Functional characterization of human NBC4 as an electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (NBCe2)

Leila V. Virkki1, Darren A. Wilson2, Richard D. Vaughan-Jones2, and Walter F. Boron1

1 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520; and 2 University Laboratory of Physiology, University of Oxford, Oxford OX1 3PT, United Kingdom


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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We have functionally characterized Na+-driven bicarbonate transporter (NBC)4, originally cloned from human heart by Pushkin et al. (Pushkin A, Abuladze N, Newman D, Lee I, Xu G, and Kurtz I. Biochem Biophys Acta 1493: 215-218, 2000). Of the four NBC4 variants currently present in GenBank, our own cloning efforts yielded only variant c. We expressed NBC4c (GenBank accession no. AF293337) in Xenopus laevis oocytes and assayed membrane potential (Vm) and pH regulatory function with microelectrodes. Exposing an NBC4c-expressing oocyte to a solution containing 5% CO2 and 33 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> elicited a large hyperpolarization, indicating that the transporter is electrogenic. The initial CO2-induced decrease in intracellular pH (pHi) was followed by a slow recovery that was reversed by removing external Na+. Two-electrode voltage clamp of NBC4c-expressing oocytes revealed large HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>- and Na+-dependent currents. When we voltage clamped Vm far from NBC4c's estimated reversal potential (Erev), the pHi recovery rate increased substantially. Both the currents and pHi recovery were blocked by 200 µM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). We estimated the transporter's HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry by measuring Erev at different extracellular Na+ concentration ([Na+]o) values. A plot of Erev against log[Na+]o was linear, with a slope of 54.8 mV/log[Na+]o. This observation, as well as the absolute Erev values, are consistent with a 2:1 stoichiometry. In conclusion, the behavior of NBC4c, which we propose to call NBCe2-c, is similar to that of NBCe1, the first electrogenic NBC.

intracellular pH; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; microelectrodes; stoichiometry; Xenopus laevis oocytes


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE EXPRESSION CLONING of the first Na+-driven bicarbonate transporter (NBC) by Romero et al. (36) led to the cloning of many other electrogenic and electroneutral Na+-driven HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporters. The original electrogenic NBC in mammals (NBCe1) is currently represented by three splice variants: NBCe1-A, which is found predominantly in kidney (8, 35); NBCe1-B, which is found in pancreas, heart, and many other tissues (1, 12); and NBCe1-C, which is found predominantly in brain (4).

The electroneutral Na+-driven HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporters in mammals share ~70% sequence identity on the amino acid level and appear to fall into two functional groups. The first group mediates Cl--independent Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport. The first member of this group has been cloned under various names, including NBC2 from human retina (22), NBC3 from human skeletal muscle (31), and NBCn1 from rat smooth muscle (11). However, the NH2-terminal 90 amino acids of NBC2 are missing, and the next 28 represent a cloning artifact.

The second group of electroneutral Na+-driven HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporters mediates the Cl--dependent transport of Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, functionally described as Na+-driven Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange. In mammals, this transporter is represented by a clone from human brain, NDCBE1 (18). A partial clone had been named NBC3 (3), representing degeneracy in the nomenclature. A related Drosophila clone (NDAE) encodes a Na+-driven anion exchanger (37).

NBC4, originally cloned by Pushkin et al. (32), is a new member of the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporter superfamily. It is ~60% identical at the amino acid level to the three electrogenic NBCe1 splice variants and 40-50% identical to the electroneutral Na+-driven HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporters. A Northern blot (32) and a similar mirror image (33) showed high levels of NBC4 mRNA in liver, spleen, and testes and moderate levels in heart, kidney, placenta, and stomach.

We mapped the NBC4 sequence onto a recent topology model developed for the anion exchanger AE1 (Fig. 1A), which is in the same superfamily as the Na+-coupled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporters. Pushkin et al. (32, 33) described two variants of NBC4, NBC4a and NBC4b, and have submitted two additional sequences, NBC4c and NBC4d, to GenBank. As shown in Fig. 1B, NBC4a and NBC4b each contain, beginning just after the predicted transmembrane segment (TM) 11, a putative exon not present in either NBC4c or NBC4d. NBC4b has a unique 16-bp insertion in its last TM. This insertion/frameshift introduces a premature stop codon and causes the deduced amino acid sequence to exhibit little homology with other members of the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporter (BT) superfamily. NBC4d is missing two exons present in NBC4c, resulting in the elimination of all amino acids between the putative end of TM 10 and near the beginning of TM 14. To date, no functional data have been reported for any of the four NBC4 variants.


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Fig. 1.   Na+-driven bicarbonate transporter (NBC)4 sequence analysis and topology. A: using sequence alignment analysis, we mapped the NBC4c amino acid sequence on a recent topology model developed for anion exchanger (AE)1 (43). The putative NH2 and COOH termini are intracellular. Gray cylinders indicate transmembrane segments (TMs) 1-14, of which TM 12 is nonhelical (stippled). Four glycosylation consensus motifs are located on the large extracellular loop connecting TMs 5 and 6. B: gray bars indicate the relative length of the amino acid sequence of the 4 NBC4 variants. The position of the putative TMs are indicated by numbered vertical light gray bars. Variant b has a 16-bp insertion (cross-hatching on dark background) in the middle of the last TM, which causes a frameshift and results in a unique COOH terminus (cross-hatching on white background). Variant c lacks a 48-bp exon (between TMs 11 and 12) present in variants a and b. Variant d lacks 2 exons in addition to the 1 missing in variant c, resulting in the loss of the region between TMs 10 and 14.

We have undertaken the present study to investigate the functional properties of NBC4. Using PCR, we obtained nine clones corresponding to the NBC4c coding region but none corresponding to the other NBC4 variants. We have expressed NBC4c in Xenopus laevis oocytes and assayed for pH regulatory function as well as membrane potential and ionic currents. The results show that NBC4c is an electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter that, at least in the oocyte, appears to operate with a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry of 2:1.

