The NHX Family of Na+-H+ Exchangers in Caenorhabditis elegans
2002; Elsevier BV; Volume: 277; Issue: 32 Linguagem: Inglês
10.1074/jbc.m203200200
ISSN1083-351X
AutoresKeith Nehrke, James E. Melvin,
Tópico(s)Planarian Biology and Electrostimulation
ResumoNa+-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. Chem. 2001; 276: 17387-17394Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 16Attaphitaya S. Park K. Melvin J.E. J. Biol. Chem. 1999; 274: 4383-4388Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 17Baird 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 (142) 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, 19Wang Z. Orlowski J. Shull G.E. J. Biol. Chem. 1993; 268: 11925-11928Abstract Full Text PDF PubMed Google Scholar, 20Orlowski J. Kandasamy R.A. Shull G.E. J. Biol. Chem. 1992; 267: 9331-9339Abstract Full Text PDF PubMed Google Scholar, 21Sardet C. Franchi A. Pouyssegur J. Cell. 1989; 56: 271-280Abstract Full Text PDF PubMed Scopus (671) Google Scholar). 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 pH, fibroblasts and lacrimal and salivary acinar cells from NHE1−/− mice appear to lack the means to recover effectively from an intracellular acid load (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, 25Nguyen H.V. Shull G.E. Melvin J.E. J. Physiol. 2000; 523: 139-146Crossref PubMed Scopus (30) Google Scholar, 26Cox 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 (239) Google Scholar, 27Evans R.L. Bell S.M. Schultheis P.J. Shull G.E. Melvin J.E. J. Biol. Chem. 1999; 274: 29025-29030Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Indeed, lack of NHE1 resulted in a dramatic reduction in salivation, consistent with a role in promoting chloride flux via functionally coupled Cl−-HCO3− exchangers by increasing the pHi during sustained secretion (28Park K. Evans R.L. Watson G.E. Nehrke K. Richardson L. Bell S.M. Schultheis P.J. Hand A.R. Shull G.E. Melvin J.E. J. Biol. Chem. 2001; 276: 27042-27050Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Loss of NHE2 led to structural abnormalities in the oxyntic mucosa of the gastric corpus as well as a reduction in net gastric acid secretions in adults (29Schultheis 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 (222) Google Scholar). It is thought that the phenotypic effects of NHE2 loss are related to survival of parietal cells rather than the ability to secrete acid per se. Loss of NHE3 led to renal and intestinal absorptive defects, such as a mild tubular acidosis and slight diarrhea as well as a decreased ability to absorb fluid and HCO3− in the proximal convoluted tubules (30Schultheis 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 (703) Google Scholar). A reduction in blood pressure, increased absorptive area provided by swelling of the cecum, and elevated levels of blood aldosterone were postulated to compensate partially for loss of NHE3. Further evidence of compensation was noted in that the levels of kidney renin, AE1 (anion exchanger type 1), and ENaC (epithileal sodium channel) mRNA as well as other mRNAs were altered substantially in homozygous null mice. These results suggest that the proteins and processes involved in Na+ reabsorption in the kidney and intestine are tightly coupled functionally and that NHE3 plays a major role in promoting sodium homeostasis in these tissues. Thus, although a great deal is known about 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 advent of compensatory mechanisms after mammalian gene ablation creates some difficulties in correlating physiological membrane transport processes with the existing null phenotypes. To broaden our understanding of how individual Na+-H+ isoforms enable epithelial membrane transport processes and contribute to maintaining cellular Na+, pH, and fluid homeostasis, we have chosen to studyCaenorhabditis elegans as a model system. This soil nematode is similar to mammals in many ways. Despite there being only roughly 1000 cells in the adult hermaphrodite, the worm possesses multiple distinct tissues that are conserved