RAPID COMMUNICATION
Cloning and functional expression of rNBC, an electrogenic Na⁺- cotransporter from rat kidney

¹ Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520; and ² Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

	ABSTRACT

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We have recently cloned the renal electrogenic Na⁺-bicarbonate cotransporter of the salamander Ambystoma tigrinum (aNBC) (M. F. Romero, M. A. Hediger, E. L. Boulpaep, and W. F. Boron. FASEB J. 10: 89, 1996; and Nature 387: 409-413, 1997). Here we report the cloning of a mammalian homolog of aNBC, named rNBC for rat Na⁺-bicarbonate cotransporter. NBC constitutes the major route for reabsorption and assists in Na⁺ reabsorption across the basolateral membrane of the renal proximal tubule (PT). We used aNBC as a probe to screen a rat kidney cortex cDNA library in gt10 and identified several clones. Each has an initiator Met and a large open-reading frame followed by a 3'-untranslated region of ~500 bp. The 7.5-kb mRNA for rNBC is present in kidney, liver, lung, brain, and heart. In situ hybridization with the rNBC probe in the rat kidney revealed staining in the S2 segment of PT. rNBC encodes a protein of 1,035 amino acids, with a predicted molecular mass of 116 kDa. Its deduced amino acid sequence is 86% identical to that of aNBC. Comparison of both the aNBC and rNBC sequences to the GenBank database reveals a low level of amino acid identity (~30%) to the AE family of Cl/ exchangers. Injection of rNBC cRNA into Xenopus oocytes leads to expression of an electrogenic Na⁺- cotransporter that is qualitatively similar to that of aNBC but at a much lower level. Placement of the rNBC cDNA into the context of a Xenopus expression vector produces a substantial increase in rNBC expression. Addition of 1.5% CO₂/10 mM elicits a hyperpolarization of >50 mV and a rapid decrease of intracellular pH (pH_i), followed by an increase in pH_i. Subsequent removal of Na⁺ in the presence of CO₂/ causes a depolarization of >50 mV and a concomitant decrease of pH_i. Thus rNBC is in the same newly identified family of Na⁺-linked transporters as is aNBC.

rat sodium-bicarbonate cotransport; homology cloning; Xenopus oocyte expression; intracellular pH; sodium transport; bicarbonate transport

	INTRODUCTION

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IN THE MAMMALIAN KIDNEY, Na⁺, , and several other solutes are filtered from the blood by the glomerulus and then reabsorbed (80-90%) by the epithelial cells of the proximal tubule (PT). Luminal Na⁺ enters the PT cell via a variety of Na⁺-coupled transport systems (e.g., Na⁺/H⁺ exchanger, Na⁺-glucose cotransporter). Once the Na⁺ is inside the PT cell, the Na⁺-K⁺ pump extrudes the Na⁺ across the basolateral membrane, maintaining a low intracellular Na⁺ concentration ([Na⁺]_i). Luminal , on the other hand, is titrated by H⁺ secreted into the tubule lumen, forming CO₂ in a reaction facilitated by luminal glycosyl-phosphatidylinositol-anchored carbonic anhydrase IV. The CO₂ then rapidly enters the PT cell, where carbonic anhydrase II converts it and OH to . In the early segments of the PT, the vast majority of the intracellular moves across the basolateral membrane into the blood via the electrogenic Na⁺- cotransporter (4).

Recently, we used Xenopus oocytes to expression clone the renal electrogenic Na⁺-bicarbonate cotransporter (NBC) from the tiger salamander Ambystoma tigrinum (aNBC) (22, 23). This was the first Na⁺-coupled transporter to be cloned. Here, we report the cloning and functional expression of the rat homolog.

Portions of this work have been previously presented in abstract form (21, 24).

	METHODS

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Cloning of Rat NBC cDNA

Library screening. The oligo(dT)-primed gt10 cDNA library (28) was plated and grown until plaques were 1 mm in diameter, and filter lifts were screened at medium stringency (35°C for 14 h) with the EcoR I fragment of Ambystoma NBC (aNBC, GenBank accession no. AF001958). The hybridization solution was 5× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0), 3× Denhardt's, 25 mM soduim-2-(N-morpholino)ethanesulfonic acid, 0.2% sodium dodecyl sulfate (SDS), 2.5 mM sodium pyrophosphate (pH 6.5), 10% dextran sulfate, 50% formamide, and 200 µg/ml yeast tRNA. Filters were washed with 1) three consecutive washes of 5× SSC, 0.05% sarcosyl, and 0.1% SDS at room temperature, 2) followed by several washes at 42°C over 1 h, and 3) several washes at 42°C with 0.1× SSC, 0.05% sarcosyl, and 0.1% SDS. Positive plaques were visualized by autoradiography, picked, and stored in SM buffer (25).