Portions of this work have been published in abstract form (44).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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cDNA Cloning

On the basis of the published cDNA sequence for NBC4a (GenBank accession no. AF243499; Ref. 33), we designed oligonucleotide primers corresponding to the 5' and 3' regions of the open reading frame to this clone. We performed PCR with sense primer 5'-CCTCGAGTCATGAAGGTGAAGGAGGAGAAGC-3' and antisense primer 5'-CCCGGGAAGAATCAGAGTGAGTAACTCCAACTGG-3' using, as a template, a mixture of human cDNAs from 37 different normal tissues, including brain, heart, kidney, liver, lung, and testes (Human Universal QUICK-Clone cDNA; Clontech Laboratories, Palo Alto, CA). The underlined portions of the primer sequences correspond to the engineered restriction enzyme sites for XhoI and XmaI, respectively. We subcloned the PCR products into a TA cloning vector (pCR II TOPO; Invitrogen, Carlsbad, CA). Individual clones were sequenced by GeneWiz (New York, NY) or the Keck Biotechnology Resource Laboratory, Boyer Center for Molecular Medicine, Yale University. The consensus cDNA sequence contained a 3,366-bp open reading frame that encodes 1,121 amino acids, which is identical to the c variant of human NBC4 (GenBank accession no. AF293337).

Expression in Oocytes

We subcloned the cDNA fragment into the KSM Xenopus oocyte expression vector by excising the insert from pCR II TOPO with XhoI and XmaI. The KSM expression vector is a derivative of pBluescript, in which the entire polylinker was replaced by a PCR product encoding (from 5' to 3') the 5' untranslated region (UTR) of the Xenopus beta -globin gene, a series of restriction sites for subcloning, the 3' UTR of the Xenopus beta -globin gene, a poly-A tail, and several additional restriction sites for 3' linearization of cDNA before in vitro transcription. The vector was a kind gift from Dr. William Joiner (Yale University). Capped mRNA was synthesized in vitro with the T3 Message Machine kit (Ambion, Austin, TX).

Stage V-VI oocytes from Xenopus laevis were isolated as described previously (35). One day after isolation, the oocytes were injected with 50 nl of a solution containing 0.5 ng/nl of mRNA encoding NBC4c. Control oocytes were injected with 50 nl of sterile water. The oocytes were used in experiments 2-5 days after injection. All experiments were performed at room temperature (~22°C).

Solutions

Nominally HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free ND96 solution contained (in mM) 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 mM HEPES at a pH of 7.50. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing solutions were prepared by replacing 33 mM NaCl with 33 mM NaHCO3 in ND96 and equilibrating the solution with 5% CO2-balance oxygen. Na+-free ND96 and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solutions were prepared by substituting N-methyl-D-glucammonium (NMDG+) for Na+. Cl--free solutions were prepared by substituting gluconate for Cl-. Osmolality of all solutions was ~200 mosmol/kgH2O. For butyrate-containing solutions, 30 mM Na-butyrate replaced 30 mM NaCl in ND96. Assuming that butyrate has a pKa of 4.8 and that the buffering power of an oocyte is 13.5 mM/pH,1 we calculated that 30 mM extracellular butyrate would give approximately the same degree of intracellular acidification as 5% CO2.

Electrophysiological Measurements

An oocyte was placed in a perfusion chamber and constantly superfused at a solution flow of 4 ml/min. Bath solutions were delivered with syringe pumps (Harvard Apparatus, South Natick, MA), and solutions were switched with pneumatically operated valves (Clippard Instrument Laboratory, Cincinnati, OH). In all experiments, the oocyte was initially superfused with the ND96 solution, which is nominally CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> free.

Measurement of intracellular pH. We assayed pH regulatory function of oocytes expressing NBC4c by measuring intracellular pH (pHi) with pH-sensitive microelectrodes. The electrodes were fabricated and used as described previously (36, 40). Briefly, the oocyte was impaled with two microelectrodes, one for measuring the membrane potential (Vm) and the other for measuring pHi. The tip of the pH electrode contained a liquid membrane across which a pH-dependent voltage was generated. pHi was obtained by subtracting the signal of the Vm electrode from that of the pH electrode. A calomel electrode was used as the reference in the bath. Voltages were measured with an FD 223 electrometer (World Precision Instruments, Sarasota, FL), and data were acquired with software written in-house. The system was calibrated with buffered pH standards at pH 6.0 and 8.0. An additional single-point calibration was performed with the standard ND96 solution of pH 7.50 in the bath before the oocyte was impaled.

Two-electrode voltage clamp. We used two-electrode voltage clamp to measure whole cell ionic currents in oocytes expressing NBC4c or injected with water (control). Oocyte currents and voltages were recorded with a model OC-725C oocyte clamp (Warner Instruments, Hamden, CT) controlled by the Clampex module of pCLAMP software (Version 8; Axon Instruments, Foster City, CA). Electrodes were pulled from thin-walled borosilicate glass and had resistances of 0.4-1.0 MOmega when filled with 3 M KCl. Oocytes were held at a potential close to the spontaneous Vm until the initiation of the voltage-clamp protocol. Current-voltage (I-V) relationships were generated by stepping the holding potential from -140 mV to +40 mV in 20-mV increments, each of which lasted 200 ms. Data were analyzed with the Clampfit module of pCLAMP.

