Molecular Cloning and Functional Expression of a Rat Na+/H+ Exchanger (NHE5) Highly Expressed in Brain*

Surat AttaphitayaDagger §, Keerang ParkDagger , and James E. MelvinDagger parallel

From the Dagger  Center for Oral Biology and the § Department of Neurobiology and Anatomy, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

    ABSTRACT
Top
Abstract
Introduction
References

We report here the cloning, primary structure, heterologous expression, tissue distribution, and localization of a cDNA encoding rat NHE5, a fifth member of the mammalian plasma membrane Na+/H+ exchanger (NHE) gene family. The full-length open reading frame as well as 34 nucleotides of 5'-untranslated and 1443 nucleotides of 3'-untranslated sequences were obtained using a polymerase chain reaction strategy involving reverse transcription-polymerase chain reaction and 5'/3'-rapid amplification of cDNA ends. The NHE5 cDNA encodes a protein of 898 amino acids with a calculated Mr of 99,044 and is predicted to contain 11-13 transmembrane domains. An amino acid comparison of the coding region of rat NHE5 reveals 95% identity with human NHE5. Northern hybridization analysis showed that high level expression of NHE5 mRNA is restricted to brain. Transfection of the coding region of rat NHE5 into NHE-deficient PS120 cells resulted in Na+/H+ exchange activity that was relatively insensitive to the amiloride analogue, 5-(N-ethyl-N-isopropyl) amiloride, with a half-maximal inhibitory concentration (IC50) of 1.53 ± 0.25 µM. In situ hybridization of rat brain sections revealed significant NHE5 mRNA levels in the dentate gyrus with lower levels observed in the hippocampus and cerebral cortex. These results suggest a specialized role for this fifth NHE isoform in neuronal tissues.

    INTRODUCTION
Top
Abstract
Introduction
References

Na+/H+ exchangers (NHE)1 comprise a family of integral plasma membrane proteins involved in a variety of physiological processes such as pH regulation, cellular differentiation, cell volume control, and electrolyte transport (reviewed in Refs. 1-3). Since the initial cloning of the human growth factor activable Na+/H+ exchanger, NHE1 (4), four additional plasma membrane NHE isoforms have been reported (5-7). Recently a human mitochondrial isoform, NHE6, has also been cloned (8). Characterization of the individual NHE isoforms has revealed differences in exchange kinetics, pHi sensitivity, mode of regulation, cellular localization, and tissue expression (1-3). As a result of this diversity, specialization of function has been postulated for the different NHE isoforms and, in some cases, confirmed in null-mutant mice (9-11).

Rat hippocampal neurons express an amiloride-insensitive Na+/H+ exchanger, the molecular identity of which is unknown (12). Pharmacological characterization of the different NHE isoforms indicates that rat NHE1 and NHE2 are very sensitive to amiloride and, therefore, not the hippocampal Na+/H+ exchanger (5, 6). In contrast, rat NHE3 and NHE4 are relatively insensitive to amiloride (5, 13, 14); however, little NHE3 mRNA is expressed in rat brain (5), and NHE4, unlike the Na+/H+ exchanger expressed in hippocampal neurons (12), is inhibited by high concentrations of amiloride (13, 14). Consequently, neither NHE3 nor NHE4 are likely to represent the activity of an amiloride-insensitive Na+/H+ exchanger previously described in hippocampal neurons (12). The partial coding sequence for a fifth isoform primarily expressed in brain, NHE5, is most closely related to NHE3 (7), and therefore NHE5 might also be expected to express amiloride resistance. To investigate further the properties of this putative brain-specific Na+/H+ exchanger, we cloned the rat NHE5 cDNA and determined its primary structure, tissue distribution, and localization. NHE5 encodes for a functional Na+/H+ exchanger that is resistant to the amiloride derivative EIPA and is highly expressed in the dentate gyrus.

    EXPERIMENTAL PROCEDURES

Cloning of the Rat NHE5 cDNA-- Total RNA was prepared from whole brain of male Wistar rats (150-200 g) using TRIzol reagent (Life Technologies, Inc.). RNA was reverse-transcribed to cDNA using oligo(dT) and random hexamer primers according to the manufacturer's recommendations (1st Strand cDNA Synthesis kit, CLONTECH, Palo Alto, CA). Rat brain cDNA was then used in subsequent RT-PCR reactions. 5'- and 3'-RACE was performed using adapter-ligated rat brain Marathon-Ready cDNAs (CLONTECH, Palo Alto, CA). RT-PCR and RACE amplification products were subcloned into the pCR 2.1 vector (Invitrogen, San Diego, CA). DNA sequence analysis of both strands was performed manually using the T7 Sequenase 7-deaza-dGTP sequencing kit (Amersham Pharmacia Biotech). Sequence analysis and alignment were performed using MacVector software (International Biotechnologies Inc., New Haven, CT).

