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¹ Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322, USA
² Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA

	Abstract

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The electrogenic Na⁺–HCO₃^– cotransporter (NBCe1) plays a central role in intracellular pH (pH_i) regulation as well as HCO₃^– secretion by pancreatic ducts and HCO₃^– reabsorption by renal proximal tubules. To understand the structural requirements for the electrogenicity of NBCe1, we constructed chimeras of NBCe1-A and the electroneutral NBCn1-B, and used two-electrode voltage clamp to measure electrogenic transporter current in Xenopus oocytes exposed to 5% CO₂–26 mM HCO₃^–(pH 7.40). The chimera consisting of NBCe1-A (i.e. NBCe1-A ‘background’) with the cytoplasmic N-terminal domain (Nt) of NBCn1-B had a reversal potential of –156.3 mV (compared with a membrane potential V_m of –43.1 mV in a HCO₃^–-free solution) and a slope conductance of 3.0 µS (compared with 12.5 µS for NBCe1-A). Also electrogenic were chimeras with an NBCe1-A background but with NBCn1-B contributing the extracellular loop (L) between transmembrane segment (TM) 5 and 6 (–140.9 mV/11.1 µS), the cytoplasmic C-terminal domain (Ct; –123.8 mV/9.7 µS) or Nt + L + Ct (–120.9 mV/3.7 µS). Reciprocal chimeras (with an NBCn1 background but with NBCe1 contributing Nt, L, Ct or Nt + L + Ct) produced no measurable electrogenic transporter currents in the presence of CO₂–HCO₃^–. pH_i recovered from an acid load, but without the negative shift of V_m that is characteristic of electrogenic Na⁺–HCO₃^– cotransporters. Thus, these chimeras were electroneutral, as were two others consisting of NBCe1(Nt–L)/NBCn1(TM6–Ct) and NBCn1(Nt–L)/NBCe1(TM6–Ct). We propose that the electrogenicity of NBCe1 requires interactions between TM1–5 and TM6–13.

(Received 7 June 2006; accepted after revision 5 October 2006; first published online 12 October 2006)
Corresponding author I. Choi: Department of Physiology, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322, USA. Email: ichoi{at}emory.edu

	Introduction

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Cells regulate their internal ionic environment via membrane proteins that mediate the transmembrane movement of ions. The SLC4A gene family, which has 10 members, consists mainly of transporters that are specialized to move HCO₃^– (or in some cases, CO₃^2–) across plasma membranes (for review see Romero et al. 2004). For many SLC4 family members, major physiological roles include the regulation of intracellular pH (pH_i) and/or the transepithelial movement of HCO₃^–. Regarding pH_i regulation, some SLC4 members mediate the efflux of HCO₃^– (or CO₃^2–) and thus act to lower pH_i (acid loaders), whereas others mediate the influx of HCO₃^– (or CO₃^2–) and thus act to raise pH_i (acid extruders). The balance between the activities of acid extruders and loaders, as well as metabolic production and consumption of H⁺ ions, determines the steady-state pH_i (Boron, 1989). For example, the electrogenic Na⁺–HCO₃^– cotransporter NBCe1 (SLC4A4) (Bevensee et al. 1995) – operating with a Na⁺:HCO₃^– stoichiometry of 1 : 2 in cells such as astrocytes – is an acid extruder. This HCO₃^–-mediated acid extrusion helps to avoid pH_i in astrocytes falling below normal levels during cerebral acidification (Giffard et al. 2000).

In HCO₃^–-transporting epithelia, at least one member of the SLC4A gene family is usually present in the basolateral membranes, mediating a crucial step in the transepithelial movement of HCO₃^–. For example, NBCe1, apparently operating with a 1 : 2 stoichiometry, mediates HCO3^– uptake across the basolateral membrane of the pancreatic duct for secretion into the lumen (Marino et al. 1999). In the renal proximal tubule, which reclaims more than 80% of filtered HCO₃^–, NBCe1 mediates the efflux of HCO₃^– across the basolateral membrane for delivery to the blood. An essential requirement for this HCO₃^– exit is that NBCe1 in the proximal tubule operates with an apparent Na⁺:HCO₃^– stoichiometry of 1 : 3, which makes the reversal potential for the transporter more positive than the basolateral membrane potential of the proximal tubule cell (Soleimani et al. 1987; Boron & Boulpaep, 1989). By moving Na⁺ out of the cell, NBCe1 in the proximal tubule may be unique in moving Na⁺ against the Na⁺ electrochemical gradient without utilizing ATP directly.

