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Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294

Correspondence to Mark O. Bevensee: bevensee{at}physiology.uab.edu

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Using pH- and voltage-sensitive microelectrodes, as well as the two-electrode voltage-clamp and macropatch techniques, we compared the functional properties of the three NBCe1 variants (NBCe1-A, -B, and -C) with different amino and/or carboxy termini expressed in Xenopus laevis oocytes. Oocytes expressing rat brain NBCe1-B and exposed to a CO₂/HCO₃^– solution displayed all the hallmarks of an electrogenic Na⁺/HCO₃^– cotransporter: (a) a DIDS-sensitive pH_i recovery following the initial CO₂-induced acidification, (b) an instantaneous hyperpolarization, and (c) an instantaneous Na⁺-dependent outward current under voltage-clamp conditions (–60 mV). All three variants had similar external HCO₃^– dependencies (apparent K_M of 4–6 mM) and external Na⁺ dependencies (apparent K_M of 21–36 mM), as well as similar voltage dependencies. However, voltage-clamped oocytes (–60 mV) expressing NBCe1-A exhibited peak HCO₃^–-stimulated NBC currents that were 4.3-fold larger than the currents seen in oocytes expressing the most dissimilar C variant. Larger NBCe1-A currents were also observed in current–voltage relationships. Plasma membrane expression levels as assessed by single oocyte chemiluminescence with hemagglutinin-tagged NBCs were similar for the three variants. In whole-cell experiments (V_m = –60 mV), removing the unique amino terminus of NBCe1-A reduced the mean HCO₃^–-induced NBC current 55%, whereas removing the different amino terminus of NBCe1-C increased the mean NBC current 2.7-fold. A similar pattern was observed in macropatch experiments. Thus, the unique amino terminus of NBCe1-A stimulates transporter activity, whereas the different amino terminus of the B and C variants inhibits activity. One or more cytosolic factors may also contribute to NBCe1 activity based on discrepancies between macropatch and whole-cell currents. While the amino termini influence transporter function, the carboxy termini influence plasma membrane expression. Removing the entire cytosolic carboxy terminus of NBCe1-C, or the different carboxy terminus of the A/B variants, causes a loss of NBC activity due to low expression at the plasma membrane.

Abbreviations used in this paper: AE, anion exchanger; DIDS, 4,4'-diisothiocyanatostilbene-2,2' disulfonate; HA, hemagglutinin; HRP, horseradish peroxidase; NBC, Na/bicarbonate cotransporter; pH_i, intracellular pH; PIP₂, phosphatidylinositol 4,5-bisphosphate; SOC, single-oocyte chemiluminescence.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Na/HCO₃ cotransporters (NBCs) are functionally diverse proteins that are involved in the regulation of intracellular pH (pH_i), absorption or secretion of HCO₃^–, and maintenance of ion homeostasis in many tissues. The first Na⁺/HCO₃^– cotransporter was identified by function in the proximal tubule of the kidney (Boron and Boulpaep, 1983), where the transporter has a 1:3 Na⁺:HCO₃^– stoichiometry (Soleimani et al., 1987) and is responsible for reabsorbing as much as 90% of filtered bicarbonate (Boron et al., 1997). In glial cells of the central nervous system, an electrogenic Na⁺/HCO₃^– cotransporter with a 1:2 Na⁺:HCO₃^– stoichiometry (Deitmer and Schlue, 1989; O'Connor et al., 1994; Bevensee et al., 1997a,b) contributes to intracellular and extracellular pH changes that can influence neuronal activity (Chesler, 2003; McAlear and Bevensee, 2004).

At the molecular level, the first cDNA encoding an electrogenic NBC was identified by expression from salamander kidney (Romero et al., 1997). The cloning of salamander kidney NBC paved the way for homology cloning of both electrogenic and electroneutral NBCs, as well as other cation-coupled anion transporters (for review see Romero et al., 2004). Cation-coupled anion transporters in conjunction with anion exchangers (AEs) are members of a superfamily of bicarbonate transporters (BTs). Using the convention introduced by Choi et al. (2000) and expanded by Romero et al. (2004), we refer to the first cloned NBC as NBCe1-A, where "e" refers to electrogenic, "1" refers to the first gene cloned of this family, and "A" refers to the first splice variant identified. The putative membrane topology of NBCe1 shown in Fig. 1 is based on sequence alignment of NBCe1 and AE1 (Romero et al., 1998b). We mapped the NBCe1 sequence on a topology model of the related bicarbonate transporter anion exchanger 1 or AE1 (Taylor et al., 2001). As described by Taylor et al. (2001), the putative topology of AE1 is determined from proteolysis and cysteine accessibility data.