Four overlapping partial cDNA clones of rat NHE5 (rNHE5a -rNHE5d) provided the complete nucleotide sequence of rat NHE5 (rNHE5) cDNA, including 5'- and 3'-untranslated regions (UTR). We then amplified and cloned the coding region of rNHE5 by RT-PCR. The rNHE5 open reading frame was placed into an eukaryotic expression vector, pCMV/SEAP (Tropix, Bedford, MA), generating the expression construct pCMV/rNHE5 used in functional studies. The following is a description detailing the procedures and primers used to amplify the above clones.

The initial 384-bp clone (rNHE5a) was amplified using human NHE5 primers based on previously published partial coding sequences (7). The primers used were 5'-TGCTGGGCCTGGTGCTA-3', corresponding to codons 82-87, and 5'-ACTCGCCAAAGACGATGAT-3', complementary to codons 204-209 of human NHE5 (7). PCR amplification was performed for 30 cycles using Amplitaq DNA polymerase (Perkin-Elmer) on a DNA thermal cycler (PTC-200 Peltier Thermal Cycler, MJ Research, Watertown, MA) with each cycle set to 94 °C for 30 s, 64 °C for 30 s, and 72 °C for 2 min. A 5-µl aliquot of the first-round PCR reaction mixture was re-amplified using the same PCR conditions.

The second 592-bp clone (rNHE5b) corresponded to the 5' end of rat NHE5 (roughly the first six transmembrane domains) and included 34 bases of the 5'-untranslated region. This clone was amplified via 5'-RACE using an AP1 primer (5'-CCATCCTAATACGACTCACTATAGGGC-3') complementary to the adapter region and a rat NHE5-specific primer (5'-CACCGCTGAGATGAGGCTTCCAAAGA-3') complementary to codons 178-186. PCR amplification was performed using CLONTECH's Advantage cDNA Polymerase Mix as follows: 94 °C for 1 min followed by 15 cycles at 94 °C for 30 s then 72 °C for 4 min, 5 cycles at 94 °C for 30 s then 70 °C for 4 min, and 20 cycles at 94 °C for 30 s then 68 °C for 4 min. A 1-µl aliquot of the first-round PCR reaction mixture was re-amplified under the same PCR conditions.

The third 2103-bp clone (rNHE5c) spanning the majority of the coding region was generated via RT-PCR using a rat NHE5-specific primer (5'-GCCGGGGACCTTCTTCCTTTTCCTG-3') corresponding to codons 106-113 and a primer (5'-GGGGACGCTAGGCTCTCCAGGGATGA-3') complementary to codons 796-804 of human NHE5.2 PCR reactions were the same as described above for rNHE5a.

The fourth 1866-bp clone (rNHE5d) corresponded to the remainder of the coding region and the entire 3'-untranslated region. This clone was attained through 3'-RACE using a rat NHE5-specific primer (5'-TGCGCGGAGAAAGAGCTACCCTGGAA-3') corresponding to codons 759-767 and AP1 as the adapter primer. PCR conditions were the same as described for rNHE5b.

The rNHE5 expression clone containing 34 bp of 5'-UTR, the entire coding region, and 7 bp of 3'-UTR was amplified by RT-PCR using high fidelity Pfu polymerase (Stratagene, La Jolla, CA), rat brain cDNA as template, and rat NHE5-specific primers. The primers used were 5'-acgaagcttGCCGGCGGCCGTGCAGTGCCCGGAG-3', corresponding to nts -34 to -10, and 5'-actctagaCCCTGGGCTACAGCCTGCCTCCTCTGTT-3', complementary to nts 2677-2704. Lowercase letters represent HindIII and XbaI restriction sites flanking each primer, placed to facilitate ligation into the eukaryotic expression vector.