In spite of the importance of the electrogenicity of NBCe1 for its physiological function, the protein domains responsible for electrogenic ion translocation are not known. Sequence analysis reveals a high level of amino acid identity (> 50%) among the five known Na⁺–HCO₃^– transporters of the SLC4 family (Romero et al. 2004), two of which are electrogenic (NBCe1/SLC4A4 and NBCe2/SLC4A5) (Burnham et al. 1997; Romero et al. 1997; Sassani et al. 2002; Virkki et al. 2002) and three of which are electroneutral (NBCn1/SLC4A7, NDCBE/SLC4A8 and NCBE/SLC4A10) (Pushkin et al. 1999; Choi et al. 2000; Wang et al. 2000; Grichtchenko et al. 2001). It is not surprising that the identity level is generally higher in the transmembrane domains of the proteins than in the cytoplasmic N- and C-terminal domains and the large, third extracellular domain between transmembrane segment (TM) 5 and TM6. This relationship is consistent with the hypothesis that the transmembrane domains mediate the actual transport event, but that differences in these less-conserved regions somehow modulate the transmembrane domains and thereby control the stoichiometry of ion transport. Several studies have shed light on the structure–function relationships of NBCe1. A large-scale mutagenesis study targeted acidic and basic amino acids near or outside transmembrane segments, and identified several that block transport (Abuladze et al. 2005). However, it remains unclear whether those residues directly participate in ion translocation or determine stoichiometry.

In the present study, we systematically constructed a series of chimeric transporters between NBCe1 and the electroneutral Na⁺–HCO₃^– cotransporter NBCn1 (for a sequence comparison of the two transporters, see Choi et al. 2000). We swapped transmembrane and non-transmembrane domains of NBCe1 and NBCn1, expressed them in Xenopus oocytes, and analysed the HCO₃^–-dependent currents mediated by chimeras in two-electrode voltage clamp. We found that neither the cytoplasmic N-terminal domain (Nt), the large, third extracellular loop (L) nor the cytoplasmic C-terminal domain (Ct) affects electrogenicity. However, both the transmembrane domain before the third extracellular loop (TM1–5) and the transmembrane domain after the third extracellular loop (TM6 to the last TM) are necessary for electrogenicity.

	Methods

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Construction of chimeras

The ‘renal’ splice variant of rat NBCe1 (NBCe1-A, GenBank accession number NM_053424) and NBCn1 (NBCe1-B, GenBank accession number NM_058211) were used for constructing chimeric proteins. Table 1 summarizes the regions that we swapped. To swap regions of interest, we introduced restriction endonuclease sites (generating silent mutations that did not affect amino acid composition) at both ends of the corresponding regions of both transporters, using the QuickChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA). Details are available upon request. PCR was carried out using Pfu DNA polymerase with high proofreading capability. Chimeric constructs were sequenced by the Biomedical Facility at Emory University or the Keck Center at Yale University. The constructs were inserted in the oocyte expression vector pGH19 (Choi et al. 1999), which contains the 5' and 3' untranslated regions of the Xenopus -globin gene. For wild-type NBCe1 and chimeras containing the N-terminal domain of NBCe1, we used the pTLN2 vector (Romero et al. 1998) because of incompatibility of the restriction enzyme sites. pTLN2 is virtually identical to pGH19 except for the polycloning sites.

The DNAs encoding chimeric transporters were linearized with Not I or Nhe I for constructs in pGH19 and in vitro transcribed with T7 RNA polymerase using the mMessage/mMachine transcription kit (Ambion, Austin, TX, USA). For constructs in pTLN2, Mlu I was used for linearization and SP6 RNA polymerase for transcription. Oocytes (stages V and VI) were prepared from Xenopus laevis. A piece of ovary containing oocytes was excised, rinsed five times with Ca²⁺-free ND96 solution containing (mM): NaCl 96, KCl 2, MgCl₂ 1 and Hepes 5 (pH 7.5), for 20 min each. The tissue was then agitated in sterile-filtered Ca²⁺-free ND96 with collagenase (Type 1 A, Sigma-Aldrich, St Louis, MO, USA) for 40 min. Defolliculated oocytes were washed with Ca²⁺-free ND96, sorted and stored at 18°C in OR3 media (50% Leibovitz L-15 media in 5 mM Hepes, pH 7.5) supplemented with 5 U ml^–1 penicillin-streptomycin. Oocytes were injected with 50 nl of either RNA (0.4 µg µl^–1). Injected oocytes were maintained for 3–7 days at 18°C.