Stoichiometry

To probe the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry of NBC4, we used two-electrode voltage clamp to determine zero-current potential (reversal potential, Erev) in oocytes expressing NBC4c. We began by applying a solution containing 98.5 mM Na+ and 5% CO2/33 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. In separate experiments, we measured pHi to verify that, after 5 min in this solution, pHi had reached its minimum value and would thereafter change only very slowly. The oocytes subsequently were exposed to CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing solutions with one of four different concentrations of extracellular Na+ (9.8, 29.6, 69.0, or 98.5 mM). After completing the voltage-clamp protocol at one Na+ concentration, we turned off the voltage clamp, switched to a Na+-free HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution for 2-3 min, then switched to another of the Na+-containing test solutions, waited 2-3 min for Vm to stabilize, clamped the oocyte to this new spontaneous Vm, and then performed the voltage-clamp protocol once again. We bracketed the entire procedure with I-V curves obtained at 98.5 mM Na+. We discarded the data if the spontaneous Vm values differed by >5 mV for the bracketing periods in 98.5 mM Na+. With these criteria, the bracketing I-V curves in 98.5 mM Na+ were virtually identical except for small shifts (<5 mV) in Erev.

The reason for returning to Na+-free solutions for 2-3 min between I-V curves was to minimize changes in intracellular Na+ concentration ([Na+]i) and [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i due to NBC4 transport activity; these Na+-free periods prompted NBC4 to run backwards, presumably depleting the Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> that had built up as NBC4 was running in the forward direction in the presence of Na+. We obtained HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent Erev from the above I-V curves by subtracting the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> curve measured in ND96 (CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> free) from each of the I-V curves measured in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> at different Na+ concentrations.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Nine Consecutive PCR Products Represented NBC4c

Our cloning strategy was designed to pick up any of the four NBC4 variants currently in GenBank. After performing end-to-end PCR and subcloning the PCR product into a cloning vector, we sequenced nine independent clones. All of these clones corresponded to the c variant of NBC4.

NBC4 is Electrogenic, Na+ Dependent, and Cl- Independent

pHi data. Figure 2A shows a recording of pHi and Vm for an oocyte injected with cRNA encoding NBC4c, and Fig. 2B shows a similar recording for a water-injected (control) oocyte. The initial pHi of the NBC4c-expressing oocytes was slightly higher (7.34 ± 0.01; n = 18) than in the controls (7.22 ± 0.03; n = 9), suggesting that the ambient HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in the incubation medium had provided enough substrate for at least some NBC4c activity since the time of the cRNA injection. We then switched the superfusate from ND96 to 5% CO2/33 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, which caused a dramatic hyperpolarization of the NBC4c-expressing oocyte, followed by a slower relaxation of Vm toward more positive values. The rapid hyperpolarization is consistent with the hypothesis that NBC4c mediates the uptake of Na+ and the equivalent of two or more HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> ions. In addition, pHi began to fall rapidly, reflecting the entry of CO2 into the oocyte, the subsequent hydration to form H2CO3, and dissociation to liberate HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and H+. In the continued presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, pHi eventually started to recover slowly (Table 1), consistent with the NBC4c-mediated uptake of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. When we removed extracellular Na+ (replacing it with NMDG+), the cell rapidly depolarized and began to acidify, indicating that the direction of transport had been reversed. Reintroducing Na+ caused the opposite set of Vm and pHi changes. At the end of the experiments, removing CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> caused the cell to depolarize and alkalinize rapidly. The rapid depolarization probably reflects the temporary reversal of NBC4c caused by the removal of extracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> while [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i is still high. The rise in pHi reflects the exit of CO2, which produces the depletion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> that is reflected by the slower negative drift of Vm.


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Fig. 2.   Recording of intracellular pH (pHi) and membrane potential (Vm). A: representative pHi and Vm trace of an oocyte expressing NBC4c. The oocyte was initially superfused with ND96. During the indicated period, the oocyte was superfused with the 5% CO2/33 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution. CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-induced hyperpolarization averaged -90 ± 6 mV in 6 experiments. While in the CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution, extracellular Na+ was removed as indicated. This resulted in a marked depolarization, averaging +95 ± 10 mV in the same experiments. B: data obtained with a similar protocol on a water-injected oocyte. Arrowheads indicate when Na+ was removed or reintroduced. The experiment was performed on 5 water-injected oocytes.


                              
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Table 1.   Rates of pHi change

In the control oocyte in Fig. 2B, the exposure to CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> did not cause the rapid, initial hyperpolarization. Also, we observed little pHi recovery during the prolonged exposure to CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (Table 1). Moreover, removing extracellular Na+ from the control oocyte caused a slight hyperpolarization (Fig. 2B), reflecting the native Na+ permeability of the oocyte membrane. This native Na+ permeability is completely masked in the NBC4c-expressing oocyte by the overwhelming NBC4c-dependent depolarization.

Voltage-clamp data. Using a two-electrode voltage clamp, we further explored the electrogenic nature of NBC4c. Figure 3A shows representative current records obtained in an NBC4c-expressing oocyte under three conditions: the nominal absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, the presence of 5% CO2/33 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and the absence of Na+ in the continued presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Introducing CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> elicits a large increase in the whole cell current, whereas removing Na+ virtually abolishes the NBC4c-dependent outward currents.


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Fig. 3.   Voltage-clamp experiments. A: representative current recordings obtained from an NBC4c-expressing oocyte. The top tracing was acquired in ND96, and the middle and bottom tracings were acquired in 5% CO2/33 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. In the bottom tracing, external Na+ was removed. B: representative current-voltage (I-V) curves for NBC4c-expressing oocytes generated from current tracings similar to those in A. Data were acquired in ND96 solution (black-lozenge ), CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution (), Cl--free CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution (open circle ), and Na+-free CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution (triangle ). C: representative I-V curves generated from water-injected oocytes (dashed lines) and NBC4c-expressing oocytes (solid lines), all in the nominal absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Data from water-injected oocytes were acquired in ND96 solution () and CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution (). Data from NBC4c-expressing oocytes were acquired in ND96 solution (black-lozenge ), Na+-free ND96 solution (triangle ), and Cl--free ND96 solution (open circle ). We performed a total of 8 such experiments for water-injected oocytes and 7 for NBC4c-injected oocytes. Results were similar to those shown.

