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J. Biol. Chem., Vol. 275, Issue 45, 35486-35490, November 10, 2000

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The Na⁺-driven Cl/HCO₃ Exchanger

CLONING, TISSUE DISTRIBUTION, AND FUNCTIONAL CHARACTERIZATION^*,

Chang-Zheng Wang, Hideki Yano^§, Kazuaki Nagashima^¶, and Susumu Seino

From the Department of Molecular Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan

Received for publication, July 13, 2000, and in revised form, September 8, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The Na⁺-driven Cl/HCO₃ exchanger is an important regulator of intracellular pH in various cells, but its molecular basis has not been determined. We show here the primary structure, tissue distribution, and functional characterization of Na⁺-driven chloride/bicarbonate exchanger (designated NCBE) cloned from the insulin-secreting cell line MIN6 cDNA library. The NCBE protein consists of 1088 amino acids having 74, 72, and 55% amino acid identity to the human skeletal muscle, rat smooth muscle, and human kidney sodium bicarbonate cotransporter, respectively. The protein has 10 putative membrane-spanning regions. NCBE mRNA is expressed at high levels in the brain and the mouse insulinoma cell line MIN6 and at low levels in the pituitary, testis, kidney, and ileum. Functional analyses of the NCBE protein expressed in Xenopus laevis oocytes and HEK293 cells demonstrate that it transports extracellular Na⁺ and HCO₃ into cells in exchange for intracellular Cl and H⁺, thus raising the intracellular pH. Thus, we conclude that NCBE is a Na⁺-driven Cl/HCO₃ exchanger that regulates intracellular pH in native cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Regulation of intracellular pH (pH_i)¹ in response to various stimuli is critical in many cellular functions (1-4). A family of bicarbonate transporters is the major pH_i regulator under physiological conditions in animal cells (5). Bicarbonate transporters are divided functionally into four groups (5): the Na⁺-independent Cl/HCO₃ exchanger (alternatively called an anion exchanger (AE)); the Na⁺-HCO₃ cotransporter (NBC); the K⁺-HCO₃ cotransporter; and the Na⁺-driven Cl/HCO₃ exchanger. Three AEs (3) and three NBCs (6-10) have been cloned and functionally characterized, but the molecular structure of the K⁺-HCO₃ cotransporter and the Na⁺-driven Cl/HCO₃ exchanger has remained unknown.

The Na⁺-driven Cl/HCO₃ exchanger was first discovered in invertebrate neurons (11) and was found later in vertebrate neurons and non-neuronal cells, including the brain (12), vascular endothelial cells (13), sperm (14), kidney (15), and pancreatic -cells (16). The Na⁺-driven Cl/HCO₃ exchanger is an intracellular pH regulator that transports extracellular Na⁺ and HCO₃ into cells in exchange for intracellular Cl and H⁺ playing an important role in cellular alkalinization (5, 11).

In pancreatic islet -cells, glucose is physiologically the most important regulator of insulin secretion. It has been shown that glucose metabolism induces an increase in pH_i in pancreatic -cells (17-21) and that this glucose-induced rise in pH_i is evoked primarily by the action of the Na⁺-driven Cl/HCO₃ exchanger (16). To determine the structure and functional roles of the Na⁺-driven Cl/HCO₃ exchanger, we attempted to clone the Na⁺-driven Cl/HCO₃ exchanger from the mouse insulin-secreting cell line, MIN6. We describe here the primary structure, tissue distribution, and functional properties of a Na⁺-driven Cl/HCO₃ exchanger, designated NCBE.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

cDNA Cloning and RNA Blot Analysis-- A partial cDNA fragment of human kidney NBC cDNA (7) was amplified by polymerase chain reaction (PCR) using a human kidney cDNA as a template. The sense and antisense primers used were 5'-TTTGGAGAAAACCCCTGGT-3' (nt 2232-2250) and 5'-TGACATCATCCAGGAAGCTG-3' (nt 2912-2931). PCR was performed for 40 cycles under the following conditions: denaturation at 94 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 45 s in a thermal cycle GeneAmp PCR system 9600 (Applied Biosystems, Foster City, CA). The 700-base pair PCR product was subjected to screening of a MIN6 cDNA library (22) as a probe under the low stringency conditions previously described (23). Positive clones were subcloned in pGEM-3Z vectors (Promega, Madison, WI) and sequenced in both directions using an ABI PRISM^TM 377 DNA sequencer (Applied Biosystems).