in mammals. Indeed, on a genomic level, many of the ion transport families in mammals are reflected in worms, with a similar diversity found among family members, suggesting that the basic physiological functions of nematode cells are conserved as well. One of the many benefits of working with C. elegansis that it is a genomically defined and genetically tractable model. Obtaining transgenic lines for expression analysis is relatively straightforward. Similarly, double-stranded RNA-mediated gene interference (RNAi) allows a rapid means of pheno-copying loss-of-function alleles. To gain an understanding of acid-base physiology in the nematode, we have cloned nine cDNAs representing the entire Na+-H+ exchanger family from C. elegans. A comparison of these sequences to the seven highly related mammalian NHE exchangers leads us to believe that they may possess evolutionarily conserved functions at the cellular level. As a first step toward elucidating the role of these putative exchangers in C. elegans, we have investigated the expression patterns of the nine exchangers and, using translational fusions to GFP, have examined protein targeting to cellular and organelle membranes. A functional characterization of the ubiquitously expressed NHX-4 protein confirms that members of this family can function as Na+-H+ exchangers and reveals some of the basic physiological characteristics of the transport process. Basic local alignment search tool (BLAST) homology searches of WormPep (a predictive data base) and the C. elegans genome with NHE1 yielded nine putative Na+-H+ exchanger cDNAs (B0395.1, B0495.4, C54F6.13, F14B8.1, F57C7.2, F58E1.6, K09C8.1, Y18D10A.6, ZK822.3); several other clones with homology to E. coli napA were not studied. A multiple sequence alignment of conceptually translated predicted cDNAs was used to design probes based upon conserved domains within the amino terminus of each of the proteins. Isoform-specific probes were generated by reverse-PCR using mixed stageC. elegans 1st strand cDNA. Using these probes, twoC. elegans cDNA libraries, an oligo(dT)-primed cDNA library (λ-ACT-RB1) and a random-primed cDNA library (λ-ACT-RB2) (kindly provided by Dr. R. Barstead, University of Wisconsin-Madison), were screened as described previously (31Nehrke K. Begenisich T. Pilato J. Melvin J.E. Am. J. Physiol. Cell Physiol. 2000; 279: 2052-2066Crossref Google Scholar). Multiple clones for 8 of the 9 NHE homologs were obtained. Additional sequence information at the 5′ end of each clone was derived from 5′-rapid amplification of cDNA ends using the SL1 trans-spliced leader sequence as an anchored primer in combination with nested isoform-specific primers. For NHX-6 mRNA, 3′-rapid amplification of cDNA ends analysis was used to establish the distal coding region and 3′-untranslated region. Each cDNA clone was sequenced fully on both strands to assess splice site variation, and the full-length cDNAs were submitted to GenBankTM and WormBase using the gene family name nhx as assigned by the C. elegans Genomics Center. These sequences are found under the accession numbers AF497823-AF497835. Transcriptional promoter fusions to GFP were created by PCR amplification of 4 kb of genomic sequence from upstream of the first start codon for each isoform using restriction site-tagged oligonucleotide primers that contained a mutated start codon complement (ATG to TTG) to prevent translational initiation. The PCR products were cloned into the complementary sites of the vector pFH6.II (courtesy of F. Hagen, University of Rochester, Rochester, NY), which is a derivative of pPD 95.81 (courtesy of A. Fire, Carnegie Institute of Washington, Baltimore, MD) to create pJP109-nhx-N (where N is 1–9). Translational promoter fusions (pJP113-nhx-N) were created similarly but instead used a downstream genomic primer that annealed immediately before the stop codon (rather than start codon), and the PCR product was cloned so that GFP would be translated in-frame with the full NHX protein-coding region. Because of size limitations and