Figure 3B shows representative I-V relationships for an NBC4c-expressing oocyte. Replacing extracellular Cl- with the impermeant gluconate does not change Erev and has little effect on either the inward or outward currents, indicating that NBC4c does not require external Cl- to operate in either direction. In contrast, replacing extracellular Na+ with NMDG+ shifts Erev to more positive values and virtually abolishes outward current (i.e., inward movement of the negative net charge carried by 1 Na+ and 2 or more HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>). In other experiments (not shown), we observed that the CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent current increased during the first 3-4 min of exposure to CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> but remained stable for several minutes afterwards. We therefore performed all ionic substitution experiments after at least 5 min of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exposure.

Figure 3C shows that, in water-injected control oocytes, there is little difference between currents obtained in the presence and absence of 5% CO2/33 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, demonstrating the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent currents in native oocytes. Figure 3C also shows that in nominally HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions, the whole cell conductance of NBC4c-expressing oocytes is markedly larger than in water-injected controls. It is conceivable that the small amount of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> present in air-equilibrated ND96 solutions (estimated at ~200 µM) is sufficient to carry measurable current in NBC4c-expressing oocytes. However, our observation that the current is unaffected by removal of external Na+ makes this explanation unlikely. Removal of external Cl- slightly reduced the outward current (i.e., inward movement of Cl-) but did not bring the residual outward currents to the same levels as in control oocytes. The increased "background" conductance seen in the present study is not unique to NBC4c. Compared with control oocytes, those expressing either rat kidney NBCe1-A (39) or rat NBCn1 (11) exhibit increased background conductances.

Electrical Gradient Can Drive Substantial Acid-Base Transport Through NBC4c

In the experiment shown in Fig. 2A, the pHi recoveries in the presence of Na+ and CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, as well as the pHi decline in the absence of Na+, were quite slow despite marked changes in Vm. We hypothesized that the reason for this apparent discrepancy was that the spontaneous Vm was only 3-4 mV more positive than the Erev for NBC4c. This would be the case if NBC4c were the dominant current carrier in the oocyte (i.e., if the oocytes had minimal background ion conductance). For example, during the slow pHi recovery in the presence of Na+ and CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in Fig. 2A, Vm was about -120 mV. This voltage is nearly the same as Erev for the I-V curve in Fig. 3B, which was obtained under similar conditions.

To test this hypothesis, we performed an experiment similar to that shown in Fig. 2A except that, at times, we not only monitored pHi but voltage clamped as well. The oocyte had a spontaneous Vm of about -50 mV when impaled only with the voltage electrode. Vm shifted to about -45 mV when we introduced the current electrode and initially fell to about -25 when we introduced the pH electrode. By the beginning of the record in Fig. 4, Vm had recovered somewhat, but this oocyte was still rather "leaky." Introducing CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> caused Vm to shift only to -100 mV (compared with about -155 mV for the tighter oocyte in Fig. 2A). At the times indicated in Fig. 4, we acquired I-V curves by using the same voltage protocol as in Fig. 3. We calculated Erev for the NBC4c-dependent currents by subtracting the I-V curve acquired in ND96 from the I-V curves acquired in the CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solutions. For the first I-V curve, Erev was -110 mV, compared with a spontaneous Vm of about -95 mV. This ~15-mV difference between Erev and Vm would explain why the spontaneous pHi recovery is faster in Fig. 4 than in Fig. 2. When we then voltage-clamped the oocyte in Fig. 4 to -30 mV, pHi increased approximately six times more rapidly than when Vm was free-floating at about -95 mV. This large increase in the pHi recovery rate is consistent with the large increase in the difference between Erev (-90 mV) and the clamped Vm of -30 mV. When we reversed the transporter by removing extracellular Na+ in Fig. 4 (Vm = -30 mV), pHi initially decreased more rapidly than in Fig. 2 (Vm = +2 mV). Thus a large electrical gradient can substantially increase acid-base transport through NBC4c. The rates of pHi change in oocytes clamped to -30 mV are compared with those of unclamped oocytes in Table 1.


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Fig. 4.   Measurement of pHi in a voltage-clamped oocyte. The oocyte was initially superfused with ND96 without clamping the voltage. During the indicated period, the oocyte was superfused with the 5% CO2/33 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution. While in the CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution, first extracellular Na+ and then also extracellular Cl- were removed as indicated. Gray area indicates the period during which the oocyte was voltage-clamped to -30 mV. Asterisks indicate the time points at which an I-V curve was acquired. We performed a total of 3 such experiments on different NBC4c-expressing oocytes, obtaining results similar to those shown.

While continuing to voltage clamp the oocyte in Fig. 4 at -30 mV, we removed extracellular Cl- in the absence of Na+ to revisit the question of whether acid-base transport by NBC4c requires extracellular Cl-. If NBC4c were a Na+-driven Cl--HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger, then it should require extracellular Cl- when running in reverse. However, even at a relatively high rate of pHi decrease in Fig. 4, Cl- removal had no effect on the pHi trajectory. Thus NBC4c does not mediate Na+-driven Cl-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange.

NBC4c is HCO<UP><SUB>3</SUB><SUP><UP>−</UP></SUP></UP> Dependent

In Fig. 2A, we saw that exposing an NBC4c-expressing oocyte to CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> caused an abrupt hyperpolarization as well as a pHi decrease from which the cell slowly recovered. To address the question of whether the Vm change and the pHi recovery depended on the application of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and not merely the intracellular acidification induced by CO2, we used butyrate to acidify oocytes in the nominal absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. We calculated that a butyrate concentration of 30 mM would produce about the same fall in pHi as 5% CO2 (see Solutions). As shown in Fig. 5A, exposing an NBC4c-expressing oocyte to a solution containing 30 mM butyrate causes virtually no change in Vm and a large, rapid acidification. However, in the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, pHi fails to recover from the acid load. After the butyrate is removed, a subsequent exposure to CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> causes the usual hyperpolarization as well as the pHi recovery from the acid load. In the control oocyte, neither butyrate nor CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> elicited large changes in Vm or a pHi recovery from the acid loads (Fig. 5B). Thus both the voltage changes and the pHi recovery require HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> per se and not merely an acidification of the cytoplasm.