NBCe1 proteins arise from different splice variants of gene SLC4A4 (Abuladze et al., 2000). The electrogenic Na/HCO₃ cotransporters can be categorized into one of three groups based on differences at the amino and/or carboxy termini (Fig. 1 ): NBCe1-A ("kidney" clone), NBCe1-B ("heart," "pancreas," and rat-brain 1 NBC clone), and NBCe1-C (rat-brain 2 NBC clone). At the amino acid level, NBCe1-B is identical to NBCe1-A except at the amino terminus where 85 residues replace the 41 residues of NBCe1-A. NBCe1-C is identical to NBCe1-B except at the carboxy terminus where 61 residues replace the 46 residues of NBCe1-B. As reported for the A and B clones from human (Choi et al., 1999), the unique amino terminus of the rat A variant is only 6% identical and 13% homologous to that of the rat B and C variants. Furthermore, while both termini are hydrophilic, 50% of the 85 amino-terminal residues of the B and C variants are charged residues in contrast to only 22% of the 41 unique amino-terminal residues of the A variant. Although NBCe1-C has only been reported from rat brain, exon excision between introns at positions 23 and 24 in human SLC4A4 (Abuladze et al., 2000) would create the unique carboxy terminus. Indeed, using RT-PCR techniques, we have recently identified the full-length cDNAs encoding both NBCe1-B and NBCe1-C from human brain (unpublished data).

View larger version (50K): [in this window] [in a new window]   Figure 1. Wild-type and truncated rat NBCe1 variants. NBCe1-A, -B, and -C differ at the amino and/or carboxy termini. The putative membrane topology of NBCe1 is based on mapping of AE1 by Taylor et al. (2001). Truncations examined in this study include hemagglutinin (HA)-tagged NBCe1s with stretches of the cytoplasmic amino or carboxy terminus removed. (Inset, top left) At the amino terminus, NBCe1-A has 41 unique amino acids that differ from 85 amino acids in the B and C variants. At the carboxy terminus, NBCe1-C has 61 unique amino acids that differ from 46 amino acids in the A and B variants.

In addition to structure–function data, there is also genetic information that highlights the importance of the cytosolic amino terminus of NBCe1 before the first predicted transmembrane domain. There are human patients with mutations in SLC4A4 who present primarily with proximal renal tubular acidosis (pRTA) and ocular abnormalities (Igarashi et al., 1999; Dinour, D., A. Knecht, I. Serban, and E.J. Holtzman. 2000. J. Am. Soc. Neurol. 11:3A; Igarashi, T., J. Inatomi, T. Sekine, Y. Yakeshima, N. Yoshikawa, and H. Endou. 2000. J. Am. Soc. Nephrol. 11:106A; Dinour et al., 2004). One patient has an inactivating homozygous missense mutation in which a Ser replaces Arg at position 298 in the cytoplasmic amino terminus of NBCe1-A (Igarashi et al., 1999). In expression studies using ECV304 cells, the authors found that this mutant NBC displayed only 50% of wild-type transporter activity. This substitution is found at position 342 in the B and C variants. A second patient has a homozygous missense mutation in which a Leu replaces Ser at position 427 at the beginning of the first predicted transmembrane domain of NBCe1-A (Dinour et al., 2004). When expressed in oocytes, the mutant NBC displayed only 10% of wild-type transporter activity. In a separate study, this mutant NBC was mistargeted to the apical instead of the basolateral membrane of polarized Madin-Darby canine kidney (MDCK) cells (Li et al., 2005).

According to the aforementioned studies, regions within the cytoplasmic amino and carboxy termini can influence the function, regulation, and expression of NBCe1 variants. However, a detailed comparison of the biophysical properties of all three variants with different amino and/or carboxy termini has yet to be performed. In the present manuscript, we used pH- and voltage-sensitive microelectrodes to characterize, for the first time, the function of rat brain NBCe1-B expressed in Xenopus oocytes. Subsequently, we used the two-electrode, voltage-clamp technique to compare the activities, as well as the ion and voltage dependencies of all three NBCe1 variants. Although all three variants have similar external ion and voltage dependencies, as well as plasma membrane expression levels as assessed by single oocyte chemiluminescence, the activity of the A variant is greater than the activities of the other two variants. According to structure–function analyses using whole-cell and macropatch recording techniques, the higher activity of A is due to its unique amino terminus. While the different amino termini influence function, one or more regions within the different carboxy termini contribute(s) to plasma membrane expression.

Portions of this work have been published in abstract form (Williams, J.B., and M.O. Bevensee. 2002. FASEB J. 16:A796; McAlear, S.D., J.B. Williams, and M.O. Bevensee. 2004. FASEB J. 18:A1022–A1023; Liu, X., S.D. McAlear, and M.O. Bevensee. 2005. FASEB J. 19:A142; McNicholas-Bevensee, C.M., X. Liu, S.D. McAlear, and M.O. Bevensee. 2006. FASEB J. 20:A1232).

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
NBC Constructs and Mutagenesis
Wild-type NBCe1 Variants.
We used cDNAs encoding NBCe1-A from rat kidney (Romero et al., 1998b), and NBCe1-C from rat brain (Bevensee et al., 2000) subcloned into the oocyte expression vector pTLNII as previously described. Full-length NBCe1-B, which was also previously identified from rat brain by RT-PCR (Bevensee et al., 2000), was constructed using convenient restriction enzymes to swap the unique carboxy terminus of full-length variant C in pTLNII with the carboxy terminus of variant B from a partial-length construct in pBluescript. For both wild-type and mutant constructs (see below), NBC expression was optimized by introducing a Kozak sequence (Kozak, 1986) at the initiator codon using PCR techniques and a Peltier thermal cycler (PTC-220 DNA Engine Dyad, MJ Research, Inc.). Sequence analysis and primer design were performed using either DNAsis (Hitachi Software) or Vector NTI Advance 9.0 (InforMax, Invitrogen), and all constructs were confirmed by bidirectional DNA sequencing (DNA Sequencing Core, Center for AIDS Research and the Genomics Core Facility, Heflin Center for Human Genetics, both at the University of Alabama at Birmingham).