Transfection and Stable Expression of rNHE5-- The rNHE5 PCR product was digested by HindIII and XbaI restriction enzymes and then subcloned into pCMV/SEAP previously digested with HindIII and XbaI. The orientation and coding region were verified by sequencing. NHE-deficient PS120 cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, L-glutamine, and penicillin (50 units/ml)/streptomycin (50 µg/ml) and selected by the H+-suicide technique as described previously (15). To establish stable expression of rat NHE5, the NHE-deficient PS120 cells were transfected with 20 µg of pCMV/rNHE5 by the CaPO4-DNA coprecipitation method (16). Cells were selected for stable expression with G418 (1000 units/ml, Life Technologies, Inc.) and further selected for expression of Na+/H+ exchanger activity by the "H+ killing" method (17). NHE5 transfected cells were maintained in medium containing G418, and the "H+ killing" procedure was repeated every 4-5 days.

Intracellular pH Measurements-- Intracellular pH was monitored using the pH-sensitive dye SNARF-1 (Molecular Probes, Eugene, OR) as described previously (18). Cells plated on glass coverslips mounted on the microscope stage of an Ultima confocal laser cytometer (Meridian Instruments, Okemos, MI) were acid-loaded using the NH4Cl pre-pulse technique (19). Briefly, coverslips were superfused with a physiological salt solution containing 60 mM NH4Cl (NaCl was replaced by NH4Cl) for 10 min and then switched to a Na+-free salt solution (Na+ was replaced by N-methyl-D-glucamine) to produce an acid load. Approximately 3 min later, extracellular Na+ was restored to initiate Na+/H+ exchanger-mediated intracellular pH recovery. The physiological salt solution contained (in mM): NaCl (135), KCl (5.4), KH2PO4 (0.4), NaH2PO4 (0.33), glucose (10), Hepes (20), CaCl2 (1.2), MgSO4 (0.8), pH 7.4. Recovery rates of the fluorescence traces were determined from the initial linear portion following the re-addition of extracellular Na+ and expressed as mean ± S.E. Calibration of the intracellular pH signal was accomplished by the high potassium-nigericin technique (20).

Northern Hybridization Analysis-- Northern analysis was performed using a rat multiple tissue Northern blot containing approximately 2 µg of poly (A)+ RNA per lane from eight different tissues (CLONTECH, Palo Alto, CA). The membrane was prehybridized at 60 °C in 5× SSPE, 2× Denhardt's reagent, 0.5% SDS, 100 µg/ml salmon sperm DNA, and 50 µg/ml tRNA. The 32P-labeled rat NHE5 probe was added, and hybridization was performed in the same solution at 60 °C for 20 h. The 384-bp cDNA probe included sequence spanning codons 84-211 of rat NHE5. The membrane was washed 4 times in 2× SSC containing 0.05% SDS at room temperature, 2 times in 2× SSC containing 0.1% SDS at room temperature, 1 time in 0.1× SSC containing 0.1% SDS at 68 °C and was exposed to film for 8 days at -86 °C. The same blot was then stripped and hybridized with a control rat beta -actin probe.

In Situ Hybridization-- In situ hybridization was used to investigate the possibility that rNHE5 may represent the amiloride-insensitive NHE variant previously reported by Raley-Susman et al. (12) in rat hippocampal neurons. Male Wistar strain rats (150-250 g) were anesthetized by intraperitoneal injection of ketamine (100 mg/kg body weight) and then perfused with 4% paraformaldehyde, pH 7.4. The isolated whole brain was placed in 4% paraformaldehyde overnight and then transferred to cryoprotectant consisting of 30% sucrose in 0.067 M phosphate-buffered saline for 2 days. Frozen sections containing the hippocampus were cut at 18 µm on a cryostat (HistoStat microtome, Scientific Instruments). Labeled antisense and sense RNA probes were prepared by in vitro transcription (SureSite II System kit, Novagen) of pCR2.1/rNHE5d using T7 RNA polymerase in the presence of 35S-UTP. Hybridizations were performed under high stringency conditions as described previously (21). Autoradiography of slides was initially done for 5 days with Kodak Biomax MR film (Eastman Kodak Co.). Slides were subsequently dipped in NT2B (Kodak) emulsion (1:1 emulsion to glycerol) and stored at 4 °C for 2 weeks before developing. Hematoxylin and eosin counterstaining was performed on selected slides.