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Fig. 5.   HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> dependence of NBC4. A: representative pHi and Vm traces of an oocyte expressing NBC4c. The oocyte was initially superfused with ND96. During the indicated time period, the oocyte was superfused with 30 mM butyrate solution at a constant extracellular pH of 7.50. After washout of the butyrate-containing solution, the oocyte was reacidified with 5% CO2-33 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution. B: data obtained with a similar protocol on a water-injected oocyte. We performed a total of 3 such experiments on different water-injected oocytes and 3 on NBC4c-expressing oocytes. Results were similar to those shown.

NBC4c Is Blocked by DIDS

pHi data. To determine whether NBC4c is sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), we exposed NBC4c-expressing oocytes and control oocytes to 200 µM DIDS during the plateau phase of a CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> pulse. Figure 6A shows that in an NBC4c-expressing oocyte, application of DIDS markedly reduced the rate of pHi recovery and also caused a modest depolarization. DIDS reduced the mean rate of pHi recovery from 21.5 ± 4.2 to 3 ± 1.2 × 10-5 pH units/s (n = 5), an inhibition of ~80%. Thus DIDS strongly inhibits the transport function of NBC4c.2 Although the inhibition of some HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporters by DIDS is poorly reversible, the pHi recovery in oocytes expressing NBC4c resumed on washout of DIDS. In the control oocyte (Fig. 6B), pHi did not recover after the initial acidification and application of DIDS had no effect on pHi. The mean rate of pHi change was 0.7 ± 2.2 × 10-5 pH units/s before application of DIDS and -1.7 ± 0.9 × 10-5 pH units/s after application of DIDS (n = 3).


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Fig. 6.   Effect of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on pHi recovery from an acid load. A: representative pHi and Vm traces of an oocyte expressing NBC4c. The oocyte was initially superfused with ND96. During the indicated time period, the oocyte was superfused with the 5% CO2/33 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution. DIDS (200 µM) was applied during the CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exposure. B: data obtained with a similar protocol on a water-injected oocyte. We performed a total of 3 such experiments on different water-injected oocytes and 5 on different NBC4c-expressing oocytes. Results were similar to those shown.

Voltage-clamp data. Using two-electrode voltage clamp, we investigated the effect of 200 µM DIDS on the current carried by NBC4c. Because work by Diakov et al. (14) has shown that DIDS (at a concentration of 250 µM) can activate endogenous ion channels in the oocyte over a period of 15-60 s, we exposed the oocytes to DIDS for no more than ~20 s, which we found to be sufficient for maximal inhibition.

Figure 7A summarizes data from a representative experiment in which we obtained sequential I-V curves at four times, with the oocyte exposed to 1) ND96, 2) CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 3) CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> + DIDS after a ~20-s pretreatment with DIDS, and 4) in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> after washout of DIDS for 2 min. DIDS reduced the currents to nearly the levels observed in ND96. Despite the short treatment period, the inhibitory effect of DIDS was not completely reversible.


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Fig. 7.   Effect of DIDS on ionic currents. A: representative I-V curves for NBC4c-expressing oocytes. Data were acquired in ND96 solution (black-lozenge ), CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution (),CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> with 200 µM DIDS (open circle ), and CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solution after washout of DIDS (triangle ). B: representative I-V curves generated from control oocytes (dashed lines) and NBC4c-expressing oocytes (solid lines), all in the nominal absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Data from water-injected oocytes were acquired in ND96 solution () and ND96 solution with DIDS (). Data from NBC4-expressing oocytes were acquired in ND96 solution (black-lozenge ), ND96 with 200 µM DIDS (open circle ), and ND96 solution after washout of DIDS (triangle ). C: average DIDS-sensitive currents in NBC4c-expressing oocytes. DIDS-sensitive current in ND96 solutions (black-lozenge ) is the average obtained by subtracting data from 5 experiments similar to those shown in B (open circle  and black-lozenge ). DIDS-sensitive current in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solutions (open circle ) is the comparable average for 5 different experiments similar to those shown in A (open circle  and ). Vertical bars indicate SE; bars are omitted when they would have been smaller than the symbol.

Figure 7B summarizes the results of an experiment in which we exposed either an NBC4c-expressing oocyte or a control oocyte to DIDS in ND96 solution (i.e., in the nominal absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>). In the NBC4c-expressing oocyte DIDS blocked some of the inward and outward currents, whereas in the control oocyte the effect of DIDS was very small. The very small DIDS-sensitive currents in the nominal absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> are not due to residual NBC4c activity; recall that in Fig. 3C, we saw that removing external Na+ in the nominal absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> did not affect the currents in an NBC4c-expressing oocyte.

Figure 7C shows the DIDS-sensitive component of the currents in NBC4c-expressing oocytes, averaged from five different experiments. The open circles in Fig. 7C represent data obtained in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (e.g., the difference between closed and open circles in Fig. 7A). The closed diamonds in Fig. 7C represent data obtained in the nominal absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (e.g., the difference between closed diamonds and open circles in Fig. 7B). It is clear that the DIDS-sensitive currents are far larger in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>.