RNA blot analysis was performed using 10 µg of total RNA from the various tissues and cells. The RNAs were denatured with formaldehyde, electrophoresed on a 1% agarose gel, and transferred to a nylon membrane. The blot was probed with NCBE cDNA (nt 1-840) under the standard conditions described previously (24). The blots were washed in 0.1× SSC and 0.1% SDS at room temperature for 1 h and then at 50 °C for 1 h before autoradiography.

Reverse Transcription (RT)-PCR-- Total RNA was prepared with TRIZOL reagent (Life Technologies, Inc.) from isolated mouse pancreatic islets. First-strand cDNA (10 ng) was generated using Superscript^TM II reverse transcriptase (Life Technologies) with random primers. PCR was performed with the Expand high fidelity PCR system (Roche Diagnostics, Mannheim, Germany) using about 1 ng of template DNA in a 20-µl reaction volume under standard conditions. The sense and antisense primers used were 5'-GTCATGTTAGACCAACAG-GT-3' (nt 4283-4302) and 5'-GTTGTAATAGCGACACTC-3' (nt 4911-4928). The product was resolved on a 1% agarose gel and confirmed by DNA sequencing.

Experimental Solutions-- The composition of the experimental solutions (solutions A to F for oocyte experiments and solutions G to O for HEK293 cell experiments) is listed in Table I.

View this table: [in this window] [in a new window]   Table I Composition of experimental solutions Solutions A, B, C, D, E, and F for experiments with oocytes were used as standard solution, Na+-free solution, Cl-free solution, HCO3-containing solution, HCO3-free solution, and HCO3-free washing solution, respectively. The pH of all solutions was adjusted to 7.4. HCO3 solutions (solutions E and F) were bubbled with 100% O2 to remove trace CO2 and HCO3. Solutions G, H, I, J, K, L, M, N, and O for experiments with HEK293 cells were used as Na+-free solution, NH4Cl-containing Na+-free solution, Na+-containing solution, Na+- and HCO3-free solution, NH4Cl-containing Na+- and HCO3-free solution, Na+-containing HCO3-free solution, Na+- and Cl-free solution, NH4Cl-containing Na+- and Cl-free solution, and Na+-containing Cl-free solution, respectively. All solutions were bubbled with 95% O2 and 5% CO2 and adjusted to pH 7.4. NMG, N-methyl-D-glucamine.