Two-electrode voltage clamp

Two-electrode voltage clamp was performed using the oocyte clamp OC-725C (Warner, Hamden, CT, USA). The electrodes were filled with 3 M KCl with a resistance of less than 1.5 M. Oocytes were clamped at a holding potential of –60 mV and subjected to staircase voltage commands stepping from –140 to +60 mV (20 mV increment) for 100 ms. Measurements of the electrogenic current were made at 1 min after switching bath solutions from modified amphibian ND96 solution containing (mM): NaCl 96, KCl 2, CaCl₂ 1.8, MgCl₂ 2 and Hepes 5 (pH 7.4), to one buffered with 5% CO₂–26 mM HCO₃^–(pH 7.4; NaCl replaced by NaHCO₃). Measurements of the ionic currents associated with NBCn1 were done in ND96, as previously described (Cooper et al. 2005). In Na⁺-free solutions, Na⁺ was replaced by NMDG⁺. The voltage signal was sampled by the Digidata 1322 interface (Axon Instruments, Union City, CA, USA) connected to an IBM-compatible PC. Data were acquired and analysed using pClamp 8 (Axon Instruments).

Measurements of pH_i in oocytes

We impaled oocytes with two electrodes, one sensitive to pH and the other a conventional voltage electrode, as previously described (Siebens & Boron, 1987; Romero et al. 1997; Lu et al. 2006). The pH electrode was made with borosilicate fibre capillaries, silanized, and filled with the proton ionophore 1 cocktail B (Sigma-Aldrich), and back filled with a phosphate buffer at pH 7.0. The voltage electrode was filled with 3 M KCl and had a resistance of 0.7–2.0 M. Electrodes were connected to a high-impedance electrometer FD-223 (WPI, Sarasota, FL, USA). The voltage signal was subtracted from the voltage from the pH electrode, which yields a voltage due solely to pH. The data were sampled by an A/D converter interfaced to a computer. Measurements of pH_i were first done in the nominally HCO₃^–-free ND96 solution (pH 7.5) and then in a solution equilibrated with 1.5% CO₂–10 mM HCO₃^– (pH 7.5). NaCl was replaced by NaHCO₃. The osmolality was adjusted to 196–200 mosmol kg^–1. All experiments were performed at room temperature (22°C).

Statistical analysis

Data were reported as means ± S.E.M. Levels of significance were assessed using the unpaired, two-tailed Student t test. P < 0.05 was considered significant. Rates of pH_i change were fitted by a line using a least-squares method.

	Results

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Electrogenic HCO₃^– currents mediated by NBCe1

In analysing the electrogenicity of chimeric transporters, our experimental approach was to express the transporters in Xenopus oocytes and determine steady-state current–voltage (I–V) relationships with the oocyte first exposed to the CO₂–HCO₃^–-free ND96 solution and then to the solution buffered with 5% CO₂–26 mM HCO₃^– (pH 7.4). Figure 1A shows average data for water-injected control oocytes. In the ND96 solution, they had a mean reversal potential (E_ND96) around –41 mV, low background currents (I_ND96) and a low slope conductance (G_ND96). After the switch to CO₂–HCO₃^–, the reversal potential (E_HCO3), currents (I_HCO3) and slope conductance (G_HCO3) were not substantially different from those in the presence of ND96 solution. The difference current I_HCO3 – I_ND96 (red triangles) nearly lies on the voltage axis. Table 2 summarizes the data from control oocytes as well as oocytes (discussed below) expressing NBCe1-A, NBCn1-B and various other chimeras.

View larger version (9K): [in this window] [in a new window]   Figure 1.  Mean steady-state current–voltage (I–V) relationships in control oocytes and oocytes expressing wild-type NBCe1 or NBCn1 A, control oocytes injected with water (n = 5). B, oocytes expressing NBCe1 (n = 7). C, oocytes expressing NBCn1 (n = 7). Oocytes were clamped at –60 mV and voltage was stepped from –140 to +60 mV for 100 ms in 20 mV increments. The recordings were done in the pH 7.40 HCO3–-free ND96 solution () or in a comparable solution buffered to pH 7.40 with 5% CO2–26 mM HCO3–(). The difference IHCO3–IND96 is an estimate of the electrogenic cotransport current ().

Figure 1C shows average data for an oocyte expressing NBCn1-B. I_ND96 values (at positive voltages) as well as G_ND96 values were higher than in H₂O-injected oocytes, and E_ND96 was relatively positive, reflecting the background Na⁺ current apparently carried by NBCn1 (Choi et al. 2000; Cooper et al. 2005). However, I_HCO3 (at positive voltages) was no higher than I_ND96, and E_HCO3 was not appreciably different from E_ND96. In other words, NBCn1 did not produce HCO₃^–-dependent currents. Nevertheless, compared to water-injected controls, oocytes expressing NBCn1-B had a greater G_ND96 (5.9 versus 1.1 µS). Previous work showed that this additional ionic conductance is mostly due to a Na⁺ current but does not require HCO₃^–. This Na⁺ current is mediated by NBCn1 expressed both in Xenopus oocytes and human embryonic kidney (HEK) cells, indicating that the conductance is probably intrinsic to the transporter (Choi et al. 2000; Cooper et al. 2005).