Apparent HCO<UP><SUB>3</SUB><SUP><UP>−</UP></SUP></UP>:Na+ Stoichiometry of NBC4c is 2:1

Slope of Erev vs. log of extracellular Na+ concentration. To determine the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry of NBC4c, we used a two-electrode voltage clamp to measure the reversal potentials of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent currents in NBC4c-expressing oocytes at four different values of extracellular Na+ concentration ([Na+]o) (see METHODS). Erev of an electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter is given by the following equation (5)
E<SUB>rev</SUB><IT>=</IT><FR><NU>1</NU><DE><IT>q−</IT>1</DE></FR> <FR><NU><IT>RT</IT></NU><DE><IT>F</IT></DE></FR> ln<FENCE><FR><NU>[Na]<SUB>i</SUB></NU><DE>[Na]<SUB>o</SUB></DE></FR> <FENCE><FR><NU>[HCO<SUB>3</SUB>]<SUB>i</SUB></NU><DE>[HCO<SUB>3</SUB>]<SUB>o</SUB></DE></FR></FENCE><SUP><IT>q</IT></SUP></FENCE> (1)
where q is the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry, [Na+]i, [Na+]o, [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i, and [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]o are the respective intracellular and extracellular concentrations, R is the gas constant, T is temperature, and F is the Faraday constant. This equation predicts that a plot of Erev vs. log [Na+]o should be a straight line, whose slope is -ln(10) × RT/[F(q - 1)]. Thus the higher the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry, the less steep the slope of the line. Thus, for a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry of 2:1, the expected slope at 22°C would be -58.5 mV/log [Na+]o, whereas for a stoichiometry of 3:1, the slope would be only half as steep, -29.3 mV/log [Na+]o. The results of five experiments (each including all 4 [Na+]o values) are summarized in Fig. 8, which is a plot of Erev against [Na+]o. Our experimental data are fitted well by a straight line with a slope of -54.8 mV/log [Na+]o, consistent with the hypothesis that NBC4c operates with a 2:1 stoichiometry.


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Fig. 8.   Dependence of the NBC4 reversal potential (Erev) on extracellular Na+ concentration ([Na+]o). Using a 2-electrode voltage-clamp technique, we measured the Erev of NBC4c-expressing oocytes in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> solutions at 4 different values of [Na+]o. The plot of Erev vs. log[Na+]o was fitted with a straight line (R2 = 0.9356), the slope of which is -54.8 mV/log[Na+]o. This slope is consistent with a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry of 2:1. Each symbol represents the mean ± SE. We performed a total of 5 experiments on different NBC4c-expressing oocytes.

The advantage of calculating transporter stoichiometry from the slope of the line in Fig. 8 is that one does not need to know any of the absolute concentrations in Eq. 1, only the fractional change in the concentration of the varied ion (i.e., [Na+]o in this case). However, in using this approach, one must assume that the values of the other three concentration terms are constant. In Fig. 8, we used only tight oocytes, like the one in Fig. 2, for which the spontaneous Vm was very close to Erev. When Vm congruent  Erev, the transporter has a low activity, and one would expect [Na+] and [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] in the intra- and extracellular unstirred layers near the membrane to be very close to the respective concentrations in the bulk fluids. Moreover, we know from Fig. 2 that pHi does not change appreciably in a tight oocyte, even when one shifts [Na+]o between 98.5 and 0 mM. We have no information on how [Na+]i might have varied during the 2-3 min during which we altered [Na+]o. However, in oocytes expressing rat kidney NBCe1-A, Sciortino and Romero (39) found that removing extracellular Na+ caused [Na+]i---measured with an intracellular microelectrode---to fall from ~9 mM to only ~8 mM over ~2 min. Moreover, if [Na+] near the two unstirred layers in our experiments had changed appreciably, we would not have obtained the linear relationship shown in Fig. 8.

Absolute value of Erev. Another approach for estimating stoichiometry is to compute q directly from each Erev value. Solving Eq. 1 for q yields
q = <FR><NU>ln <FR><NU>[Na]<SUB>i</SUB></NU><DE>[Na]<SUB>o</SUB></DE></FR> + <FR><NU><IT>F</IT></NU><DE><IT>RT</IT></DE></FR><IT> E</IT><SUB>rev</SUB></NU><DE>ln <FR><NU>[HCO<SUB>3</SUB>]<SUB>o</SUB></NU><DE>[HCO<SUB>3</SUB>]<SUB>i</SUB></DE></FR> + <FR><NU><IT>F</IT></NU><DE><IT>RT</IT></DE></FR><IT> E</IT><SUB>rev</SUB></DE></FR> (2)
We calculated [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i in our experiments from average pHi data and the Henderson-Hasselbalch equation, obtaining a mean value of 7.6 mM. Sciortino and Romero (39) found that oocytes expressing rat kidney NBCe1 and bathed in 5% CO2/33 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> had a mean [Na+]i of 10.4 mM. Using these values for [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i and [Na+]i, 33 mM for [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]o, and the four different [Na+]o values from our experiments, we computed the four q values summarized in Table 2. The mean value for all data, 2.1 ± 0.1, is consistent with a 2:1 stoichiometry.

                              
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Table 2.   Apparent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-to-Na+ stoichiometry ratios


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of this study was to determine the functional properties of NBC4, originally cloned from human heart by Pushkin et al. (32) but uncharacterized physiologically. Currently GenBank contains the sequences for four different NBC4 variants (NBC4a, b, c, and d). Our own cloning efforts resulted in the cloning of the NBC4c variant, which we have expressed in Xenopus oocytes. We report here that NBC4c carries out electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport. Furthermore, NBC4c is independent of extracellular Cl-, is blocked by DIDS, and operates with an HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry of 2:1 when expressed in oocytes. These results correlate well with sequence alignment analysis, which shows that, on the amino acid level, NBC4 is most closely related to electrogenic NBCe1. On the basis of the function of NBC4c, we propose to call it NBCe2-c.