Functional Analysis of NCBE in Xenopus laevis Oocytes-- The coding sequence of NCBE in pSD5 was linearized by digestion with FspI and in vitro transcribed with SP6 RNA polymerase as described previously (24). Defolliculated oocytes were injected with NCBE cRNA (50 nl, 0.5 µg/µl) or water and incubated in 1× MBS medium (88 mM NaCl, 1 mM KCl, 0.8 mM MgCl₂, 0.4 mM CaCl₂, 0.3 mM Ca (NO₃)₂, 2.4 mM NaHCO₃, and 7.5 mM Tris, pH 7.4) for 3-5 days at 18 °C before the studies. For the study of ²²Na⁺ or ³⁶Cl uptake, the oocytes were preincubated for 1 h at 18 °C in solution A (ml/oocyte) and then incubated in 1.4 ml of solution A, B, or C bubbled with 1.5% CO₂ or solution D or E without CO₂ containing 0.074 MBq of ²²NaCl or H³⁶Cl (PerkinElmer Life Science Products). A 10-µl aliquot was removed from the incubation solution for later determination of ²²Na⁺- or ³⁶Cl-specific activity. ²²Na⁺ or ³⁶Cl uptake was terminated after 15 min by three washes with the respective ice-cold solutions, with the exception of solutions D and E, which were replaced with ice-cold solution F. The oocytes were then dissolved in 0.5 ml of 5% SDS, and 4.5 ml of aqueous counting scintillant (Amersham Pharmacia Biotech) was added. ²²Na⁺ uptake for 15 min in solution A was examined next in the presence of 300 µM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, Sigma), an inhibitor of anion transporters. For the study of ³⁶Cl efflux, oocytes were preincubated in solution A with ³⁶Cl for 30 min for loading. The oocytes were washed three times with solution A, B, C, D, or E at 18 °C and then placed in 1.5 ml of each solution, respectively, at 18 °C for 15 min to determine ³⁶Cl efflux. The ³⁶Cl activities of the oocytes and each solution were measured. The ³⁶Cl efflux is presented as the percent relative to incorporated ³⁶Cl. The ³⁶Cl efflux in solution A for 15 min was also examined in the presence of 300 µM DIDS. The ²²Na⁺ and ³⁶Cl activities were measured with a beta scintillation counter (Aloka, Tokyo).

Functional Analysis of NCBE in HEK293 Cells-- HEK293 cells were plated at a density of 3 × 10⁵ cells/3.5-cm diameter dish containing CELLocate coverslips (Eppendorf) and were cultured in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% fetal bovine serum, streptomycin (100 µg/ml), and penicillin (60.5 µg/ml) at 37 °C under a humidified condition of 95% air and 5% CO₂. Cells were cotransfected with 1 µg/well of the full-length NCBE cDNA in the pcDNA3.1 vector (Invitrogen, Groningen, The Netherlands) and 0.05 µg/well of enhanced green fluorescent protein (GFP) vector, pEGFP-N1 (CLONTECH, Palo Alto, California), for transfection marker, using LipofectAMINE, LipofectAMINE Plus, and Opti-MEM I reagents (Life Technologies, Inc.) according to the manufacturer's instructions. The GFP-expressing cells from more than 20 monolayers were studied 48-72 h after transfection. HEK293 cells were loaded with 1 µM 2',7'-bis-(2-carboxyethyl)-5-(6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM, Molecular Probes, Eugene, OR) for 1 h and monitored for changes in pH_i (7) by a dual-excitation wavelength method with a computerized image processor (490 nm/450 nm excitation, 520-560 nm emission) (Argus-50, Hamamatsu Photonics, Hamamatsu, Japan) (7, 21). pH_i was estimated as the difference between pH_i before and 10 min after switching to the test solution. The pH_i calibration was generated using the KCl/nigericin technique (25). In all experiments, the cells were first acidified by NH₄⁺ prepulse with 40 mM NH₄Cl-containing solutions (solutions H, K, and N) for 5 min before switching to the Na⁺-containing respective test solutions (7). To examine Na⁺ dependence of the pH_i recovery (pH_i) from intracellular acidification by solution H, solutions G and I were used, and to test for HCO₃ dependence, solutions J, K, and L were used. To determine Cldependence, solutions M, N, and O (in this case, cells transfected with NCBE were preincubated in solution M for more than 1 h to decrease intracellular Cl) were used, and the results were compared with control and NCBE-transfected cells. All solutions were bubbled with 95% O₂ and 5% CO₂, adjusted to pH 7.4. The osmolarity of each solution was adjusted with sucrose. The assays were carried out at 37 °C.