    RESULTS

PCR Cloning of a Rat Brain NHE5 cDNA-- Human NHE5 is highly expressed in brain tissue (7). We amplified a 384-bp product from rat brain cDNA, rNHE5a, using primers based on the partial coding sequences provided in this earlier report. The nucleotide sequence and translation of rat NHE5 is depicted in Fig. 1. Nucleotide sequence analysis of rNHE5a yielded >90% identity with human NHE5 suggesting that this cDNA was the rat homolog of human NHE5. We subsequently used sequence information gained from rNHE5a to design primers for 5'-RACE to amplify the amino-terminal coding sequence, clone rNHE5b. The 592-bp, rNHE5b clone included 34 bp of 5'-UTR containing a purine at the -3 position as described for numerous eukaryotic genes by Kozak (22). Together, rNHE5a and rNHE5b provided sequence encoding the first six transmembrane domains.


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Fig. 1.   Nucleotide and deduced amino acid sequence of rNHE5. Nucleotides are numbered on the right and amino acids are numbered below the sequence. Underlined nucleotides from -34 to -10 and 2677 to 2704 represent sense and antisense primers used for amplification of the cDNA forming the expression construct pCMV/rNHE5. The consensus polyadenylation signal is indicated by the double underline.

A third clone, rNHE5c, was derived by RT-PCR amplification using a rat NHE5-specific sense primer and a human NHE5 antisense primer.2 Amplification with these primers produced a 2103-bp product corresponding to the majority of the coding region of rat NHE5. The remaining 3'-coding region and the 3'-UTR were then obtained through 3'-RACE. This fourth clone, rNHE5d, was 1866 bp and included 423 bp of coding region upstream of the stop codon and 1443 bp of 3'-UTR. A consensus AATAAA polyadenylation signal preceded by 20 nucleotides the 3' poly(A) tract, indicating an intact 3' terminus (double underline in Fig. 1). Thus, the overlapping clones, rNHE5a-d, provided sequence information encoding the entire rat NHE5 cDNA.

Deduced Amino Acid Sequence of NHE5-- The amino acid sequence alignment of rat and human NHE5 is shown in Fig. 2. The 898-amino acid sequence of rat NHE5 is 95% identical to human NHE5. In comparison with other isoforms, rat NHE5 is most closely related to rat NHE3 (51% identity). Highest similarity between NHE5 and NHE3 is in the amino-terminal transmembrane domain (62%), whereas the cytoplasmic carboxyl-terminal domain is less similar (31%). The amino acid identity to other rat NHE isoforms is considerably less, with 39% identity to rat NHE1 and NHE2, and 31% identity to rat NHE4.


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Fig. 2.   Amino acid sequence alignment of rat NHE5 and human NHE5. The amino acid sequence of rat NHE5 is compared with human NHE5 (24). Alignment of rat and human NHE5 reveals 95% amino acid identity. Identical amino acids are boxed.

Hydropathy analysis, using the algorithm of Kyte and Doolittle (23), predicts that the amino terminus contains 11-13 hydrophobic domains, with an extensive carboxyl-terminal hydrophilic domain. This general structure is consistent with the predicted topology of other NHE isoforms. Three potential N-glycosylation sites are present at Asn-201, Asn-321, and Asn-794; however, only Asn-321 is common to the other potential N-glycosylation sites observed in the different rat NHE isoforms (5, 6). One mode of regulation for rat Na+/H+ exchanger activity is mediated through phosphorylation of the cytoplasmic domain (24). A number of potential phosphorylation sites are present in rNHE5 including protein kinase C phosphorylation sites located at Ser-593 and Ser-652, and cAMP-dependent kinase and Ca+/calmodulin-dependent kinase II sites found at Ser-649, Ser-732, Ser-855, and Ser-857. All of these potential regulatory sites are also present in human NHE5 (25).

Tissue Distribution of NHE5-- Northern hybridization analysis of a rat multiple tissue Northern blot under high stringency conditions indicated that size as well as tissue distribution of rat NHE5 mRNA paralleled that reported for human NHE5 (7). The highest levels of mRNA were observed in brain (Fig. 3). The dominant mRNA was ~4.4 kb, although as observed for human NHE5, larger bands ranging between 9 and 9.5 kb were also detected. These larger transcripts may represent retained intron sequences as observed by Baird et al. (25) for human NHE5. Long exposure times were required to detect NHE5 mRNA expression in spleen, lung, skeletal muscle, and testis.