Different NBC4 Variants

Considerable progress has been made in the last few years on the topology of anion exchangers (AEs), which are ~30% identical to the NBCs on the amino acid level. The membrane domain of AE1 (i.e., the portion lacking the presumably cytoplasmic NH2 and COOH termini) is ~40% identical to that of the Na+-coupled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporters and is, by itself, sufficient to carry out transport (23, 26, 27). Reasoning that the topology of the membrane domains of the BTs are probably conserved, in Fig. 1A we mapped NBC4c onto a recent folding model developed in the laboratory of Joe Casey (43). The model predicts 14 membrane-spanning segments, of which the twelfth segment is proposed to be nonhelical.

Comparing the cDNA sequence of NBC4c with the human genome database reveals the presence of 25 exons that span the length of the coding region. Variants a and b share an additional exon not present in c and d. The presence of this additional exon results in a 16-amino acid insertion into the very short region between putative TMs 11 and 12, a region that is highly conserved among all superfamily members. In addition, the third-to-last exon in variant b is longer than in the other variants because, at its 5' end, it contains 16 bp of intronic sequence (Fig. 1B). This 16-bp insertion creates a frameshift in the middle of the last TM, resulting in a truncated COOH terminus (Fig. 1B) that has no homology to other members of the BT superfamily. Variant d is lacking two additional exons between TMs 10 and 14, resulting in a 294-bp deletion in a region that is very highly conserved in all members of the BT superfamily. Thus variant c is the only one of the four NBC4 variants whose deduced amino acid sequence maps onto the superfamily consensus sequence without major gaps or insertions. Indeed, nine consecutive cDNA clones in our study---obtained by PCR using, as a template, a mixture of human cDNAs from different tissues---proved to be of the c variety. If the four original NBC4 mRNA species had been evenly distributed, and if the PCR efficiencies for the respective cDNA species were identical, then the odds of picking the c variant nine times in a row would be 1 in 49. It will be informative to learn whether variants a, b, and d encode functional proteins.

DIDS

Our results show that 200 µM DIDS strongly blocks both the pHi recovery and ionic currents mediated by NBC4c. DIDS is a classic inhibitor of anion transport via AEs, various Na+-coupled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporters, and anion channels. Cabantchik and Rothstein (9) established that DIDS interacts with the protein now known to be AE1 in two steps: a rapid ionic interaction that is reversed by scavenging the DIDS with albumin and a slower covalent reaction. However, whether DIDS binds ionically or covalently, it blocks transport. Aligning the amino acid sequences of AE1, AE2, and AE3, Kopito et al. (23) proposed a consensus motif at the extracellular end of TM 5 for the covalent interaction of DIDS with the AEs: KLXK (X = I, Y). After cloning the first Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter, Romero et al. (36) pointed out that NBCe1 has KMIK at the site homologous to KLXK in the AEs. Therefore, they suggested a consensus DIDS-reaction motif of KX1X2K (X1 = M, L; X2 = I, V, Y). Indeed, NBCn1, which is not sensitive to DIDS, has a disrupted consensus motif: KLFH (11).

In NBC4c, the sequence at the homologous TM 5 site is KMIG. Thus DIDS blocks the function of NBC4c even though NBC4c lacks the second K of the consensus motif. However, NBC4c is not the first DIDS-sensitive BT family member with a disrupted consensus motif. NDAE1 from Drosophila (37) and human NDCBE (18) are both DIDS sensitive, even though the sequences at their respective homologous sites are NVMV and KLIH. One explanation for these observations is that DIDS may inhibit transport by NBC4c (and also by NDAE and NDCBE1) via an ionic interaction that does not require the consensus DIDS reaction motif. Alternatively, DIDS may covalently react at another site on the transporter molecule. Distinguishing among these possibilities will require a structure-function analysis of the three different aspects of the interaction of DIDS with the Na+-coupled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporters: 1) inhibition of transport, 2) ionic or reversible binding, and 3) covalent or irreversible binding.

NBC4 in Liver

Probing human Northern blots with NBC4, Pushkin et al. (32) found that the strongest signal appears in the liver. So far, no other Na+-coupled HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporters have been shown to be strongly expressed in liver: NBCe1 (1, 35), NBCn1 (11, 22, 31), NDCBE1 (3, 18), and NCBE (45). Hepatocytes carry out a diversity of metabolic and transport functions that may severely challenge pHi homeostasis in these cells. To counteract the effects of changes in pHi produced by metabolism and transport, hepatocytes have evolved canalicular (apical) Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchangers as well as sinusoidal (basolateral) Na+/H+ exchangers and electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters (reviewed in Ref. 7). The importance of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport for hepatocyte pHi regulation is underscored by three observations: 1) steady-state pHi is lower in isolated hepatocytes incubated in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (17); 2) the acid extrusion rate during pHi recovery from an acid load is markedly lower in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (16); and 3) at the higher pHi prevailing in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, the Na+/H+ exchanger is virtually inactive (34, 42). In addition, intracellular acidification in hepatocytes produces a depolarization (presumably via pH-sensitive K+ channels) that would increase the driving force for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> entry via an electrogenic Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (15). Inasmuch as NBC4 is electrogenic, DIDS sensitive, and strongly expressed in liver, it is a good candidate for the major hepatic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter.

NBC4 in Kidney

The vast majority of renal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption is mediated by electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport in the proximal tubule. Using light microscopy, Schmitt et al. (38) showed that NBCe1 is restricted to the proximal tubule, specifically to the basolateral membrane of the S1 and S2 segments of the rat and rabbit proximal tubule. Maunsbach et al. (28) confirmed and extended these observations on the rat with immunoelectron microscopy, demonstrating that the antibody (raised to the COOH terminus of NBCe1) labels an epitope on the cytoplasmic side of the membrane. According to thermodynamic calculations, the stoichiometry of the Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter would have to be at least 3:1 for the transporter to mediate the net efflux of Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> across the basolateral membrane of the proximal tubule and oppose the large electrochemical gradient favoring the entry of Na+ into the cell (6, 46). Indeed, Soleimani et al. (41) determined that in isolated rabbit renal membrane vesicles the stoichiometry of Na-HCO3 cotransport is 3:1. However, when the kidney variant of NBCe1 is expressed in Xenopus oocytes, it appears to operate with a 2:1 stoichiometry (21). There is, however, evidence that the stoichiometry of NBCe1 is not fixed at a particular ratio but can change (30). Gross et al. presented evidence that the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry might shift from 2:1 to 3:1, depending on the cell type in which it is expressed (19) and on the phosphorylation state of the protein (20). In addition, the conditions within a particular cell (such as intracellular Ca2+) may affect the stoichiometry (29).

