this fluorescent of the from is expressed in the seam cells and and the protein to in a expression of and is to an intracellular as in this through the excretory cell is expressed processes of the excretory cell in of but the intracellular more restricted and in the expression was by the of the or was found exclusively in the of the The was in and however, the was an as was in and animals, with not The and were in the with a the and In the in and exclusively in the cells of the expression was restricted to a single cell or cell in a of for and expression in the hypodermal cells of the main body 2, whereas expression from the in exclusively in the hypodermal seam cells 2, and in the of the excretory cell, which as the nematode of the and Although a for the to the pan-neural expression of and suggesting that this play a role in cell function. In several of cells was as in by and large, the of cell expression patterns that the Na+-H+ exchanger isoforms specialized functions for cell the the from the gene the expression of in a of cells, in a 2, and Although the expression of the cells, was in in cell not NHX-4 the housekeeping Na+-H+ to the intracellular of in as to the function of Na+-H+ exchangers can be from for mammalian an important role in and and is found associated with the membrane in the and constructs were by the of exchanger with expression of these was by shows the fluorescent and from these In the expression patterns found for the translational fusion constructs were to for the transcriptional were however, as we these be to in the or of the gene or to the of an be the size of some of the found in these and the that were identified for of these the site was in the transcriptional fusion The in the expression of which was to be restricted to The translational fusion was expressed in a hypodermal and cells the length of the nematode and was in cells and and with and the fusion protein to be a that the of the cell the of this as well as we not to of intracellular but only determined the or at plasma that the of in the NHX-1 from an the as a translational fusion to we were able to from the site as well as that to the of a protein. The site for NHX-1 which an upon our is and the exchanger patterns of expression that that expression was restricted to the membrane of the and whereas expression was found on the membrane and was in that the of this protein to a membrane to be and that the and of the exhibited whereas the in the to contain at the the the of a the remaining only was to the plasma which was of was in all cells and cell and of the and and in the hypodermal cells, in associated with the and In the of can be to in a at the cell membrane of body with the of the to some of to a in the perhaps with cells in cells, the protein is the with the cell of and through the neuronal processes not expressed in polarized epithelial cells, to be to the membrane In this NHX-4 to be to the ubiquitous mammalian Na+-H+ and on the to be associated with intracellular membranes expression was in translational and transcriptional and in the hypodermal cells of the main body in addition, in the cells in the to the were K and as were the cells K The cells are a cell to the In addition, was in cells and the excretory cell, specialized that the not The fluorescent expression the cells these were to be with In to the or these hypodermal cells function as a major for and the be of that are to hypodermal cells, or which play in the functions of of or the of expression in neuronal cell with cells in the and and the in the and in the and the and of the in an was as were the cells of the and the excretory cell not see for transcriptional was in the and through the neuronal cell was expressed as expected in the seam cells but was expressed in the the and cells, and in cells of the and in these cells have been a result of or in the of the gene that are not present in the transcriptional fusion the in the translational that expression in these The protein was in the seam cells in a that the as in the with some by fluorescent hypodermal Finally, the fusion protein was to the excretory cell, but the was in the of the cell the protein in and using that these and were not associated with the plasma In the excretory cell processes were the whereas at the more in a on a as by the in that from the from the cell of the and as fusion proteins from translational that the found of the splice for these cDNAs are not the major and cell in which is expressed is in is not to be but only to as a is a of a nematode using to in the cells it is In addition, the distributions of and are by on the or of the In that the cells of the express and on the cell In to is expressed in the excretory cell to size and the of NHX-4 is as by the green To display expression from cells that in a the seam cells, hypodermal cells, and the excretory a of a nematode is in the In this NHX-1 is as expressed in the body it is the only the ubiquitous NHX-4 that we found we that the expression of NHX-1 is more and we that there are single cells, as the cell, cells, and the that express or and are not To the for functional Na+-H+ the nematode family members were expressed in the Na+-H+ cell and recovery from an intracellular was using the fluorescent Although of the exchangers to mediate recovery in this not cells the ubiquitous exhibited Na+-dependent pH recovery after acidification recovery was in cells The recovery was not on extracellular and the K a for was ∼32 In addition, activity was relatively insensitive to the amiloride analog and a of than from the NHX-4 not function in the recombinant expression not of the recombinant protein that the protein to it to the plasma with the of a on the splice not is that the plasma membrane isoforms and to be in a of these isoforms an to function The in this study on a nematode family of Na+-H+ exchangers that in the of have cloned nine cDNAs from the nematode C. that exhibit to the mammalian family of Na+-H+ These proteins have been NHX-1 through The that recombinant can mediate Na+-dependent recovery from an intracellular that the similarity the nematode NHX family and the mammalian NHE family at the functional as well as amino of the NHX proteins -4, and are to the plasma the that the mammalian exchanger can in intracellular that to the plasma membrane upon cell S. A. K. K. Orlowski J. Grinstein S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google that intracellular isoforms have the to mediate plasma membrane In addition, the of a relatively of putative intracellular in C. with to with a to the and that have been in to the role that these proteins play in and function. was that of the nematode isoforms were expressed exclusively in a single cell or cell that the of be to the functions that are to that by cell we into the NHX proteins the expression of in cell in the nematode and at the membrane that this be for pH the expression of that it to the function of the excretory cell, which is to the in ablation of the excretory cell to and J. Exp. Zool. PubMed Scopus Google Scholar), with a role in regulation of the a intracellular exchanger be in this is an to these of is to the and function by of processes or The of this was quite by some by in the of of the C. is by an which in the M.A. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). for that the body the in the of a mutant of and E. The restricted of at the of cells in the and this Na+-H+ exchanger it with of the on These a role for Na+-H+ exchange in have to be a model for the study of In to genomically defined and genetically studies are the and of cell are and only are and as gene to a null in the of the of of S. D. J.E. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J.E. 1998; PubMed Scopus Google Scholar), it be to study pH regulation by the exchangers in an nematode. The of the cellular and intracellular expression patterns for of the NHX proteins is an important the and that we have in this we can to regulation and at single cell in this model and for and for a of this