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Fig. 3.   Tissue distribution of rNHE5 mRNA. Northern blot containing approximately 2 µg of poly(A)+ RNA per lane from eight different tissues was probed under high stringency conditions with a 384-bp cDNA probe. The probe contained specific sequence corresponding to codons 84-211 of rat NHE5. The film was exposed for 8 days. A 4.4-kb mRNA was detected at the highest levels in brain.

Localization of NHE5 in Rat Brain-- The high levels of NHE5 mRNA present in rat brain may correlate with a unique Na+/H+ exchange activity expressed in hippocampal neurons (12). To explore this possibility, in situ hybridization of rat brain sections was performed. Probes were prepared by in vitro transcription of pCR2.1/rNHE5d. The rNHE5d clone corresponds to the 3'-untranslated region of rat NHE5 that has no similarity to other cloned NHE isoforms. Fig. 4A shows that NHE5 mRNA was strongly expressed in the dentate gyrus, with lower levels of expression seen in the CA1 field of the hippocampus and in the cerebral cortex. In comparison, no significant hybridization signals were observed when adjacent brain slices were probed with the sense probe (Fig. 4B). Higher magnification within the dentate gyrus (see arrow in Fig. 4A) revealed that NHE5 mRNA expression localized to the region containing the cell bodies of neurons (Fig. 4, C and D).


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Fig. 4.   Localization of rNHE5 by in situ hybridization. Alternating sections of rat brain were hybridized with antisense and sense RNA probes containing the 3'-UTR and 423 bp upstream of the stop codon of rat NHE5. A, highest rNHE5 signal is localized to the dentate gyrus (DG), with lower levels detected in the CA1 field of the hippocampus (Hip) and the cerebral cortex (CT). B, an adjacent section hybridized with the sense probe. C, detailed view of region indicated by the open arrow in A showing hematoxylin- and eosin-stained neuronal cell bodies. Samples were viewed and analyzed on a Zeiss Axioplan microscope using a 20 × neofluor objective (Carl Ziess, Inc., Germany). D, same region as in C detailing silver grains deposited by radioactive probe on neuronal cell bodies under dark-field illumination.

Functional Expression of rNHE5 in NHE-deficient PS120 Cells-- To determine whether rNHE5 is a functional Na+/H+ exchanger, RT-PCR was used to obtain a clone containing the entire coding sequence. The rNHE5 clone contained 34 bp of the 5'-UTR, the coding region, and 7 bp of 3'-UTR. Underlined nucleotides in Fig. 1 indicate the position of the sense and antisense primers used for amplification. Intracellular pH recovery from an acid load was not observed in nontransfected control cells (Fig. 5). In contrast, an intracellular pH recovery was activated in transfected cells following the addition of Na+ at the time indicated by the arrow. The Na+-dependent pH recovery observed in rNHE5 expressing cells was inhibited by the amiloride analogue EIPA (Fig. 6). Like NHE3, rat NHE5 was relatively resistant to inhibition by this compound. The half-maximal concentration for inhibition of rat NHE5 (1.53 ± 0.25 µM) was comparable to the previously reported (19) EIPA sensitivity of rat NHE3 (2.4 µM).


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Fig. 5.   Na+-dependent recovery of the intracellular pH in cells expressing rNHE5. SNARF-1 loaded cells were acidified using the NH4+ pre-pulse technique. Recovery from an acid load was initiated by the re-addition of external Na+ at time indicated by the arrow. Shown are typical results from rNHE5-expressing cells (down-triangle) and non-transfected (black-square) control cells. Each data point represents the mean ± S.E. of n > 30 cells.


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Fig. 6.   EIPA sensitivity of rNHE5. The concentration dependence of EIPA inhibition was tested in a range from 100 nM to 50 µM EIPA. The continuous line represents a sigmoidal fit of the data providing a half-maximal inhibitory concentration (IC50) of 1.53 ± 0.253 µM. Each data point represents the mean percent inhibition, relative to controls, of n > 20 different cells from three separate experiments for the various EIPA concentrations. Data shown are mean ± S.E. In all cases the standard error was less than 5% of the mean.


    DISCUSSION

In this report we describe the molecular cloning and complete nucleotide sequence of a rat cDNA encoding the fifth member of the mammalian plasma membrane NHE family, NHE5. The cDNA contains 34 and 1443 bp of 5' and 3' untranslated sequences, respectively. The 2694 bp open reading frame encodes a protein of 898 amino acids with a predicted Mr of 99,044, making NHE5 the largest member of the Na+/H+ exchanger gene family (5, 6).