The only thing that is known about the renal localization of NBC4 is that the message is present in the kidney lane of human Northern blots. Thus it is not clear whether NBC4 participates in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption. Our results suggest that NBC4c operates with a 2:1 stoichiometry in oocytes, so that NBC4c would be expected to mediate net HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> uptake (i.e., acid extrusion) in most cells. In this context, NBC4c could help to protect against intracellular acidification in cells throughout the kidney. If present at an apical membrane, and operating with a 2:1 stoichiometry, NBC4c could thus contribute to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption. If NBC4c were present at the basolateral membrane and functioned with a 3:1 stoichiometry in the proximal tubule, then it could contribute to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in that nephron segment. In principle, even with a 2:1 stoichiometry, basolateral NBC4 could contribute to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption elsewhere in the nephron, provided that [Na+]i and/or [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i were sufficiently high and/or provided that the basolateral membrane potential were sufficiently negative.

NBC4 in Heart

Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport plays an important role in pHi regulation in mammalian cardiac myocytes, in which it mediates the equivalent of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> uptake (2, 10, 24, 25). In guinea pig ventricular myocytes (24) and sheep cardiac Purkinje fibers (13), this HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> uptake appears to be voltage insensitive, consistent with an electroneutral cotransporter. However, other reports on rat myocytes (2) and cat papillary muscle (10) indicate a strong voltage sensitivity, consistent with an electrogenic cotransporter, possibly with a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>:Na+ stoichiometry of 2:1. The molecular identity of the cotransporter(s) that mediate pHi regulation in these different cell types and species is not known. At the mRNA level, the two electrogenic transporters, NBCe1-B (12) and NBC4 (32), as well as the electroneutral NBCn1 (31) are all present in human heart. At least NBCn1 is expressed in rat heart (11, 22). Thus further work will be required to correlate functional Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport with specific NBC proteins in different cardiac cell types from various species.


    ACKNOWLEDGEMENTS

We thank Dr. William Joiner for the gift of the Xenopus expression vector.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-30344. L. V. Virkki was supported by a fellowship from the American Heart Association.

1 The buffering power beta  of an oocyte was calculated from the change in [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i produced in control oocytes by exposure to 5% CO2/33 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, divided by the change in pHi, so that beta  = Delta [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]/Delta pHi.

2 A question that arises is why DIDS inhibits acid-base transport by ~80% but has only a modest effect on Vm. Because the oocyte is very "tight" electrically, a small NBC4c current can drive Vm very close to Erev. Thus, whereas the NBC4c current and the rate of pHi recovery depend linearly on the turnover rate of NBC4c, Vm does not---because of the tightness of the oocyte, only a relatively small turnover is required to drive Vm very close to Erev.

Address for reprint requests and other correspondence: L. V. Virkki, Dept. of Cellular and Molecular Physiology, Yale Univ. School of Medicine, PO Box 208026, 333 Cedar St., New Haven, CT 06520-8026 (E-mail: leila.virkki{at}yale.edu).

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.

First published January 16, 2002;10.1152/ajpcell.00589.2001

Received 12 December 2001; accepted in final form 9 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abuladze, N, Lee I, Newman D, Hwang J, Boorer K, Pushkin A, and Kurtz I. Molecular cloning, chromosomal localization, tissue distribution, and functional expression of the human pancreatic sodium bicarbonate cotransporter. J Biol Chem 273: 17689-17695, 1998[Abstract/Free Full Text].

2.   Aiello, EA, Petroff MG, Mattiazzi AR, and Cingolani HE. Evidence for an electrogenic Na-HCO3 symport in rat cardiac myocytes. J Physiol 512: 137-148, 1998[Abstract/Free Full Text].

3.   Amlal, H, Burnham CE, and Soleimani M. Characterization of the Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter isoform NBC-3. Am J Physiol Renal Physiol 276: F903-F913, 1999[Abstract/Free Full Text].

4.   Bevensee, MO, Schmitt BM, Choi I, Romero MF, and Boron WF. An electrogenic Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain. Am J Physiol Cell Physiol 278: C1200-C1211, 2000[Abstract/Free Full Text].

5.   Boron, WF, and Boulpaep EL. Intracellular pH regulation in the renal proximal tubule of the salamander: basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport. J Gen Physiol 81: 53-94, 1983[Abstract].

6.   Boron, WF, and Boulpaep EL. The electrogenic Na/HCO3 cotransporter. Kidney Int 36: 392-402, 1989[ISI][Medline].

7.   Boyer, JL, Graf J, and Meier PJ. Hepatic transport systems regulating pHi, cell volume, and bile secretion. Annu Rev Physiol 54: 415-438, 1992[ISI][Medline].

8.   Burnham, CE, Amlal H, Wang Z, Shull GE, and Soleimani M. Cloning and functional expression of a human kidney Na+:HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter. J Biol Chem 272: 19111-19114, 1997[Abstract/Free Full Text].

9.   Cabantchik, ZI, and Rothstein A. The nature of the membrane sites controlling anion permeability of human red blood cells as determined by studies with disulfonic stilbene derivatives. J Membr Biol 10: 311-328, 1972[ISI][Medline].

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