stability issues, the NHX-5, -7, and -8::GFP translational fusion constructs contained only 1.5 kb of promoter sequence upstream of the initiator codon. Nematodes (Bristol N2 strain) were cultured at 14 or 22 °C on nematode growth medium plates seeded with HB101 or OP50 bacteria from an overnight culture using standard techniques. GFP fusion construct and pRF4, which produces a rol-6 roller phenotype (32Kramer J.M. French R.P. Park E.C. Johnson J.J. Mol. Cell. Biol. 1990; 10: 2081-2089Crossref PubMed Scopus (262) Google Scholar), were mixed at 75 mg/ml each in injection buffer (composition) then co-injected into the gonad of young adult Bristol-N2 nematodes as described (33Mello C.C. Kramer J.M. Stinchcomb D. Ambros V. EMBO J. 1991; 10: 3959-3970Crossref PubMed Scopus (2443) Google Scholar). After 4 days, rollers were picked from at least 10 injections to separate plates to look for germ-line transmission. The nematodes were imaged on 2% M9-agarose pads using a Nikon Eclipse E800 microscope equipped with a Nikon 10, 20, 60, or 100× Apo series objectives under 100-W mercury illumination and a GFP or 4,6-diamidino-2-phenylindole filter set, as appropriate. The images were captured using a Spot2 camera and analyzed in Adobe Photoshop (Adobe Systems, San Jose, CA). Alternatively, images were captured using a Leica confocal microscope under 488 nm laser illumination and a fluorescein isothiocyanate filter set. The full coding region for NHX-4a was amplified from a λ cDNA clone and ligated into the vector pcDNA3.1/V5/His/topo (Invitrogen). The NHX-4a expression vector pPM1 and the CD8 expression plasmid pID3-CD8 (courtesy of Dr. Brian Seed, Harvard University) were transiently transfected at a 10:1 ratio into the Na+-H+ exchanger-deficient cell line AP1 (courtesy of Dr. Sergio Grinstein, University of Toronto) using Superfect reagent (Qiagen, Valencia, CA). Immediately after transfection, the cells were trypsinized and plated onto 10 mm glass coverslips. Twenty-four hours later, cells were loaded with the fluorescent pH indicator BCECF (2 μm) at room temperature for 30 min in 1 ml of a normal, low potassium buffer (135 mm NaCl, 5.4 mm KCl, 0.4 mmKH2PO4, 0.33 mmNaH2PO4, 10 mm glucose, 20 mm HEPES, 1.2 mm CaCl2, 0.8 mm MgSO4; the pH was adjusted to 7.4 with Tris base. The coverslips were then moved to a perfusion chamber mounted on the microscope and superfused with normal buffer. Individual CD8+ cells were identified by incubating with anti-CD8 antibody-coated Dynabeads M-450 (Dynal, Oslo, Norway). A dual wavelength excitation system (Axon Instruments, Foster City, CA) rigged to a Nikon inverted microscope equipped with a CCD camera detection system (Cooke, Germany) was used to monitor pH changes. The fluorescent emissions were measured at 530 nm after excitation at 440 and 490 nm. An acute acid load was induced by the NH4+/NH3 prepulse technique (34Roos A. Boron W.F. Physiol. Rev. 1981; 61: 296-434Crossref PubMed Scopus (2288) Google Scholar). Briefly, 60 mm NaCl in the above normal solution was replaced by NH4Cl, and after several minutes of perfusion, the cells were then switched to a solution containing 135 mmn-methyl-d-glucamine-HCl in place of sodium. Recovery upon sodium readdition was monitored by image acquisition using AXON Image2.1 software, then analyzed using Microcal Origin 6.0. The derivative fluorescent ratio was converted to pHi by in situ calibration using the high K+/nigericin technique. In all experiments, CD8+ cells were matched with non-transfected cells, which never exhibited pHi recovery. Approximately 8–10 CD8+ cells were imaged per coverslip. To address Na+ dependence, NaCl in the above normal buffer was replaced with the appropriate amount ofn-methyl-d-glucamine-HCl, and to address Cl− dependence, KCl and NaCl in the above normal solution were replaced with potassium and sodium gluconate, respectively. We isolated cDNAs encoding nine different proteins with homology to the mammalian NHE family of Na+-H+ exchangers, which we have called NHX-1 through NHX-9; the nhx gene designation has