Design of chimeric transporters

Rat NBCe1-A and NBCn1-B share overall 55% amino acid identity (more than 65% in the transmembrane domains), and have similar hydropathy plots (Choi et al. 2000). We presumed that this similarity would facilitate the construction of chimeric proteins between the two transporters without major problems in retaining protein conformation. Although both NBCe1 and NBCn1 exist as multiple splice variants, we are aware of no marked functional differences among variants of NBCn1. In the absence of protein binding partners such as IRBIT (Shirakabe et al. 2006), NBCe1-A, with its unique Nt, is substantially more active than either NBCe1-B or NBCe1-C (McAlear et al. 2006). Therefore, we made our constructs with rat NBCe1-A, which is expressed in the proximal tubules of the kidney (Romero et al. 1998), and rat NBCn1-B, which is predominantly present in the cardiovascular system (Cooper et al. 2006). One specific prediction is that replacing the Nt of NBCe1-A with that of NBCn1-B may be analogous to replacing the Nt of NBCe1-A with that of NBCe1-B, and thus render an electrogenic transporter less active. Chimeras might also be less active than the wild-type molecules because of ‘mismatches’ of various domains, which might lead to decreased surface expression, or to decreased activity of individual molecules. Here we use the shorthand terminology ‘e1’ for NBCe1-A and ‘n1’ for NBCn1-B. We made 10 chimeric constructs by swapping different regions of NBCe1-A with the corresponding ones of NBCn1-B (Fig. 2 and Table 1). In establishing boundaries of regions to be swapped, we followed the 13-TM topology model proposed for the Cl^––HCO₃^– exchanger AE1 (Fujinaga et al. 1999; Kuma et al. 2002).

View larger version (26K): [in this window] [in a new window]   Figure 2.  Cotransporters used in the present study We generated chimeras between NBCe1-A (grey areas) and NBCn1-B (orange areas). N, N-terminal domain; TM1–5, transmembrane segments 1–5; L, third extracellular loop; TM6–13, transmembrane segments 6–13; C, C-terminal domain.

Figure 3 summarizes the function of the chimeric transporter n1(Nt)/e1 (Fig. 3A), which has the cytoplasmic N-terminal domain of NBCn1-B, with the remainder of the molecule contributed by NBCe1 (i.e. NBCe1 ‘background’). In voltage-clamp experiments (e.g. Figure 3B), mean I_ND96 (at positive voltages) and G_ND96 (Table 2) were relatively low compared to NBCe1-A (Fig. 1B), but higher than for H₂O-injected controls (Fig. 1A). E_ND96 for n1(Nt)/e1 was about –43 mV. In the presence of 5% CO₂–26 mM HCO₃^–, I_HCO3 (at positive voltages) and G_HCO3 were much higher than the corresponding values in the presence of ND96. Moreover, E_HCO3 was markedly more negative than E_ND96. From the difference I_HCO3–I_ND96, we see that I_NBC (at positive voltages) and G_NBC were much greater than the corresponding values for the chimera in ND96, but much less than G_NBC for wild-type NBCe1-A. For the chimera, E_NBC was more negative than –150 mV. These results indicate that replacing the N-terminal domain of NBCe1-A with the corresponding region of NBCn1-B results in a transporter that is electrogenic. The relatively low G_NBC of the chimera, which presumably reflects mismatching the N terminus to the rest of the transporter, could be due to either reduced protein delivery to the cell surface or to reduced activity of individual transporters.

View larger version (11K): [in this window] [in a new window]   Figure 3.  Replacing the N-terminal domain of NBCe1-A with the N-terminal domain of NBCn1-B A, chimeric transporter n1(Nt)/e1. B, (I–V) relationships obtained from oocytes expressing n1(Nt)/e1 (n = 7) in the absence () and presence () of 5% CO2–26 mM HCO3– (pH 7.4). The difference IHCO3–IND96 is an estimate of the electrogenic cotransport current (). C, representative recordings of pHi and membrane potential (Vm) in an oocyte expressing n1(Nt)/e1. The pHi recovery from a CO2-induced acidification (arrow) and the hyperpolarization upon application of 1.5%CO2–10 mM HCO3– (arrowhead) are hallmarks of electrogenic Na+–HCO3– transport. One of four experiments.