HA-tagged NBCe1 Constructs.
To insert the nine-residue hemagglutinin (HA) epitope (YPYDVPDYA) into NBCe1-C, we used the QuikChange PCR-based mutagenesis kit (Stratagene) to create a unique restriction site (Bsu36I) at base pair 1941 (residue 647) within the extracellular loop between transmembrane domains 5 and 6. A double-stranded pair of 5'- and 3'-phosphorylated primers encoding the HA epitope was flanked by Bsu36I sticky ends. Equal concentrations of the two primers were first denatured in a thermocycler (Genius FGEN05TP, Techne) by heating to 94°C (15 s), and then allowed to anneal by slowly cooling the reaction in increments of 2°C (15 s each) to 37°C. This double-stranded primer pair was then ligated into the engineered Bsu36I site in NBCe1-C. Because of the introduced Bsu36I cut sites, the inserted HA epitope in the protein was flanked by Asp and Gly. HA-tagged NBCe1-A and -B were constructed from the tagged NBCe1-C using convenient restriction enzymes.

Truncated NBCe1 Variants.
Truncated NBCe1 constructs (see Fig. 1) were generated using PCR techniques and HA-tagged NBCe1 variants subcloned into pTLNII as templates. In generating amino-terminal truncations, we targeted residue 43 of the A variant and the homologous residue 87 of the C variant to optimize the Kozak sequence. NBCe1 constructs truncated at the carboxy terminus were generated by introducing a targeted stop codon using site-directed mutagenesis (QuikChange kit, Stratagene).

Functional Studies on NBCs
Generation of cRNA.
pTLNII plasmids containing NBCe1 constructs were linearized with the restriction enzyme MluI. The linearized cDNA was then transcribed from the SP6 promoter using the SP6 transcription kit (Ambion), and the resulting cRNA was purified using the RNeasy kit (QIAGEN).

Isolation and Injection of Oocytes.
Oocytes were harvested from female Xenopus laevis frogs using an approach very similar to that previously described (Romero et al., 1998b; Bevensee et al., 2000). A small incision was made in the abdominal cavity of the frog, and oocyte-containing segments of the ovarian lobe were removed. The segments were teased apart into small pieces and digested for 1.5–2 h in sterile Ca²⁺-free ND96 containing 2 mg ml^–1 collagenase A (Roche Applied Science). Subsequently, the dissociated oocytes were first washed in Ca²⁺-free ND96, and then in Ca²⁺-containing ND96 before healthy-looking stage V/VI oocytes were separated under a dissecting microscope (GZ6, Leica). The oocytes were incubated at 18°C in sterile ND96 containing 10 mM Na/pyruvate and 10 mg ml^–1 gentamycin (Mediatech Inc.).

Oocytes were injected with 48 nl of either RNase-free H₂O or a cRNA solution using a "Nanoject II" microinjector (Drummond Scientific). Injected cells were incubated at 18°C in the aforementioned oocyte media, and experiments were performed at room temperature at least 2 d after injection.

pH_i and V_m Experiments.
Injected oocytes were placed in a flowthrough chamber connected to a custom-designed, dual-bank, solution delivery system. Main solution lines from two banks, which are each connected to six solution lines via a six-way rotary manifold, were directed to a two-position, Eagle four-way miniature solenoid valve with five ports (Clippard Instrument Laboratory). During experiments, solution from one bank was directed to the chamber, and solution from the other bank was directed to waste for priming purposes. Changing solutions delivered to the chamber occurred by pneumatically alternating between the two valve positions.

For simultaneous pH_i and voltage recordings, microelectrodes were pulled from borosilicate glass capillaries (G200F-4, Warner Instruments) with a Brown-Flaming micropipette puller (P-80 or P-97, Sutter Instruments). For pH electrodes, pulled acid-washed capillaries were subsequently baked at 200°C and silanized with bis-(methylamino)dimethylsilane (Fluka). Electrode tips were filled with hydrogen ionophore I-cocktail B (Fluka), and then the electrodes were backfilled with a pH 7.0 solution containing (in mM): 150 NaCl, 40 KH₂PO₄, and 23 NaOH. pH electrodes were then connected to one channel of a high-impedance electrometer (FD223, WPI). Voltage electrodes were filled with a saturated KCl solution and connected to a second channel. The microelectrodes typically had resistances of 1–3 M. The pH signal was obtained with a four-channel electrometer (Biomedical Instrumentation Laboratory, Department of Cellular and Molecular Physiology, Yale University, New Haven, CT) that subtracts the potential of the voltage electrode from the potential of the pH electrode. A miniature calomel electrode (Accumet, Fisher Scientific) filled with saturated KCl served as the reference for the voltage electrode. Data were acquired and plotted using custom-designed software written by Mr. Duncan Wong for the Boron laboratory (Department of Cellular and Molecular Physiology, Yale University).