been registered with the Caenorhabditis Genetics Center. In general, each predicted NHX protein was from 600 to 750 amino acids in length, with the exception of NHX-1, which was the smallest member of the family, at 480 amino acids, and each protein was predicted to contain from 10 to 12 transmembrane domains with a large exposed C terminus, as is found in all members of the mammalian NHE family. An alignment of the cDNA and corresponding genomic sequences resulted in the schematic shown in Fig.1 A. Multiple variants were identified for four of the isoforms. NHX-4 mRNA was unique in that two distinct SL1 splice sites were used, separated by three exons and ∼3 kb of genomic sequence, resulting in NHX-4a and NHX-4b mRNA variants (Fig. 1 A). NHX-5 mRNA also exhibited splice variation and was the only nematode Na+-H+ exchanger that had a variable C terminus (NHX-5a and NHX-5b). NHX-8a mRNA contained a coding sequence derived from an additional upstream exon and lacked an SL1 leader compared with NHX-8b mRNA, suggesting that it may represent an unprocessed mRNA, whereas the NHX-9a mRNA variant contained an alternate exon 1 that had an SL1 trans-spliced leader (Fig. 1) and, thus, appeared to be the result of true alternative splicing. However, exon1 from NHX-9b mRNA was not preceded by an SL1 leader. And in an interesting twist, both the NHX-8b and NHX-9b mRNA variants contained out-of-frame ATGs that preceded the start of the putative open reading frames. This is unusual in worms, where the first ATG after the SL1 leader typically initiates translation. We do not know the reason behind this anomaly. Based upon homology, the seven known mammalian NHE proteins can be grouped into two distinct branches. The first of these contains the plasma membrane exchangers NHE1 through NHE5, whereas the second contains the intracellular ion transport proteins NHE6 and NHE7. We aligned the predicted proteins NHX-1 through NHX-9 with human (NHE1, -2, -3, -5, -6, and -7) and rat (NHE4) Na+-H+exchanger proteins and used the resulting alignment to create a phylogram showing relatedness among family members (Fig.1 B). There was generally between 20 and 40% identity and from 40 to 60% homology across the core conserved region both within the family and when compared with the mammalian Na+-H+ exchangers, with several pairs of exchangers exhibiting higher levels of homology (for example, as mentioned above, see NHX-2 versus NHX-6 and NHX-3versus NHX-9, or in the case of the mammalian exchangers, NHE3 versus NHE5). As in mammals, the C. elegansexchangers appear to be grouped into at least two distinct branches, with NHX-1, -2, -3, -6, and -9 on one branch and NHX-4, -5, -7, and -8 on the other; NHX-5 and -8 of the latter branch appear to be closely related to the human intracellular exchangers NHE6 and NHE7. It is important to note, however, that NHX-3 is not the worm ortholog of NHE3, and so on, for the entire family. We first created transgenic animals expressing GFP from promoter-transgene reporter fusion constructs driven by a 4-kb genomic fragment from upstream of the first putative start codon for each NHX family mRNA (see Fig. 1). In each case, the start codon was modified by mutagenesis such that it would not be utilized. Transgenic animals were identified based upon co-injection of a plasmid encoding the rol-6 marker, which results in a roller phenotype (32Kramer J.M. French R.P. Park E.C. Johnson J.J. Mol. Cell. Biol. 1990; 10: 2081-2089Crossref PubMed Scopus (262) Google Scholar). One caveat in the interpretation of these results is that the injected plasmids are inherited as non-physiological multi-copy extra-chromosomal arrays and may lack sequences that are necessary for cell-specific regulation; thus, these results should be considered an approximation of the nhx gene family cell expression patterns until verified by the use of specific antibodies or in situ analysis. In addition, the loss of the array during cell division produces a phenomenon known as mosaicism; individual animals may not exhibit the full repertoire