In this series of experiments, we analysed three other chimeras. The first, n1(L)/e1, had the third extracellular loop of NBCn1-B on a background of NBCe1-A (not shown). The second, n1(Ct)/e1, had the cytoplasmic C-terminal domain of NBCn1-B on a background of NBCe1-A (not shown). The third chimera, n1(NtLCt)/e1, had the N terminus, extracellular loop and C terminus from NBCn1-B on a background of NBCe1 (Fig. 4A). Voltage-clamp data for all three chimeras (summarized in Table 2) were similar to those shown for the triple chimera in Fig. 4B. G_HCO3 was substantially higher than G_ND96, G_NBC was much higher than G_ND96 in H₂O-injected oocytes (Fig. 1A), and E_NBC for n1(NtLCt)/e1 was more negative. In other words, all three had the functional characteristics of an electrogenic transporter. These data show that the major non-transmembrane domains (i.e. cytoplasmic N and C termini and third extracellular loop, which are less-well conserved than the transmembrane domains) of NBCe1-A are not critical for electrogenicity. In other words, the transmembrane domains of NBCe1-A are the only components required for electrogenic function.

View larger version (14K): [in this window] [in a new window]   Figure 4.  Replacing the major non-transmembrane domains of NBCe1-A with the corresponding domains of NBCn1-B A, chimeric transporter n1(NtLCt)/e1. B, (I–V) relationships obtained from oocytes expressing n1(NtLCt)/e1 in the absence () and presence () of HCO3––CO2 (n = 5).

To further confirm the above findings, we constructed the four opposite chimeras in which the major non-transmembrane domains of NBCe1-A replaced the homologous domains of NBCn1-B. The first, e1(Nt)/n1, had the cytoplasmic N terminus of NBCe1-A on a background of NBCn1-B (Fig. 5A). The second and third, e1(L)/n1 and e1(Ct)/n1, respectively, had the third extracellular loop or cytoplasmic C terminus of NBCe1-A on a background of NBCn1-B (not shown). Finally, e1(NtLCt)/n1 had the N and C termini as well as the third extracellular loop of NBCe1-A on a background of NBCn1-B (Fig. 6A). Voltage-clamp data for all four chimeras (summarized in Table 2) were similar to data shown in Figs 5B and 6B. I_ND96 (at positive voltages) and G_ND96 values were higher than for H₂O-injected oocytes. E_ND96 values were around –30 mV. The chimeras share these characteristics with wild-type NBCn1-B, which has a background conductance. I_HCO3 (at positive voltages) and G_HCO3 for the chimeras were somewhat lower than corresponding values in the presence of ND96; this is also a characteristic of wild-type NBCn1-B (Fig. 1C). The I–V relationship of the triple chimera e1(NtLCt)n1 exhibited slight outward rectification (Fig. 6B). Clearly, none of these four chimeras exhibited the currents characteristic of electrogenic cotransport. Conversely, the presence of the background conductance in all four chimeras is consistent with previous conclusions that the ionic conductance is likely to be a property of the transmembrane domains of NBCn1-B.

View larger version (10K): [in this window] [in a new window]   Figure 5.  Replacing the N-terminal domain of NBCn1-B with the N-terminal domain of NBCe1-A A, chimeric transporter e1(Nt)/n1. B, (I–V) relationships obtained from oocytes expressing e1(Nt)/n1 in the absence () and presence () of HCO3––CO2 (n = 7). C, representative recordings of pHi and Vm in an oocyte expressing e1(Nt)/n1. The pHi recovery from a CO2-induced acidification (arrow) without affecting membrane potentials (arrowhead) are hallmarks of electroneutral Na+–HCO3– transport. A substantial hyperpolarization caused by Na+ removal is due to an associated Na+ conductance. One of four experiments.

View larger version (14K): [in this window] [in a new window]   Figure 6.  Replacing the major non-transmembrane domains of NBCn1-B with the corresponding domains of NBCe1-A A, chimeric transporter e1(NtLCt)/n1. B, (I–V) relationships obtained from oocytes expressing e1(NtLCt)/n1 in the absence () and presence () of HCO3––CO2 (n = 7).

Taken together, the data in this section demonstrate that the major non-transmembrane domains (i.e. cytoplasmic N and C termini and third extracellular loop) of NBCe1-A do not confer electrogenicity on chimeras in which the transmembrane domains come from NBCn1-B. Conversely, the transmembrane domains of NBCn1-B are the only components required for the background conductance.