Isolation of rat NBC. Lambda phage DNA was isolated from plate lysates of pure plaques using the Lambda Midi kit (Qiagen, Santa Clarita, CA). Inserts were excised from -DNA using Not I, fragments were gel purified, and inserts were subcloned into pBluescript KS() at the Not I site. Sequencing of three independent clones revealed an initiator Met, a complete open-reading frame, and ~500 bp of 3'-untranslated region (UTR), as well as a deduced amino acid sequence 86% identical to that of aNBC.

Sequence analysis. Sequence analysis was performed with either the DNAsis (Hitachi Software, San Bruno, CA) or DNAstar programs (Lasergene, Madison, WI) as previously reported (23).

Northern Blot

A rat multiple tissue Northern blot was purchased from Clontech (Palo Alto, CA). The blot was probed with r715na, i.e., the entire 3.6-kb rat NBC (rNBC) cDNA in ExpressHyb (Clontech) at 65°C for 3 h, washed with 2× SSC-0.05% SDS at room temperature for 40 min, and finally washed with 0.1× SSC-0.1% SDS at 50°C for 40 min. Bound probe was visualized by autoradiography.

In Situ Hybridization

Digoxigenin-labeled antisense and sense runoff transcripts were synthesized from a polymerase chain reaction fragment, flanked by promoter sites for SP6 and T7 polymerases using a Genius Kit (Boehringer-Mannheim, Indianapolis, IN). Two different rNBC fragments were used as probes for in situ hybridization: nucleotides 143-2113 or nucleotides 2234-3495. After synthesis, the resulting RNAs were alkali-hydrolyzed to 200-400 nucleotide lengths. In situ hybridization was performed on cryosections (12 µm) of fresh-frozen rat kidney as previously described (26). We used a hybridization buffer of 50% formamide, 5× SSC, 2% blocking reagent (Boehringer-Mannheim), 0.02% SDS, and 0.1% sarcosyl. The probe concentration was ~200 ng/ml. Sections were hybridized at 68°C for 18 h, washed three times in 2× SSC (68°C), and washed twice for 30 min each time in 0.2× SSC (68°C). The hybridized, labeled probes were visualized using anti-digoxigenin Fab fragments coupled to alkaline phosphatase (Boehringer-Mannheim) and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate (26). Sections were developed in substrate solution for 20 h, then rinsed in 10 mM tris(hydroxymethyl)aminomethane, 1 mM EDTA, pH 8.0, and covered with crystal mount (Fisher Scientific, Pittsburgh, PA). Adjacent sections were hybridized with a probe for the Na⁺-glucose transporter SGLT1, which hybridizes to the late S2 and S3 segments (15), to determine the tubule localization of rNBC mRNA.

Oocyte Experiments

Oocyte isolation. Experimental solutions are detailed in Table 1. For oocyte preparation, a female Xenopus laevis was anesthetized with fresh 0.1-0.2% tricaine (3-aminobenzoic acid ethyl ester, methanesulfonate salt, catalog no. A-5040; Sigma, St. Louis, MO) in 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5, for 5 min and then packed in ice to cause hypothermia. After ovarian lobes were removed and placed in the 0 Ca²⁺ ND-96 solution, the skin incision was closed using 6-0 silk sutures. The frog was then placed in 0.1 M NaCl at room temperature until fully recovered from the anesthesia (usually 2-5 min); the frog remained in this NaCl solution for at least 12 h to facilitate healing. Oocytes in the pieces of ovary were enzymatically dissociated using 2 mg/ml type IA collagenase (catalog no. C-9891; Sigma) diluted in sterile-filtered (0.22-µm filter; Millipore, Bedford, MA) 0 Ca²⁺ ND-96 solution. After dissociating the oocytes, we selected stage V-VI oocytes and maintained them in sterile-filtered OR3 media at 18°C. Our OR3 media contained (in 2.0 liters) one pack of powdered Leibovitz L-15 media with L-glutamine (GIBCO-BRL no. 41300-039; Life Technologies, Gaithersburg, MD), 100 ml of 10,000 U penicillin and 10,000 U streptomycin solution in 0.9% NaCl (catalog no. P-0781, Sigma), 5 mM HEPES (final concentration). We titrated this solution to pH 7.5 at room temperature using NaOH. The solution had an osmolality of 195-200 mosmol/kg.