Statistics-- The results are expressed as means ± S.E. The statistical significance between each experiment was determined by Student's t test.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Cloning of the NCBE cDNA and Predicted Protein-- Using a partial human kidney NBC (7) cDNA as a probe, we cloned a cDNA encoding NCBE by screening a MIN6 cDNA library. The composite nucleotide sequence of 5385 base pairs contains an open reading frame following an in-frame termination signal upstream of the ATG, which encodes a protein of 1088 amino acids with a predicted molecular mass of 122 kDa (GenBank^TM/EBI/DDBJ data bank with accession no. AB033759, available on-line as Fig. 1S). A hydropathy analysis suggests that NCBE has 10 putative membrane-spanning segments (26). There are three potential N-linked glycosylation sites in the extracellular loops between the third (TM3) and fourth (TM4) membrane-spanning region (Asn-647, Asn-657, and Asn-667). Comparison of the amino acid sequences between NCBE and other bicarbonate transporters shows that NCBE has 74, 72, 55, 49, 38, and 34% amino acid identity to human skeletal muscle NBC (9), rat smooth muscle NBC (8), human kidney NBC (7), Drosophila Na⁺-driven anion exchanger (NDAE1) (30), mouse brain AE3 (31), and mouse erythrocyte AE1 (32), respectively, indicating that NCBE represents a novel bicarbonate transporter. NCBE is also homologous to several members of a bicarbonate transporter superfamily, the functional properties of which have not yet been characterized. NCBE has 76, 74, 52, and 49% amino acid identity to an NBC-related clone from human brain (SLC4A8, GenBank^TM accession no. NM004858), putative human retinal NBC (33), Drosophila gene product alt 1 (GenBank^TM accession no. AAF52496), and Drosophila gene product alt 2 (GenBank accession no. AAF52497), respectively. The amino acid sequence in the putative transmembrane regions is well conserved between NCBE and members of the bicarbonate transporter superfamily, whereas the intracellular amino- and carboxyl-terminal regions and a large extracellular loop between the third and fourth membrane-spanning region are rather divergent.

Tissue Expression of NCBE-- RNA blot analysis revealed a 5.5-kilobase NCBE mRNA expressed at high levels in brain and insulin-secreting cell line MIN6 cells and expressed at low levels in the pituitary, testis, kidney, and ileum (Fig. 1a). RT-PCR analysis shows that NCBE mRNA is also expressed in pancreatic islets (Fig. 1b).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Expression of NCBE mRNA. a, tissue distribution of NCBE mRNA. RNA blot analysis of NCBE mRNA in various rat tissues and hormone-secreting cell lines is shown. The size of the hybridized transcripts is indicated. Sk., skeletal; Pan., pancreatic; kb, kilobases. b, RT-PCR detection of NCBE mRNA in mouse pancreatic islets. DNA length markers and RT-PCR products are shown in lanes 1 and 2, respectively.