Two-electrode, Voltage-clamp Experiments.
Voltage-sensitive and current-passing microelectrodes were pulled from borosilicate glass capillaries (G200F-4 or G83165T-4, Warner Instruments) with a micropipette puller (P-80 or P-97, Sutter Instruments or PC-10, Narishige, Tokyo, Japan). The electrodes were filled with saturated KCl and attached to the OC-725C voltage-clamp apparatus (Warner Instruments). Electrode resistances were typically 1–3 M for the voltage electrodes, and 0.1–0.6 M for the current electrodes. For experiments at a fixed holding potential of –60 mV, data were obtained at a filtering frequency of either 8–10 or 800 Hz with an 8-pole Bessel filter (LFP-8, Warner Instruments) and a sampling frequency of 30 Hz or 2 kHz, respectively. For the current-voltage (I-V) relationships shown, the data were obtained at a filtering frequency of 800 Hz and a sampling frequency of 2 kHz. The voltage-step protocol for the I-V plots shown included 12 sweeps in which the voltage was held at –60 mV for 60 ms, then stepped to one of 12 voltages (-200 mV to 20 mV in increments of 20 mV) for 20 ms, and finally returned to –60 mV for 20 ms before the next sweep. After obtaining each I-V plot, we confirmed that the oocyte was electrically tight by turning off the voltage clamp and verifying that the spontaneous V_m was close to the acquired reversal potential (Virkki et al., 2002). Data acquired with the ClampEx software (Axon Instruments pClamp 8.2, Molecular Devices, San Jose, CA) were digitized with a 1322A interface (Axon Instruments), and then analyzed with ClampFit software (pClamp 8.2, Axon Instruments).

Inside-out macropatch experiments.
Macropatch studies were performed on oocytes using a modification of the technique described by Hilgemann (1995). Patch pipettes were pulled from N-51-A borosilicate glass capillaries (O.D. 0.084 in. or 0.064 in., Drummond) using a PC-10 micropipette puller (Narishige). Tips gently broken to 10–12 µm were plunged into a bead of melted 8161 Corning glass (G86165T-4, Warner Instruments) fixed to 30-gauge, MF-9 platinum wire (Technical Products International Inc.), which was transiently heated by passing current. A break at the tip resulted from the wire retracting as it cooled. The process of heating the glass bead, plunging the tip, and cooling the wire was repeated until a satisfactory jagged-free tip of 14 µm in diameter was obtained. Pipette resistances were 4 M.

Experiments were performed at room temperature (23°C) in a flowthrough chamber on the stage of an inverted microscope (DMIRB, Leica). All solutions contained low Cl^– (2 mM) to minimize contaminating endogenous Cl^– currents in the oocyte (Machaca et al., 2002; Weber, 2002). Immediately before experiments, pipettes were backfilled with solution containing 5% CO₂/33 mM HCO₃^–. Access to the plasma membrane was obtained by shrinking the oocyte in a hypertonic solution (see below) and then removing the vitelline membrane with fine forceps. G seals were obtained at a negative holding potential (V_p = –50 mV), and solution flow was initiated after patch excision. NBC currents were similar from patches obtained from either the animal or vegetal pole of the oocyte.

Currents were obtained using an Axopatch 200B patch-clamp amplifier (Axon Instruments). Low-pass (1 kHz, internal filter) currents were digitized with a Digidata-1322A interface (Axon Instruments) at a sampling frequency of 5 kHz. Clampex software (pClamp 8.2, Axon Instruments) was used for data acquisition and analysis. Seal stability was routinely monitored throughout the experiment by measuring both membrane capacitance (using the membrane test function of Clampex) and seal resistance. For the figures shown, current recordings were further filtered at 100 Hz (8-pole Bessel filter), and subjected to data reduction (substitute average by a factor of four) using Clampfit (pClamp 8.2, Axon Instruments). Data were exported to Microsoft Excel 2002 for analysis and Origin 7.5 (OriginLab Corporation) for graphing.

Solutions
Two-electrode, Voltage-clamp Experiments.
The standard ND96 solution (pH 7.5) contained (in mM): 96 NaCl, 2 KCl, 1 MgCl₂, 1.8 CaCl₂, 5 HEPES, 2.5 NaOH. In the standard 5% CO₂/33 mM HCO₃^– solution, 33 mM NaCl was replaced with an equimolar amount of NaHCO₃, and the solution was equilibrated with 5% CO₂/95% O₂ to pH 7.5. In the low Cl^– (7.6 mM)-containing ND96 and 5% CO₂/33 mM HCO₃^– solutions used in the bicarbonate and voltage dependence experiments, the NaCl was replaced with equimolar amounts of sodium gluconate. 7.6 mM Cl^– was kept constant in solutions containing different amounts of HCO₃^– by replacing sodium gluconate with equimolar amounts of NaHCO₃. pH of the different HCO₃^–-containing solutions was maintained at 7.5 by equilibrating the solutions with appropriate mixtures of CO₂/O₂. In the Na⁺ dependence experiments, equimolar Na⁺ was replaced with NMDG⁺.

Inside-out Macropatch Experiments.
The hyperosmotic solution used to remove the vitelline membrane contained (in mM): 220 NMDG⁺, 220 aspartic acid, 2 MgCl₂, 10 EGTA, and 10 HEPES. The solution was titrated to pH 7.2 with NMDG⁺.