View this table: [in this window] [in a new window]   Table 1.   Experimental solutions

Oocyte injections. The original rNBC-cRNA (clone r715na) was transcribed in vitro from the T7 promoter of pBluescript KS() using the mCap mRNA Capping Kit (Stratagene, La Jolla, CA). We also subcloned rNBC into the Xenopus expression vector pTLN2, which is a modified version¹ of pTLN (16), and used the SP6 promoter to transcribe the rNBC-pTLN2 cRNA (mMessage mMachine; Ambion, Austin, TX). Each oocyte was injected with 10 ng of cRNA (50 nl of a solution containing 0.2 µg/µl). Oocytes were studied 3-10 days after injection of cRNA.

Electrophysiology experiments. An oocyte, visualized with a dissecting microscope, was held on a nylon mesh in a chamber having a volume of ~250 µl. The oocyte was continuously superfused with a saline solution (3-5 ml/min) that was delivered through Tygon tubing. Solutions were switched using a daisy-chain system of computer-actuated five-way valves with zero dead space. Solution changes in the chamber occurred within 15-20 s. Membrane voltage (V_m) and intracellular pH (pH_i) of X. laevis oocytes were measured simultaneously using microelectrodes, as described previously (23). Briefly, V_m electrodes were pulled from borosilicate fiber-capillary glass (Warner Instruments, West Haven, CT); these were backfilled with 3 M KCl and had resistances of 3-5 M. The pH electrodes were pulled in a similar manner, silanized by exposing them for 5-10 min to 40 µl of bis-di-(methylamino)-dimethylsilane (Fluka Chemical, Ronkonkoma, NY), deposited in an enclosed container at 200°C, and then baked overnight. These pH micropipettes were cooled under vacuum, and their tips were filled with hydrogen ionophore I-cocktail B (Fluka Chemical). The pH micropipettes were then backfilled with a buffer containing 0.04 M KH₂PO₄, 0.023 M NaOH, and 0.015 M NaCl (pH 7.0). The pH microelectrodes had slopes ranging from 54 to 59 mV/pH unit. The V_m and pH_i electrodes were connected to high-impedance electrometers (6, 29), and the voltage due to pH was obtained by electronically subtracting the signals from the pH and V_m electrodes. V_m was obtained by subtracting the signals from the V_m electrode and the external reference (calomel) electrode. Electrometer outputs were directed to an 486 computer via a 12-bit analog-to-digital converter. Our expression assay had two steps (23). First, we switched from a HEPES-buffered extracellular solution to one containing CO₂/. Second, after pH_i stabilized in CO₂/, we removed extracellular Na⁺.

	RESULTS AND DISCUSSION

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Sequence Features of rNBC

With the expression cloning and sequencing of aNBC (23), we realized that the renal, electrogenic Na⁺- cotransporter and the anion exchangers (i.e., AE1, AE2, AE3) are both part of a superfamily of transporters. Figure 1A is a multiple alignment of the deduced amino acid sequences of rat and Ambystoma NBC, as well as representative AEs. The sequences aligned in Fig. 1 are the AE sequences that were found to be most similar to the NBC sequences. Not only are the predicted membrane-spanning domains (MSDs) closely related, but the ~340-amino acid NH₂ terminal to the first MSD also exhibit a high degree of homology (only identity is shown in Fig. 1A). Regions in which several consecutive amino acids are identical among NBC and the AEs may represent the signature of superfamily members. rNBC and aNBC are 86% identical at the amino acid level. A dendrogram summarizing the divergence among the rNBC, aNBC, rat cardiac AE3, human AE2, and human AE1 sequences is shown in Fig. 1B. The rNBC sequence diverges from aNBC by 13% but diverges from AE1, AE2, and AE3 (isoforms above) by 61%, 61%, and 58%, respectively. Thus the "percent divergence" for this group indicates that rNBC is most closely related to aNBC, more distantly related to rat cardiac AE3, and most distantly related to human AE1 and AE2. While we were physiologically characterizing rNBC, a related human amino acid sequence was published (5); this hNBC sequence is nearly 97% identical to that of rNBC.