Functional Expression of NCBE in Xenopus Oocytes and HEK293 Cells-- We first examined the functional properties of NCBE using a Xenopus oocyte system. ²²Na⁺ and ³⁶Cl uptake or efflux were measured 3-5 days after injection of the cRNAs or water as control. By bubbling with 1.5% CO₂ or using 16 mM butyric acid without CO₂ to acidify the oocytes (6), we examined the time course of ²²Na⁺ uptake. Because oocytes injected with water showed almost no increase, but NCBE-expressing oocytes showed a linear increase in ²²Na⁺ uptake from 5 to 45 min (data not shown), we measured the uptake at 15 min to calculate ²²Na⁺ uptake (nmol/oocyte/h). As shown in Fig. 2a, in standard solution (solution A) ²²Na⁺ uptake in NCBE-expressing oocytes was 31.4 ± 2.1 nmol/oocyte/h (n = 21), whereas uptake in water-injected oocytes was 1.6 ± 0.3 nmol/oocyte/h (n = 22), indicating that the ²²Na⁺ uptake in NCBE-expressing oocytes was 20-fold greater than in control oocytes (p < 0.05). In contrast, the ²²Na⁺ uptake in NCBE-expressing oocytes was almost zero (n = 10) in Na⁺-free solution (solution B), but it increased in an extracellular Na⁺ concentration-dependent manner (data not shown), indicating that the activity of NCBE depends on extracellular Na⁺. When oocytes were acidified with butyric acid rather than CO₂ (8), the ²²Na⁺ uptake in NCBE-expressing oocytes in HCO₃-containing solution (solution D) was 19 ± 2.8 nmol/oocyte/h (n = 8). Thus, acidification with butyric acid is also effective on uptake of ²²Na⁺. To determine the effect of HCO₃, we used HCO₃-free solution (solution E) with butyric acid without CO₂. Under this condition, ²²Na⁺ uptake was significantly decreased (3.1 ± 0.5 nmol/oocyte/h, n = 8, p < 0.05), indicating that extracellular HCO₃ is required to transport Na⁺ into cells. We then examined the effect of Cl on ²²Na⁺ uptake. ²²Na⁺ uptake in NCBE-expressing oocytes in solution C was 18.6 ± 1.6 nmol/oocyte/h (n = 16), decreased to 50% of that in standard solution, indicating that extracellular Cl accelerates NCBE activity and that there is ²²Na⁺ uptake even under extracellular Cl-free conditions (also see on-line supplemental information, Table IIS). To determine whether extracellular Cl accelerates NCBE activity and whether intracellular Cl is exported by NCBE, we measured the uptake and efflux of ³⁶Cl. ³⁶Cl uptake was measured in NCBE-expressing oocytes or control oocytes injected with water in solution A, B, C, D, or E. Although control oocytes showed no increase in ³⁶Cl uptake, NCBE-expressing oocytes showed a significant increase in ³⁶Cl uptake in all solutions except solution C (Fig. 2b). The values were 24 ± 2.8 (n = 9), 32 ± 0.8 (n = 10), 0.44 ± 0.1 (12), 13 ± 0.1 (n = 11), and 48 ± 1.2 nmol/oocyte/h (n = 7) in solutions A, B, C, D, and E, respectively. The increase in ³⁶Cl uptake of NCBE-expressing oocytes in these solutions indicates that NCBE transports extracellular Cl into the cell and that the importing activity of Cl is significantly increased in the absence of extracellular Na⁺ (solution A versus solution B, p < 0.05) or HCO₃ (solution D versus solution E, p < 0.05). We then measured the Cl efflux of the NCBE-expressing oocytes (Fig. 2c). The rate (%) of ³⁶Cl efflux was 74.7 ± 2.8 (n = 8), 43.0 ± 2.0 (n = 9), 42.3 ± 1.3 (n = 17), 48.0 ± 4.0 (n = 8), and 17.0 ± 4.0% (n = 8) in solutions A, B, C, D, and E, respectively (solution A versus other solutions, p < 0.05). These results indicate that: 1) 75% of intracellular ³⁶Cl is exported out of the cell in standard solution, 2) extracellular Na⁺ is necessary for exporting intracellular Cl, 3) extracellular HCO₃ is essential for exporting intracellular Cl, and 4) extracellular Cl accelerates Na⁺ uptake and Cl efflux. Because Cl can be transported into and out of the cell when NCBE-expressing oocytes are acidified, the transporting activity appears to be bidirectional. The functional properties of NCBE under different conditions in Xenopus oocytes are summarized in Fig. 2d. The direction of ion movement through NCBE under the physiological condition (Fig. 2d-1) or the extracellular Cl-free condition (Fig. 2d-2) during the period of decrease in pH_i is forward, whereas the ion movement through NCBE under the extracellular Na⁺-free (Fig. 2d-3) or HCO₃-free (Fig. 2d-4) condition is reverse. Taken together, these findings suggest that NCBE exchanges extracellular Na⁺ and HCO₃ with intracellular Cl under the physiological condition, that is, in the presence of extracellular Na⁺, HCO₃, and Cl, when the cells are acidified. We also examined the effect of DIDS on ²²Na⁺ uptake and ³⁶Cl efflux. ²²Na⁺ uptake in NCBE-expressing oocytes in standard solution was 6.0 ± 0.7 nmol/oocyte/h (n = 14) in the presence of 300 µM DIDS, indicating that DIDS decreased ²²Na⁺ uptake to 20% of that in solution A (Fig. 2a). The ³⁶Cl efflux in NCBE-expressing oocytes in solution A was 41 ± 2% (n = 9) in the presence of 300 µM DIDS. Although DIDS decreased the ³⁶Cl efflux to 55%, the value of ³⁶Cl efflux was at almost the same level as in solution C (Fig. 2c).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Functional analysis of NCBE in Xenopus laevis oocytes. a, effects of different ions on 22Na+ uptake in oocytes injected with NCBE cRNA or water. 22Na+ uptake (expressed as nmol/oocyte/h) in oocytes injected with water (open columns) or NCBE cRNA (filled columns) 3-5 days after injection was measured in standard solution (solution A), Na+-free solution (solution B), Cl-free solution (solution C), HCO3-containing solution (solution D), HCO3-free solution (solution E) and standard solution in the presence of 300 µM DIDS (solution A+DIDS). b, effects of different ions on 36Cl uptake in oocytes injected with NCBE cRNA or water. c, effects of different ions on 36Cl efflux in oocytes injected with NCBE cRNA. The percent 36Cl efflux is shown. d, functional properties of NCBE during the period of decrease in pHi. The direction of ion movement through NCBE under various conditions is shown: 1, physiological; 2, extracellular Cl-free; 3, extracellular Na+-free; 4, HCO3-free. Thin arrows indicate decreased activity. Note that solutions A, B, and C were bubbled with 1.5% CO2, but solutions D and E were without CO2. Results were obtained from 2 to 3 independent experiments. The values represent the mean ± S.E. of 7-22 oocytes from each experiment.