For inside-out macropatch experiments, the 2 mM-Cl^–, ND96 solution contained (in mM) 96 sodium cyclamate, 2 mM KOH, 2 mM cyclamic acid, 1 mM MgCl₂, 1.8 mM calcium cyclamate, 5 mM HEPES, and was adjusted to pH 7.5 with NaOH. In the 2-mM Cl^– solution containing 5% CO₂/33 mM HCO₃^–, 33 mM sodium cyclamate was replaced with an equimolar amount of NaHCO₃, and the solution was equilibrated with 5% CO₂/95% O₂ to pH 7.5. For all experiments, the 2 mM Cl^– solution containing 5% CO₂/33 mM HCO₃^– served as the patch pipette solution.

All chemicals were obtained from Sigma-Aldrich unless otherwise specified. According to packaging information, 4,4'-diisothiocyanatostilbene-2,2' disulfonate (DIDS) was at least 80% pure, a value factored into making DIDS-containing solutions.

Expression Studies on NBCs
Immunoblotting.
Similar to previously described (Schmitt et al., 1999; Bevensee et al., 2000), each oocyte was homogenized in a buffer containing protease inhibitors, and the suspension was centrifuged to remove cell debris and nuclei. Proteins were separated by SDS-7.5% PAGE (Ready Gels Precast Gels, Bio-Rad Laboratories) and transferred to an Immobilin-P PVDF membrane (Millipore). The membrane was probed for 1–2 h with a 1:200 dilution of a rabbit polyclonal antibody (Rab3A) to the amino terminus of NBCe1 (Schmitt et al., 1999), and then for 1 h with a 1:10K dilution of the secondary antibody, goat rabbit-IgG coupled to horseradish peroxidase (HRP) (Jackson ImmunoResearch Laboratories). Bound HRP was detected by chemiluminescence (SuperSignal, Pierce Chemical Co.) before being exposed to HXR Film (Hawkins X-Ray Supply).

Single-oocyte Chemiluminescence.
To evaluate the expression of NBCs at the oocyte plasma membrane, we used the single-oocyte chemiluminescence (SOC) technique pioneered by the Jan lab (Zerangue et al., 1999; Margeta-Mitrovic et al., 2000) and used by others (Yoo et al., 2003) to quantitate a hemagglutinin (HA)–tagged protein expressed at the cell surface. This technique employs enzyme amplification with a chemiluminescence substrate and sensitive, linear detection with a luminometer. As reported by Yoo et al. (2003), there is a linear relationship between surface expression detected by SOC and functional activity of the K⁺ channel, ROMK (Kir 1.1).

The following protocol with sterile ND96 solutions was performed on injected oocytes incubated at 4°C. Oocytes were fixed with 4% paraformaldehyde in ND96 for 15 min, rinsed 3x5 min with equal volumes of ND96, and then incubated for 30 min (or overnight) in a 1% BSA-ND96 blocking solution (used in subsequent antibody incubation steps). Fixing the oocytes with paraformaldehyde does not appreciably increase their permeability to the HA antibody. As shown in Results, there was no detectable labeling of truncated, HA-tagged NBCe1 proteins that fail to traffic to the plasma membrane, but were clearly present in a microsomal fraction based on immunoblot analyses. Fixed oocytes were incubated for 1 h in a 1:100 dilution of the rat monoclonal -hemagglutinin antibody (Roche), and then for 1 h in a 1:400 dilution of the secondary antibody, goat -rat IgG-HRP (Jackson ImmunoResearch Laboratories). For chemiluminescence readings, each oocyte was transferred to an eppendorf tube, and the transferred solution was replaced with 50 µl SuperSignal Elisa Femto substrate (Pierce Chemical Co.). The eppendorf tube was placed in a luminometer (TD-20/20, Turner Designs) and luminescence was measured 15 s later.

Statistics
Means between groups of data were compared using one-factor analysis of variance (ANOVA), as well as paired or unpaired forms of the Student's t test (Microsoft Excel 2002). P < 0.05 is considered significant. Rates of pH_i recoveries were determined by linear fits to pH_i vs. time data using a least-squares method. With Origin 7.5 software (OriginLab), Na⁺ dependence data were fit using Michaelis-Menten enzyme kinetics, and HCO₃^–-dependence data were fit using a modified form of the Michaelis-Menten equation that includes a linear component (Grichtchenko et al., 2000). Comparisons of fits were performed using Origin 7.5 software (OriginLab).

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Functional Expression of Rat Brain NBCe1-B in Oocytes
At the time the cDNA encoding rat brain NBCe1-C was cloned by homology and the protein characterized as an electrogenic NBC when expressed in oocytes, rat brain NBCe1-B was also identified (Bevensee et al., 2000). The amino acid sequence of rat brain NBCe1-B is 96% identical to that of human heart NBCe1-B, which has been characterized as a stilbene-sensitive, electrogenic NBC when expressed in oocytes (Choi et al., 1999). In the present manuscript, we expressed the rat brain version of NBCe1-B in oocytes and evaluated NBC function using pH- and voltage-sensitive microelectrodes.