View larger version (123K): [in this window] [in a new window]   View larger version (10K): [in this window] [in a new window]   Fig. 1.   Amino acid sequence and model of rNBC. A: multiple amino acid sequence alignment of NBCs with the AEs. A 3.6-kb cDNA encodes the rat renal electrogenic Na+-bicarbonate cotransporter (rNBC). The cDNA has an open-reading frame from nucleotides 123 to 3230 encoding a 1,035-amino acid protein, with last ~500 bp being 3'-untranslated region (UTR). The resulting rNBC protein is predicted to have a molecular mass of 116 kDa. There is a consensus Kozak sequence (A/G)-ATGCC (11) with stop codons 5' to the start Met. Membrane-spanning domains of rNBC are indicated by a line over the rNBC sequence. In the multiple sequence alignment, AE sequences [GenBank accession nos. S03074 (human AE1), S21086 (human AE2), A42497 (rat cardiac AE3)] identical to NBC are highlighted in reverse type. Glycosylation sites for NBC are indicated by solid dots () over the amino acid sequences that are boxed. NBC is similar to several sequences in the expressed sequence tag (EST) database (e.g., W31917, W39298, N27899, and N58147) and a Caenorhabditis elegans genomic clone (F52B5.1). The GenBank accession no. of rNBC is AF004017. B: dendrogram showing the "percent divergence" of the amino acid sequences of NBC (aNBC for Ambystoma NBC, rNBC for rat NBC) and the most homologous AEs (same as in A). We used DNAstar software to calculate percent divergence as a quantification of sequence relatedness. Our use of this software was described previously (23). The total length of the horizontal line segments from one label (e.g., "aNBC") to another label indicates the percent divergence. Thus the divergence between rNBC and AE1 is 61%, and the divergence between rNBC and aNBC is 13%. These calculated values indicate that rNBC is more related to aNBC than to AE1, AE2, or AE3. On the basis of the same DNAstar software, NBC is 35%, 33%, and 33% similar [similarity as defined and calculated by the DNAstar software is not always but may be synonymous with identity (see Ref. 23 for detailed definition).] to AE3, AE2, and AE1, respectively. Note that the sum of the "% divergence" and the "% similarity" is not necessarily 100%.

The hydropathy plot of rNBC is virtually identical to that of aNBC and is consistent with the presence of at least 10 MSDs and a large extracellular loop between MSDs 5 and 6. The major divergences between rNBC and aNBC amino acid sequences occur at the NH₂ terminus (first 20 amino acids) and at the 5-6 loop region. In this 5-6 loop, aNBC is predicted to have four N-linked glycosylation sites, whereas rNBC is predicted to have only three sites (boxes with dots in Fig. 1; sites in Table 2). Similarly, there are consensus sites for protein kinase A, protein kinase C, casein kinase II, and tyrosine phosphorylation (see Table 2). Finally, as is the case for aNBC, rNBC contains several predicted myristylation sites (Table 2).

View this table: [in this window] [in a new window]   Table 2.   Putative modification sites of rNBC

With rNBC added to the superfamily, two sequence features become more apparent. 1) The signature sequences for the superfamily are confirmed. 2) An additional site is suggested at which 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) might covalently react with the protein. Biochemical experiments identified two lysine residues in AE1 with which DIDS reacts (2, 7, 19). The alignment of AE1, AE2, and AE3, as well as the knowledge of the first of the two DIDS reaction sites (human K539 or mouse K558), suggested a consensus amino acid sequence KL(X)K (X = I, V, Y) that might be characteristic of a DIDS reaction site (10). The cognate sequence in both Ambystoma and rat NBC is KMIK (558-561). In addition, at a more COOH-terminal site, both NBC clones have the original AE consensus motif sequence KLKK (768-771). If both the more NH₂-terminal and COOH-terminal NBC sequences prove to be actual DIDS-reaction sites, then a more generalized DIDS-reaction motif would be K-(Z)(X)-K, where Z = M, L and X = I, V, Y.

mRNA Distribution of rNBC

Northern analysis. Using high-stringency Northern analysis, we observed rNBC-mRNA at ~7.5 kb (Fig. 2). rNBC message is abundant in kidney (source of rNBC-cDNA) but also present in substantial amounts in liver and brain and at lower levels in lung, spleen, and heart. The r715na-cDNA that encodes the rNBC protein is ~3.6 kb, indicating that the message has additional UTRs of more than 4 kb. In Ambystoma, not only was the message smaller, but there was no reactivity in the liver, lung, and spleen, and an even smaller message (~2 kb) was in the heart (23).