To clarify the role of NCBE in regulating pH_i, pH_i changes under the various conditions were measured in HEK293 cells transiently transfected with NCBE. All experiments were carried out in acidified pH_i conditions with NH₄ prepulse (with solutions H, K, or N). To determine whether the change in pH_i is dependent on extracellular Na⁺, the environment of the cells was changed from Na⁺-free solution (solution G) to Na⁺-containing solution (solution I). In the presence of 1 mM 5-(N-ethyl-N-isopropyl-amiloride (EIPA), a specific inhibitor of the Na⁺/H⁺ exchanger, rapid pH_i recovery (pH_i) was observed only in the NCBE-transfected cells (pH_i was 0.239 ± 0.028 in NCBE-transfected cells (n = 97) and 0.003 ± 0.015 in control (n = 70), p < 0.05) (Fig. 3a). This pH_i recovery was partially inhibited by 300 µM DIDS (pH_i was 0.023 ± 0.042 (n = 89), p < 0.05) (Fig. 3a). To determine whether the change in pH_i is bicarbonate-dependent, the environment of NCBE-transfected cells was changed from HCO₃-free and Na⁺-free solution (solution J) to HCO₃-free but Na⁺-containing solution (solution L) in the presence of 1 mM EIPA. No pH_i recovery was detected (pH_i was 0.002 ± 0.014 (n = 71)) (Fig. 3b). We also examined Cl dependence. NCBE-transfected cells were kept in Cl-free solution (under the intracellular Cl-depleted condition) throughout the experiments. Under this condition, the environment of the cells was changed from Na⁺-free (solution M) to Na⁺-containing solution (solution O). In the presence of 1 mM EIPA, pH_i recovery was not detected (pH_i was 0.067 ± 0.012 (n = 95)) (Fig. 3c). These results show that pH_i recovery from intracellular acidification is detected only in the presence of extracellular Na⁺, HCO₃, and intracellular Cl.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Functional analysis of NCBE in HEK293 cells. HEK293 cells were transfected with NCBE and assayed for Na+, HCO3, and Cl dependence in pHi recovery. a, representative trace of control (nontransfected) cells and NCBE-transfected cells with or without 300 µM DIDS as indicated. The environment of the cells was changed from Na+-free solution (solution G) to Na+-containing solution (solution I). Each trace is from a different cell; three separate traces are superimposed. b, the environment was changed from Na+-free (solution J) to Na+-containing solution (solution L) under HCO3-free conditions. c, the environment was changed from Na+-free (solution M) to Na+-containing solution (solution O) under Cl-free conditions.