At the onset of the experiment shown in Fig. 2 A , an oocyte injected with rat brain NBCe1-B and bathed in a nominally CO₂/HCO₃^–-free, HEPES-buffered solution (ND96, pH 7.5) had a resting pH_i of 7.32 (top trace) and a V_m of –55 mV (bottom trace). Exposing the oocyte to a solution containing 1.5% CO₂/10 mM HCO₃^– (pH 7.5) elicited an initial decrease in pH_i (ab) due to CO₂ entry and subsequent formation of H⁺ in the cell. The pH_i then increased (bc) due to NBC-mediated HCO₃^– transport into the oocyte. The mean pH_i recovery rate at a pH_i of 7.12 ± 0.02 (after the initial CO₂-induced acidification) was 7.1 ± 0.8 x10^–5 pH units s^–1 (n = 6). This pH_i recovery was blocked (cd) by 200 µM DIDS, an inhibitor of bicarbonate transporters. As shown in Table I , stilbenes reduced the rate of pH_i recovery under different CO₂/HCO₃^– conditions by a mean of 76 ± 7.5% (range: 58–100%). Inhibition of the pH_i recovery was at least partially reversible (de) when DIDS was removed. Returning the oocyte to ND96 caused pH_i to increase rapidly (ef) due to the net conversion of intracellular H⁺ and HCO₃^– to H₂O and CO₂, which exited the cell. The final resting pH_i was higher than the initial value, an observation consistent with net NBC-mediated HCO₃^– influx during segment be.

View larger version (13K): [in this window] [in a new window]   Figure 2. Activity of rat brain NBCe1-B expressed in an oocyte. (A) pHi (top trace) and voltage (bottom trace) were measured simultaneously in an NBCe1-B–injected oocyte that was initially bathed in a nominally HCO3–-free, HEPES-buffered solution. The oocyte was exposed to a solution containing 1.5% CO2/10 mM HCO3– during segment ae. Electrogenic NBC activity was evident from the instantaneous hyperpolarization (b'), and the pHi recovery (bc) following the initial CO2-induced acidification (ab). DIDS blocked the pHi recovery (cd), and partially reversed the hyperpolarization (c'). (B) The same experimental protocol in panel A was performed on an H2O-injected oocyte. Exposing the oocyte to CO2/HCO3– had no effect on Vm (a'b'), and elicited no pHi recovery (bc) following the initial decrease in pHi (ab). 200 µM DIDS generated only a small hyperpolarization (c').

The experimental maneuvers shown in Fig. 2 A had markedly different effects on pH_i and V_m of the H₂O-injected control oocyte shown in Fig. 2 B. As shown in the top trace, the 1.5% CO₂/10 mM HCO₃^– solution elicited the initial pH_i decrease (ab), but no subsequent pH_i recovery (bc). At a mean pH_i of 7.08 ± 0.07, the mean pH_i recovery rate of 2.1 ± 1.2 x10^–5 pH units s^–1 (n = 4) was 3.4-fold less (P < 0.01) than the rate seen in NBCe1-B-expressing oocytes. Furthermore, 200 µM DIDS in the presence of CO₂/HCO₃^– had little effect on the low, sustained pH_i (cd). Returning the oocyte to ND96 caused pH_i to increase and return to approximately the initial resting value (ef). The voltage changes in the control oocyte were equally unimpressive (bottom trace). CO₂/HCO₃^– had no effect on V_m (a'b'), and DIDS only caused a small hyperpolarization (c'), consistent with inhibition of an endogenous Cl^– conductance. In the same four experiments summarized above, the mean HCO₃^–-induced hyperpolarization of 1.5 ± 0.3 mV was 17-fold less than the hyperpolarization seen in NBCe1-B–expressing oocytes.

To increase the activity of NBCe1-B, we performed additional pH_i/V_m experiments using solutions containing 33 mM HCO₃^– and equilibrated with 5% CO₂ to maintain pH at 7.5. The mean pH_i-recovery rate at a pH_i of 6.87 ± 0.03 was 12.0 ± 2.5 x10^–5 pH units s^–1 (n = 11). At a similar pH_i (6.84 ± 0.03) in H₂O-injected oocytes, the mean pH_i recovery rate of 4.3 ± 1.9 x10^–5 pH units s^–1 (n = 6) was 2.8-fold less (P = 0.01). In these same experiments, the mean hyperpolarization elicited by 5% CO₂/33 mM HCO₃^– was 53 ± 4.8 mV in NBC-expressing oocytes, and only 0.5 ± 0.5 mV in H₂O-injected oocytes. In summary, rat brain NBCe1-B expressed in oocytes is electrogenic, stilbene-sensitive, and activated by CO₂/HCO₃^–. As described below in voltage-clamp experiments, the transporter is also Na⁺ dependent. All these characteristics are hallmarks of an electrogenic Na/HCO₃ cotransporter (see Romero et al., 2004).

Voltage Dependencies of the NBCe1 Variants
To study the electrogenicity of the NBCe1 variants in more detail, we used the two-electrode, voltage-clamp technique to examine current–voltage (I-V) relationships of all three NBCe1 variants. In each experiment, I-V relationships were obtained from oocytes first bathed in a low Cl^–, ND96 solution, and then after 10 min in 5% CO₂/33 mM HCO₃^– (to allow for intracellular equilibration of the physiological buffer). In some experiments, I-V plots were subsequently obtained after 2 min in the CO₂/HCO₃^– solution containing 200 µM DIDS. The HCO₃^–-dependent I-V plot for an NBC is the difference between the I-V plots obtained in the presence and absence of CO₂/HCO₃^–, after subtracting the corresponding HCO₃^–-dependent I-V plot obtained from batch-matched H₂O-injected eggs.