Replacing either the front or back half of NBCe1-A with the homologous region of NBCn1-B abolishes electrogenic Transport

Front half n1.  Our data described above show that the major non-transmembrane domains of NBCe1-A, despite their unique amino acid composition, are not necessary for, nor do they by themselves confer, electrogenicity of Na⁺–HCO₃^– cotransport. Thus, the better-conserved transmembrane domains must determine whether cotransport is electrogenic. Based on the work of others on AE1 (for review see Alper et al. 2001), it is reasonable to divide the transmembrane domains of NBCe1-A into two regions: TM1–5 and TM6–13. Separating the two regions is the third extracellular loop that, as demonstrated by the above data, has no significant role in electrogenicity of NBCe1-A or the background conductance of NBCn1-B. To examine the functional role of these two transmembrane regions, we made two final chimeras. The first, n1(1–821)/e1, had the composition of NBCn1-B from the N terminus to the end of the third extracellular loop (‘front half’), and the composition of NBCe1-A from TM6 to the C terminus (‘back half’, Fig. 7A). Voltage-clamp data for oocytes expressing this chimera (summarized in Table 2) show that G_ND96 was very low, suggesting that the chimera does not have the background conductance characteristic of NBCn1-B (Fig. 7B). Moreover, G_HCO3 was slightly lower than G_ND96 and E_HCO3 was not shifted to negative values, suggesting that the chimera does not have the electrogenic cotransport characteristic of NBCe1-A.

View larger version (10K): [in this window] [in a new window]   Figure 7.  Replacing the front half (N-terminus, TM1–5 and loop) of NBCe1-A with the homologous parts of NBCn1-B A, chimeric transporter n1(1–821)/e1. B, (I–V) relationships obtained from oocytes expressing n1(1–821)/e1 in the absence () and presence () of HCO3––CO2 (n = 5). C, representative recordings of pHi and Vm in an oocyte expressing n1(1–821)/e1. The number in parenthesis (1–821) represents amino acids of NBCn1-B. The pHi recovery from an acidification (arrow) without hyperpolarization (arrowhead) is shown. One of three experiments.

Nevertheless, oocytes expressing n1(1–821)/e1 clearly exhibited a more rapid pH_i recovery than water-injected controls (–0.6 ± 0.5 x 10^–5 pH units s^–1, n = 5), and the difference is highly significant (P = 0.001, unpaired two-tailed t test). Thus, n1(1–821)/e1 engages in significant acid–base transport. Because even very slow pH_i recovery rates produce large changes in V_m (Romero et al. 1997) (i.e. V_m is a very sensitive indicator of electrogenicity), we are in a position to judge the electrogenicity of the n1(1–821)/e1 chimera. However, application of CO₂–HCO₃^– did not cause the large, abrupt hyperpolarization characteristic of NBCe1-A or even of the n1(Nt)/e1 chimera shown in Fig. 3C (change in V_m, –33.3 ± 2.8 mV, n = 4), but instead led to a slow, small depolarization (change in V_m, +14.7 ± 1.7 mV, n = 5) that we often observed in H₂O-injected, control oocytes. Moreover, Na⁺ removal caused neither the large abrupt depolarization characteristic of NBCe1-A or even of the n1(Nt)/e1 chimera (+38.3 ± 10.0 mV, n = 4), nor the large hyperpolarization (reflecting the background Na⁺ conductance) of the n1(822–1218)/e1 chimera (–29.1 ± 4.9, n = 3), but only a small hyperpolarization (–6.4 ± 0.7, n = 5) similar to the observation in H₂O-injected, control oocytes (–2.9 ± 1.6, n = 5). These data show that the chimera n1(1–821)/e1, although active in terms of pH_i changes, has neither electrogenic cotransport nor a background Na⁺ conductance.

Back half n1.  The absence of electrogenic cotransport in n1(1–821)/e1 shows that TM1–5 of NBCe1 is essential for electrogenicity. To test whether TM6–13 is also involved, we then made the reciprocal chimera n1(822–1218)/e1, which had the composition of NBCe1-A from the N terminus to the end of the third extracellular loop (‘front half’), and the composition of NBCn1-B from TM6 to the C terminus (‘back half’, Fig. 8A). Voltage-clamp data for oocytes expressing n1(822–1218)/e1 (summarized in Table 2) show that G_ND96 was relatively high, suggesting that the chimera has the background conductance characteristic of NBCn1-B (Fig. 8B). On the other hand, G_HCO3 was slightly lower than G_ND96 and E_HCO3 was not shifted towards more negative values, suggesting that this chimera lacks the electrogenic cotransport characteristic of NBCe1-A.

View larger version (11K): [in this window] [in a new window]   Figure 8.  Replacing the second half (TM6-13 and C-terminus) of NBCe1-A with the homologous parts of NBCn1-B A, chimeric transporter n1(822–1218)/e1. B, I–V relationships obtained from oocytes expressing n1(822–1218)/e1 in the absence () and presence () of HCO3––CO2 (n = 7). C, representative recordings of pHi and Vm. The pHi recovery from an acidification (arrow) without hyperpolarization (arrowhead) is shown. One of three experiments.

In conclusion, our chimera studies indicate that the electrogenicity of NBCe1-A simultaneously requires both TM1–5 and TM6–13. On the other hand, the background Na⁺ conductance of NBCn1-B requires only TM6–13.