Functional studies of NCBE heterologously expressed in Xenopus oocytes and HEK293 cells show that NCBE causes pH_i recovery from acute intracellular acidification by transporting extracellular Na⁺ and HCO₃ in exchange for intracellular Cl in the presence of extracellular Na⁺, HCO₃, and Cl. NCBE is functionally distinct from the anion exchangers (3) and the Na⁺-HCO₃ cotransporters (9, 10), because NCBE-expressing oocytes show an increase in ²²Na⁺ uptake that is dependent on Cl and HCO₃, and NCBE-expressing HEK293 cells show an increase in pH_i that is dependent on extracellular Na⁺, HCO₃, and Cl. These properties are similar to those of the Na⁺-driven Cl/HCO₃ exchanger described in native cells (14, 16, 27-29), indicating that the cloned NCBE is a Na⁺-driven Cl/HCO₃ exchanger. The functional properties of NCBE are different from those of the recently identified Drosophila NDAE1, which does not require HCO₃ for transport activity (30).

Expression of NCBE mRNA in insulin-secreting cell line MIN6 and pancreatic islets implies its physiological relevance. It has been shown that glucose-induced insulin secretion is accompanied by a rise in pH_i in pancreatic islet -cells (17-21). Although several pH_i regulators have been suggested to be present in pancreatic -cells (16, 21), the molecular basis of these regulators is not known. NCBE is the first pH_i-regulating exchanger of which the primary structure and functional properties have been determined. NCBE most likely contributes to the pH_i recovery process in pancreatic -cells that have been acidified by glucose metabolism. NCBE mRNA also is present in the testis, although its level is low. It has been shown that pH_i regulates many sperm functions including sperm capacitation (1, 2, 14). Because sperm capacitation results in pH_i increases that require a functional Na⁺, Cl, and HCO₃-dependent acid efflux pathway (14), NCBE could participate in the process. NCBE mRNA is also expressed in the brain at high levels. Based on physiological studies (12, 27), NCBE may be present in hippocampal neurons and astrocytes, but its physiological significance in such cells is not known at present. Further investigation is necessary to clarify the structure and function relationships of NCBE and its physiological roles in various tissues.

	ACKNOWLEDGEMENTS

We thank Dr. K. Minami for preparing mouse pancreatic islets and Dr. P. Beguin for providing the expression vector of pSD5. Part of this study was performed in the Radioisotope Center of Chiba University.

	FOOTNOTES

^* This work was supported by Grant-in-aid 10NP0201 for Creative Basic Research from the Ministry of Education, Science, Sports and Culture and by grants from the Ministry of Health and Welfare, Japan, Novo Nordisk Pharma Ltd., Yamanouchi Foundation for Research on Metabolic Disorders, and Suzuken Memorial Foundation.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 on-line version of this article (available at http://www.jbc.org) contains Fig. 1S and Table IIS.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AB033759 (for NCBE).

Supported by a Japan Society for the Promotion of Science (JSPS) postdoctoral fellowship for foreign researchers.

^§ To whom correspondence should be addressed. Tel.: 81-43-226-2188; Fax: 81-43-226-2191; E-mail: hyano@molmed.m.chiba-u.ac.jp.

^¶ Supported by a JSPS research fellowship for young scientists.

Published, JBC Papers in Press, September 18, 2000, DOI 10.1074/jbc.C000456200

	ABBREVIATIONS

The abbreviations used are: pH_i, intracellular pH; NCBE, Na⁺-driven Cl/HCO₃ exchanger; NBC, sodium bicarbonate cotransporter; AE, anion exchanger; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; EIPA, 5-(N-ethyl-N-isopropyl)-amiloride; GFP, green fluorescent protein; nt, nucleotide(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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