An experiment performed on an oocyte expressing NBCe1-C is shown in Fig. 3 A . NBC currents obtained at potentials from –200 to +20 mV were larger in the presence of 5% CO₂/33 mM HCO₃^– (squares) than in ND96 (diamonds). The larger currents were particularly evident at voltages more positive than –80 mV. In the presence of DIDS, the I-V plot (triangles) reverted back to that seen with ND96. DIDS-sensitive HCO₃^– currents were not observed in an H₂O-injected control oocyte (Fig. 3 B). I-V plots looked similar for the control oocyte exposed to ND96 and CO₂/HCO₃^– ± DIDS.

View larger version (14K): [in this window] [in a new window]   Figure 3. Current–voltage (I-V) relationship of NBCe1-C expressed in oocytes. (A) I-V plots were obtained from an NBCe1-C–expressing oocyte initially bathed in ND96 (diamonds), followed by 10 min in 5% CO2/33 mM HCO3– (CB) (squares), and then after 2 min in the CO2/HCO3– solution containing 200 µM DIDS (triangles). (B) The same experimental protocol in A was performed on an H2O-injected oocyte.

View larger version (12K): [in this window] [in a new window]   Figure 4. I-V relationships of the three NBCe1 variants. (A) HCO3–-dependent I-V plots were obtained from oocytes expressing NBCe1-A (closed diamonds), NBCe1-B (open squares), and NBCe1-C (closed triangles). For each data point, n 11. (Inset) The I-V plots are magnified near the reversal potentials (IM = 0) for NBCe1-B and C. (B) HCO3–-dependent I-V plots from oocytes injected with 25 ng of NBCe1-A cRNA (closed diamonds, redrawn from A), 2 ng of NBCe1-A cRNA (open diamonds, n = 3), or 25 ng of HA-tagged NBCe1-AHA cRNA (closed squares, n = 6). (C) HCO3–-dependent I-V plots from oocytes injected with 0.5 ng of NBCe1-AHA cRNA (closed diamonds, n = 3), 25 ng of NBCe1-BHA cRNA (open squares, n = 4), or 25 ng of NBCe1-CHA cRNA (closed triangles, n = 4). Error bars smaller than symbols are not shown.

Expression of the NBCe1 Variants at the Plasma Membrane of the Oocyte
Activity of Hemagglutinin-tagged NBCe1 Variants.
The higher activity of NBCe1-A compared to the B and C variants shown in Fig. 4 A could be due to differences in plasma membrane expression. We therefore used the SOC technique with HA-tagged NBCe1 constructs to assess surface expression. We first tested the function of the tagged constructs expressed in oocytes. As shown in Fig. 4 B, there is no difference between the voltage dependencies of the untagged NBCe1-A (closed diamonds) redrawn from Fig. 4 A, and the HA-tagged transporter (NBCe1-A_HA, closed squares). We performed additional studies to compare the function of tagged NBCe1-C. In pH_i/V_m and voltage-clamp studies in which oocytes expressing either tagged or untagged NBCe1-C were exposed to 5% CO₂/33 mM HCO₃^–, both groups displayed similar NBC-mediated hyperpolarizations, pH_i recovery rates following the initial CO₂-induced acidification, HCO₃^–-induced outward currents under voltage-clamp conditions, and I-V relationships. In conclusion, the introduced HA epitope does not alter NBCe1 activity.

Comparing Function and Surface Expression of NBCe1 Variants.
We expressed HA-tagged NBCe1-A and NBCe1-C in oocytes, and then evaluated both transporter function with the two-electrode, voltage-clamp technique, and expression with the SOC technique. In our functional assay, we monitored NBC-mediated outward currents in oocytes voltage clamped at –60 mV and exposed to 5% CO₂/33 mM HCO₃^–. In contrast to an oocyte injected with H₂O, an oocyte injected with either the A or C variant displayed an outward current when exposed to 5% CO₂/33 mM HCO₃^– (Fig. 5 A ). The A and C currents differed in the following three ways. First, the A-mediated current was 4.5-fold larger than the C-mediated current. Second, the A-mediated current peaked faster than the C-mediated current. Finally, in contrast to the C-mediated current, the A-mediated current decayed after its peak. The current decay is consistent with reduced transport in response to the buildup of substrate or a pH_i increase at the inner surface of the oocyte membrane (and opposite changes at the outer surface of the membrane). According to subsequent data presented, the more active the transporter (as judged by the magnitude of the peak HCO₃^–-induced current), the faster the decay. As summarized in Fig. 5 B on two batches of day-matched oocytes, the mean H₂O-subtracted, peak CO₂/HCO₃^–-induced current was 4.3-fold larger in oocytes expressing NBCe1-A (874 ± 82 nA, n = 8) compared to NBCe1-C (201 ± 4 nA, n = 7). The larger NBC-mediated current seen with NBCe1-A compared to C is consistent with the I-V plot data shown in Fig. 4 A.