	Discussion

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Overview

In this study, we provide the first molecular details of the structural requirements for electrogenic transport of Na⁺–HCO₃^– cotransporters. By exchanging elements between NBCe1 and NBCn1, we determined that the relatively well-conserved transmembrane domains of NBCe1 are necessary and sufficient for electrogenic cotransport. That is, electrogenicity requires the simultaneous presence of TM1–5 and TM6–13 of NBCe1-A. However, electrogenicity is independent of the three, large, less-well conserved hydrophilic domains, namely, the cytoplasmic N terminus, the third extracellular loop and the cytoplasmic C terminus. We also found that the background Na⁺ conductance of NBCn1-B requires TM6–13 but not TM1–5 or any of the three large hydrophilic elements. These conclusions could be a valuable staring point for future studies aimed at determining the domains responsible for electrogenic versus electroneutral function as well as the background Na⁺ conductance of NBCn1-B.

Structural domains and motifs

Electrogenic transport.  Among the chimeras we constructed, those containing, and only those containing, both TM1–5 and TM6–13 of NBCe1 were electrogenic. Breaking the interaction between TM1–5 and TM6–13 of NBCe1-A (i.e. pairing an electroneutral TM1–5 with an electrogenic TM6–13, or vice versa) abolishes electrogenicity and yet maintains cotransport. In other words, both TM1–5 and TM6–13 of NBCe1-A have the machinery necessary for performing electroneutral transport, which is perhaps the basal state of NBCe1.

Electrogenic cotransport requires an interaction between at least one motif in TM1–5 and at least one motif in TM6–13. The identity of these motifs remains unclear. Up to now, only a handful of studies have focused on structure–function relationships of NBCe1. Abuladze et al. (2005) recently mutated a large number of acidic and basic amino acids in NBCe1 – in putative transmembrane segments and intra- or extracellular loops – to oppositely charged and/or neutral amino acids. Examining the effects on the Na⁺-dependent recovery of pH_i from an acid load in HEK-293T cells, they found that many mutations caused substantial decreases in acid-extrusion rate, some resulting in severe functional defects. Whereas the report by Abuladze et al. (2005) provides useful information on residues that are important for transport activity, it was not intended to address the electrogenicity of Na⁺–HCO₃^– transport and, indeed, the assays did not involve electrophysiological measurements. Moreover, the authors focused on amino acid residues that are conserved among members of the SLC4A family including Cl^––HCO₃^– exchangers and electroneutral Na⁺-coupled HCO₃^– transporters. Thus, it is unlikely that these conserved residues are critical for determining the electrogenicity of NBCe1.

Inferences from the AE subfamily.  It is possible that some insight into the structural elements of Na⁺–HCO₃^– transporters may be inferred from the AE-type Cl^––HCO₃^– exchangers, which have been more thoroughly studied than the NBCs. Traditionally, AE was divided into two structurally and functionally distinct domains: the cytoplasmic N-terminal domain (43 kDa) that binds a variety of other proteins (Low, 1986) and the transmembrane domain (55 kDa) that mediates anion transport (Passow, 1986). Studies of AE1/AE2 chimeras reveal that the transmembrane domain determines the fundamental transport characteristics (i.e, Cl^––HCO₃^– exchange and chloride–nitrate exchange), whereas the cytoplasmic N-terminal domain modulates the transport (i.e. pH dependence) (Alper et al. 2001). The membrane domain works as a ‘sensor’, whereas the cytoplasmic N-terminus is a ‘modifier’. The study of AE2/AE3 chimeras supports this functional compartmentalization (Fujinaga et al. 2003). Muller-Berger et al. (1995b) have proposed that TM5, 8, 9, 10 and 13 of AE1 are functional segments for anion translocation. Several amino acids in these TMs have been proposed to serve as a proton coupling site (Jennings & Smith, 1992; Sekler et al. 1995), an anion selectivity filter (Zhu & Casey, 2004) or a pore-entry site (Muller-Berger et al. 1995a; Tang et al. 1998, 1999; Kuma et al. 2002; Jennings, 2005). These reports emphasized the structural importance of multiple amino acids in several TMs. It is possible that homologous amino acids in NBCs may also play similar roles in ion translocation (McAlear & Bevensee, 2006).