of cells that are known to express the transgene. Thus, the results shown in Figs.2 and 3represent images of multiple animals taken from at least three separate transgenic lines.Figure 3Fluorescent profiles of transgenic nematodes expressing NHX:: GFP translational fusion proteins. A genomic fragment containing the promoter and entire open reading frame of each nhx gene was fused in-frame to GFP (see “Experimental Procedures”). The subcellular location of each NHX::GFP fusion protein was determined by either conventional or confocal fluorescent microscopy. Fluorescent images are shown next to the corresponding differential interference contrast photomicrographs. A and B, a transgenic line expressing a translational NHX-1::GFP fusion protein fluoresced in a wide variety of tissues, including muscle and hypodermal cells as well as in the pharynx. The fusion protein had an intracellular distribution (inset of panel A).C and D, NHX-2::GFP is targeted to the luminal surface of intestinal cell membranes, as demonstrated by confocal microscopy. The granular staining in the intestine is comprised of auto-fluorescent granules; using a system where color resolution is available, these granules will fluoresce yellow (see below).E and F, NHX-7:GFP is located on the basolateral surface of the posterior intestinal cells. G andH, NHX-6::GFP is expressed through the entire intestine, but the targeting of NHX-6::GFP is basolateral at the posterior and anterior ends of the intestine and apical in the middle, where the expression level is relatively weak. I and J, NHX-4::GFP is expressed ubiquitously and is localized to the plasma membrane (inset of panel I). K andL, NHX-3::GFP is expressed in the hypodermal cells of the main body syncitium as well as the ut1 cells of the vulva and the spermathecal junction cell (inset of panel K). Expression of the NHX-3::GFP protein appears to occur in a punctate pattern that is evenly distributed throughout the hypodermal cells (panels M and N). O and P, NHX-5 is a pan-neural Na+-H+ exchanger that resides intracellularly, as shown in this fluorescent micrograph of the lumbar ganglion from NHX-5::GFP transgenic worms. Q andR, NHX-8::GFP is expressed in the seam cells and other tissues (see “Results”), and the NHX-8::GFP protein appears to accumulate in a punctate, perinuclear expression pattern (inset of Q). S and T, NHX-9::GFP is targeted to an intracellular domain, as shown in this confocal micrograph taken through the excretory cell body. NHX-9::GFP is expressed uniformly along both processes of the excretory cell in young larva (inset of S), but the intracellular localization becomes more restricted and appears like beads-on-a-string in the adults.View Large Image Figure ViewerDownload (PPT) When GFP expression was driven by the promoter sequences upstream of the NHX-1-, NHX-2-, NHX-6-, or NHX-7-coding sequences, fluorescence was found nearly exclusively in the intestine of the worm (Fig. 2,panels A–H). The entire intestine was labeled evenly in thenhx-1:: GFP and nhx-2:: GFP transgenic animals (panels A–D); however, the nhx-1:: GFP labeling was most likely an artifact, as discussed below. nhx-2:: GFPlabeling was strong in both L1 and L2 larval animals, then diminished with age (data not shown). The anterior and posterior intestinal segments were labeled in the nhx-6:: GFP animal, with a distinct gradient observed along the anterior segment (panels G and H). In contrast, the nhx-7:: GFPreporter resulted in fluorescence specifically and exclusively in the posterior cells of the intestine (panels E andF). Similarly, GFP expression was restricted primarily to a single cell or cell type in a surprisingly large number of transgenic strains (see Fig. 4 for schematic and synopsis). Thenhx-3 promoter drove GFP expression in the hypodermal cells of the main body syncytium (Fig. 2, panels I andJ), whereas expression from the nhx-8:: GFPtransgene resulted in fluorescence exclusively in the hypodermal seam cells (Fig. 2, panels O and
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