View larger version (61K): [in this window] [in a new window]   Fig. 2.   Rat Northern blot probed with rNBC. High-stringency Northern analysis of poly(A)+ RNA from rat tissues probed with 32P-labeled rNBC cDNA and exposed for 14 h. Skel. Mus., skeletal muscle.

In situ hybridization. In the kidney, two antisense rNBC probes showed identical hybridization patterns. With sense probes, no hybridization was evident (data not shown). rNBC mRNA is strongly expressed in PTs at the corticomedullary junction (Fig. 3A). Comparison of the in situ hybridization patterns of rNBC (Fig. 3B) and SGLT1 (Fig. 3C), which localizes to the straight portion of the S2 as well as to the S3 segment of the PT (15), suggests that rNBC localizes to the straight portion of the S2 segment. rNBC mRNA expression was not detected in any other tubule segment.

View larger version (135K): [in this window] [in a new window]   Fig. 3.   In situ hybridization of rat kidney. Distribution of rNBC mRNA in rat kidney as detected by digoxigenin-labeled cRNA probes. A: a strong signal is present at the junction between cortex (CO) and outer medulla (OM). B and C: adjacent sections hybridized with a rNBC probe (B) or a SGLT1 probe (C). The rNBC signal overlaps the SGLT1 signal in the outermost part of the medullary ray, which corresponds to the distal straight part of S2 segments. IM, inner medulla; asterisks, blood vessels. Bars = 1.5 mm in A and 100 µm in B and C.

The in situ localization of NBC is more restricted spatially than the well-documented Na⁺- cotransporter activity, which has been detected in S1 and convoluted segments of S2 (1, 3, 8, 9, 12, 20, 30, 31), as well as S3 (8, 14, 18). Several polyclonal antibodies developed in one of our laboratories (W. F. Boron) demonstrate strong and specific basolateral immunoreactivity in S1 and S2 segments of rabbit and rat kidneys (Ref. 27; M. O. Bevensee and B. M. Schmitt, unpublished observations). The differences between the in situ hybridization and immunocytochemistry data could reflect differences in the sensitivities of the methods, real differences in the relative abundances of NBC mRNA vs. NBC protein, or differences in detected NBC isoforms.

Functional Properties of rNBC in Xenopus Oocytes

Expression of "native" rNBC. To confirm that the isolated rat kidney clone encodes an electrogenic Na⁺- cotransporter, we injected cRNA from the clones into Xenopus oocytes to evaluate cotransporter activity (see METHODS). Figure 4A shows that, unlike the salamander clone, expression of rNBC subcloned into pBluescript was weak, as judged by the responses to the expression assay (METHODS). First, adding 5% CO₂/33 mM (pH 7.5) caused the rNBC-expressing oocytes to hyperpolarize by only 15-20 mV (due to Na⁺ and influx). By contrast, in oocytes expressing the salamander clone, one-third of the [], 1.5% CO₂/10 mM , typically caused a hyperpolarization of ~50 mV. In water-injected controls, addition of 5% CO₂/33 mM caused a depolarization of about +20 mV, rather than a hyperpolarization. Second, removing Na⁺ (choline replacement) in the continued presence of 5% CO₂/33 mM depolarized rNBC-expressing oocytes (due to electrogenic Na⁺/ efflux), but only by 5-10 mV. Once again, expression of salamander NBC was more robust than that of rNBC. Removing extracellular Na⁺ from the 1.5% CO₂/10 mM solution bathing oocytes expressing the salamander NBC elicited a 50-mV depolarization. In water-injected control oocytes, removing Na⁺ from even the 5% CO₂/33 mM solution hyperpolarized the oocytes by 3-5 mV, rather than depolarizing them. Removing Cl (gluconate replacement) did not alter pH_i or V_m in oocytes expressing rNBC.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Expression of rNBC in Xenopus oocytes. Expression of rNBC (10 ng/50 nl cRNA solution) was first detectable on day 3 after injection of cRNA and continued until at least day 13. A: clone r715na (pBluescript) expression in oocytes. External solution was switched from ND-96 solution (CO2/ free) to 5% CO2/33 mM (pH 7.5). Na+ was then removed two times and Cl once in the presence of CO2/. In comparison to aNBC, r715na (pBluescript) expresses functionally very poorly in oocytes even when bathed with threefold increased . B: rNBC-pTLN2 expression in oocytes. An experiment is shown in which the external solution was switched from ND-96 solution (CO2/ free) to only 1.5% CO2/10 mM (pH 7.5). Na+ was then removed three times in CO2/. Measured functional activity of rNBC is increased substantially. That is, using a lower level of CO2/ (1.5% CO2/10 mM vs. 5% CO2/33 mM ), we observed a much larger change in membrane voltage (Vm) when applying CO2/ to oocytes injected with rNBC in the pTLN2 vector (~60 mV) than with rNBC in the pBluescript vector. Thus placing the mammalian, r715na-NBC-cDNA within this Xenopus context is apparently beneficial for expression in Xenopus oocytes. C: intracellular pH (pHi) experiment on rNBC-pTLN2 expressing oocyte (top and bottom). An experiment similar to B is shown while also monitoring pHi.