Natural NBCe1 mutations.  At least eight naturally occurring mutations in human NBCe1 have been reported (Igarashi et al. 1999, 2002; Inatomi et al. 2004; Horita et al. 2005): Q29, R298S, S427L, R510H, T485S, A799V, R881C and a frameshift at codon 721. Q29 is a non-sense mutation, whereas the others, with the exception of the frameshift at 721, are missense. All of these mutations have no more than 50% of the wild-type activity, causing a severe proximal renal tubular acidosis with the arterial blood pH of below 7.10. Additional symptoms may include ocular abnormalities such as glaucoma, cataracts, band keratopathy and blindness. Many of these point mutations cause targeting defects of the transporters, whereas others appear to have normal function (Inatomi et al. 2004; Horita et al. 2005; Toye et al. 2006). Dinour et al. (2004) has recently reported that S427L mutation in TM1 alters voltage- and Na⁺-dependent HCO₃^– movement, resulting in approximately 10% of HCO₃^– currents compared to wild-type renal NBCe1-A. The change in voltage- and Na⁺-dependence in the S427L mutant may be due to the absence of the ‘voltage-sensing’ process of the transporter. Li et al. (2005) reported that S427L has an apical targeting process in polarized kidney cells. It is interesting to note that all electroneutral Na⁺–HCO₃^– transporters have an alanine residue at the site corresponding to S427 in NBCe1-A.

The Na⁺ conductance of NBCn1.  Among the chimeras we constructed, only those containing TM6–13 of NBCn1 exhibited the Na⁺ conductance typical of NBCn1 (Choi et al. 2000; Cooper et al. 2005). Thus, only TM6–13 is required for the Na⁺ conductance. However, the identity of the critical motifs remains unclear.

Another electroneutral member of the SLC4 family, AE1, can under some circumstances exhibit an ion conductance. AE1 may be responsible for a small, DIDS-sensitive anion conductance observed in human erythrocytes (Knauf et al. 1977; Kaplan et al. 1983). Although normally electroneutral in its natural environment, trout AE1 (tAE1) responds to cell swelling by exhibiting an anion conductance (for review see Motais et al. 1997) that contributes to regulated volume decrease. When this same tAE1 is expressed in Xenopus oocytes, it exhibits a Cl^– conductance even in the absence of cell swelling (Fievet et al. 1995). Studies with trout–mouse AE1 chimeras indicate that this anion conductance of tAE1 requires residues in the extracellular loop between TM5 and TM6, as well as residues in the remaining C-terminal portion of the molecule (i.e. the ‘back half’ of tAE1) (Fievet et al. 1995; Borgese et al. 2004). It is interesting to note that, when expressed in Xenopus oocytes, the human AE1 construct AE1(6:7), which lacks transmembrane segments 6 and 7, also mediates a large anion conductance. In addition, oocytes expressing AE1(6:7) exhibit a Na⁺ conductance (Parker et al. 2006). As suggested by Parker et al. (2006), the TM6 and 7 module in human AE1 may be necessary to ensure that transport is electroneutral.

Thus, as is the case for AE1, data on NBCn1 point to the ‘back half’ of the molecule as being critical for ion conductance.

Preservation of cotransport activity

Our study was faciitated by the strong homology between NBCe1 and NBCn1, which permitted us to construct functional chimeras. Improper folding often results in failure of membrane proteins to target to the plasma membrane and instead to accumulate in the endoplasmic reticulum or Golgi. The C-terminal domains of NBCe1 and NBCn1 play an essential role in targeting and sorting of the protein. Using a series of deletion mutants with the C-terminal end of NBCe1-A, Li et al. (2004) have identified the sequence QQPFLS (amino acid residues 1010–1015) which contributes to the targeting of NBCe1-A to the basolateral membrane of the kidney epithelial cells. Nonetheless, the deletion of this motif does not abolish the targeting of the transporter to the plasma membrane in Xenopus oocytes. It is thus likely that QQPFLS would play a role in membrane polarity of the transporter. Our sequence analysis shows no significant amino acid homology in this motif among the SLC4A family. For NBCn1-B, the PDZ motif at the C-terminal domain is more important for targeting to the membrane (Loiselle et al. 2003). Deletion of the PDZ motif of the human homologue inhibited the transporter from being targeted to the plasma membrane in HEK cells and instead caused an intracellular accumulation of the transporter (Loiselle et al. 2003; Li et al. 2004). The information on the targeting role of the N-terminal domain is very rare. Despite this, our success in producing functionally active chimeras may be due at least in part to the presence of the native cytoplasmic N- and C-terminal domains of NBCe1 and NBCn1 in our chimeras.

Limitations

Strictly speaking, our results pertain only to NBCe1-A and NBCn1-B expressed in Xenopus oocytes, where NBCe1-A has a stoichiometry for Na⁺:HCO₃^– of 1 : 2 (Heyer et al. 1999). It is impossible, from the present work, to draw conclusions about the structural requirements for the 1 : 3 stoichiometry that is believed to prevail in the renal proximal tubule.

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	Acknowledgements

 
The work was supported by NIH grant DK30344 (W.F.B.) and an NKF Young Investigator Grant (I.C.).

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