The data presented in Fig. 3 demonstrated that high level expression of NHE5 mRNA was restricted to rat brain, comparable to the results described previously by Klanke et al. (7) for human tissues. Na+/H+ exchanger activity has been described in various neuronal tissues (26-28), including an amiloride-insensitive Na+/H+ exchanger in cultured hippocampal neurons (12). Neuronal expression of the different NHE isoforms has been demonstrated for the ubiquitous housekeeping isoform, NHE1, as well as the epithelial isoforms NHE2 and NHE4, although the latter two isoforms are apparently expressed to a lesser extent (5, 6). We found by in situ hybridization that NHE5 mRNA was most abundant in the dentate gyrus. Lesser expression of NHE5 was observed in the cerebral cortex and the CA1 field of the hippocampus. In agreement with these observations, Baird et al. (25) detected human NHE5 mRNA in hippocampus and several other regions within the brain. Bookstein et al. (13) previously showed by in situ hybridization that rat NHE4 is localized to the CA fields of the hippocampus. Coexpression of rat NHE4 and NHE5 in the CA1 field of the hippocampus suggests possible overlap of function. However, NHE4 is uniquely activated by hyperosmolar conditions (13), suggesting that it may have a highly specialized function in this region of the brain.

Fig. 6 shows that expression of rat NHE5 resulted in Na+/H+ exchanger activity that was relatively resistant to the amiloride derivative EIPA. Similarly, human NHE5 mediates amiloride-sensitive Na+/H+ exchange (25). Thus, amiloride and its derivatives inhibit all five members of the plasma membrane Na+/H+ exchanger gene family. The EIPA sensitivity of rat NHE5 was most like rat NHE3 (19), as might be predicted from the structural similarity of the ion transporting domains of these two proteins (62% identity). In contrast, Raley-Susman et al. (12) found that the Na+/H+ exchanger activity expressed in hippocampal neurons was not inhibited by amiloride or the amiloride derivative 5-(N,N-hexamethylene)amiloride. The simplest interpretation of this lack of sensitivity to amiloride is that NHE5 does not mediate the Na+/H+ exchanger activity that was detected in cultured hippocampal neurons. Alternatively, in hippocampal neurons the amiloride sensitivity of NHE5 may be modified by a mechanism not present in fibroblasts (Fig. 6).

In summary, we have identified the rat homologue of the human Na+/H+ exchanger, NHE5. The deduced amino acid sequence and predicted membrane organization is similar to other members of the plasma membrane Na+/H+ exchanger gene family, being most similar to the epithelial Na+/H+ exchanger, NHE3. Its pharmacological properties appear to be most like NHE3 as well. In contrast to NHE3 expression, NHE5 mRNA is restricted almost exclusively to the brain suggesting that this isoform may perform a specialized role in neuronal tissues. Investigations are currently under way to test whether the sequence similarity between NHE3 and NHE5 correlates with comparable functional properties.

    ACKNOWLEDGEMENTS

We thank Drs. Nancy Baird and Gary Shull for helpful discussions during the course of this work and sharing the sequence of human NHE5; Dr. John Olschowka for assistance in perfusion of rats and histological identification of neuronal structures; and Linda Richardson for technical assistance in the expression studies. We also thank Drs. Linda Callahan and Paul Coleman for allowing use of histological equipment and suggestions for in situ hybridization.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant DE08921 (to J. E. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF100172.

Supported by NIDR Dentist Scientist Fellowship Award DE00159 from the National Institutes of Health.

parallel To whom correspondence should be addressed: Center for Oral Biology, Box 611, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-8705; Fax: 716-473-2679; E-mail: james_melvin{at}urmc.rochester.edu.

The abbreviations used are: NHE, Na+/H+ exchanger; NHE1-6, Na+/H+ exchanger isoform 1-6; kb, kilobase; EIPA, 5-(N-ethyl-N-isopropyl)amiloride; RT-PCR, reverse transcription-polymerase chain reaction; RACE, rapid amplification of cDNA ends; CA, cavi amnoni; nt, nucleotide; bp, base pair; kb, kilobase pair; UTR, untranslated regions.

2 Nancy Baird and Gary E. Shull, personal communication.

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Abstract
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