Expression of rNBC in a Xenopus expression vector. Despite the 86% identity to the aNBC coding region, oocytes expressed the rNBC clone poorly when compared with oocytes expressing aNBC (23). We entertained the hypothesis that Xenopus oocytes would express rNBC better if the 5'- and 3'-UTRs were more typical of those present in Xenopus. Therefore, we subcloned the original r715na cDNA, which included the coding sequence and ~500 bp of the rat 3'-UTR, into the Xenopus expression vector, pTLN2. As shown in Fig. 4, B and C, expression is increased substantially when this r715na cRNA is flanked by the Xenopus -globin UTRs. Thus, in an oocyte expressing rNBC in the pTLN2 vector, adding one-third the [] elicited a hyperpolarization three- to fourfold greater (60 mV with 10 mM rather than 15-20 mV obtained with 33 mM and the other vector). After the initial CO₂-induced acidification, the experiment in Fig. 4C shows a pH_i recovery, due to continued Na⁺/ influx into the oocyte. Subsequently removing Na⁺ elicited a depolarization of some 60 mV and rapidly decreased pH_i (Fig. 4C). This depolarization was far larger than the 5- to 10-mV depolarization observed with the r715na-pBluescript clone, obtained with a threefold higher [] (Fig. 4, A and B).

In light of our results with rNBC in two different vectors, it is interesting to note that we had found it impossible to expression clone NBC from rabbit kidney (23). It may be that the expression in the Xenopus oocyte system of mammalian NBC depends critically on the presence of 5' and/or 3' amphibian UTRs, which may enhance the initiation of translation and/or the stability of the cRNA.

In summary, we have cloned an electrogenic Na⁺- cotransporter from the rat kidney, rNBC. Not only is rNBC 86% identical to our original Ambystoma NBC, aNBC, but the mRNA is predominantly expressed in the kidney. Functional expression of rNBC in Xenopus oocytes illustrates that rNBC is electrogenic, dependent on Na⁺, and dependent on .

	ACKNOWLEDGEMENTS

We thank Dr. Chairat Shayakul for providing the rat kidney cortex library. We thank Prof. Thomas Jentsch for the gift of the pTLN vector. We also thank Drs. Mark O. Bevensee and Bernhard M. Schmitt for sharing unpublished antibody data and for helpful comments on the manuscript.

	FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-30344 (to W. F. Boron), DK-40163 (to M. A. Hediger), and DK-09342 (to M. F. Romero) and by the American Heart Association (to M. F. Romero).

Present address of M. F. Romero and address for reprint requests: Dept. Physiology and Biophysics, Case Western Reserve Univ., School of Medicine, 2119 Abington Rd., Cleveland, OH 44106-4790.

¹ pTLN is a modification of pSP6 (13, 17), with the multiple cloning sites between the 5'- and 3'-UTRs of Xenopus -globin sequences. pTLN was cut at Xba I and Xho I and treated with calf intestinal alkaline phosphatase. The modified polylinker was constructed by annealing of primers designed to produce the following sites: 5' Spl I-Rsr II-Sst II-Not I/Eag I-Nde I-Asu I-Avr II-Bgl II-BstE II 3'. The Xho I site was not regenerated in pTLN2. Linearization sites (3' of the -globin 3'-UTR) were unaltered.

Received 12 September 1997; accepted in final form 11 November 1997.

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