View larger version (12K): [in this window] [in a new window]   Figure 5. Larger HCO3–-induced currents in oocytes expressing NBCe1-A than NBCe1-C. (A) A 5% CO2/33 mM HCO3– solution elicited an outward current in the NBCe1-A–expressing oocyte (AWT) that was 4.5-fold higher than the current in the NBCe1-C-expressing oocyte (CWT). An H2O-injected oocyte displayed no HCO3–-induced outward current. (B) Summary of H2O-subtracted, HCO3–-induced currents from experiments similar to those shown in A. n 7 from two batches of oocytes. (C) The mean normalized luminescence (Norm. Lum.) was similar for the A and C variants. n 12 from two batches of oocytes.

Similar functional and expression studies were performed on the HA-tagged NBCe1-B variant. The mean CO₂/HCO₃^–-induced current was threefold smaller in oocytes expressing NBCe1-B (258 ± 10 nA, n = 6) compared to NBCe1-A (787 ± 57 nA, n = 7). According to SOC analysis, the mean Norm. Lum. was identical for oocytes expressing the B variant (1.01 ± 0.07, n = 5) and A variant (1.00 ± 0.09, n = 5). Therefore, the lower activity of B or C vs. A cannot be explained by lower plasma membrane expression.

External Ion Dependencies of the NBCe1 Variants
The increased activity of NBCe1-A compared to the B and C variants may be due to a higher affinity for HCO₃^– and/or Na⁺. In the following two sections, we compare –for each NBCe1 variant– the external HCO₃^– and Na⁺ dependencies.

External HCO₃^– Dependencies.
We used an approach introduced by Grichtchenko et al. (2000) to determine the HCO₃^– dependencies of NBCe1s expressed in oocytes voltage clamped at –60 mV. NBC-mediated outward currents were recorded when oocytes were exposed to either 5% CO₂/33 mM HCO₃^– (standard HCO₃^–), or solutions containing different HCO₃^– concentrations and equilibrated with appropriate CO₂/O₂ mixtures to maintain a pH of 7.5. All solutions contained 7.6 mM Cl^– as described above.

Results from an oocyte expressing NBCe1-C are shown in Fig. 6 A . The oocyte was initially bathed in the normal Cl^– (103.6 mM), HEPES-buffered solution and voltage clamped at –60 mV. Exposing the oocyte to a solution containing 7.6 mM Cl^– generated a slow outward current (ab), possibly due to a small gluconate conductance. Exposing the oocyte to solutions containing different [HCO₃^–]s elicited rapid NBC-mediated outward currents (c-i). Currents were larger at progressively higher [HCO₃^–]s (h vs. d vs. e vs. f). Each non-33 mM HCO₃^– exposure was flanked by standard HCO₃^– exposures. A similar experiment performed on an H₂O-injected oocyte is shown in Fig. 6 B. The CO₂/HCO₃^– solutions elicited only slow inward currents (c-i).

View larger version (10K): [in this window] [in a new window]   Figure 6. External HCO3–-dependencies of NBCe1-B and -C. (A) A voltage-clamped oocyte (Vh = –60 mV) expressing NBCe1-C was exposed to different HCO3–-containing solutions at a constant [Cl–] of 7.6 mM. (B) The experimental protocol used in A was performed on an H2O-injected oocyte. (C) Normalized HCO3–-induced currents as a function of external [HCO3–] for oocytes expressing NBCe1-B (r2 = 0.99). HCO3–-induced outward currents in experiments similar to that in A were normalized to the mean currents elicited by the flanking exposures to the standard 5% CO2/33 mM HCO3– solution. (D) Normalized HCO3–-induced currents as a function of external [HCO3–] for oocytes expressing NBCe1-C (r2 = 0.99).

We also examined the bicarbonate dependence of an NBCe1-A/C chimera that contains the 41 amino-terminal residues of the A variant and the 61 carboxy-terminal residues of the C variant. In these studies, the A/C chimera exhibited a mean outward current (1240 ± 103 nA, n = 5) similar to that of wild-type NBCe1-A (1096 ± 296 nA, n = 3) when the oocytes were exposed to 5% CO₂/33 mM HCO₃^–. This chimera also had a similar apparent K_M for HCO₃^– of 5.33 ± 0.54 mM and normalized V_max of 1.00 ± 0.04 (n = 17 from five oocytes). In conclusion, the external HCO₃^– dependencies are no different (P 0.89) for all three NBCe1 variants and the NBCe1-A/C chimera. Therefore, the larger NBC-mediated current seen with NBCe1-A compared to the B and C variants is not due to a higher HCO₃^– affinity.

External Na⁺ Dependencies.
We used a similar approach to the one described above to determine the external Na⁺ dependencies of the NBCe1 variants. In our assay, we examined the magnitudes of the NBC-mediated outward currents elicited by 5% CO₂/33 mM HCO₃^– solutions containing various [Na⁺]s. Our experimental protocol minimized changes in intracellular [Na⁺] ([Na⁺]_i) by minimizing the time oocytes spent in reduced [Na⁺]s.

Results from an oocyte expressing NBCe1-C are shown in Fig. 7 A . The voltage-clamped oocyte was initially bathed in ND96 and then exposed to 5% CO₂/33 mM HCO₃^– solutions containing different amounts of Na⁺. These HCO₃^– exposures elicited rapid NBC-mediated outward currents (a-i), which were smaller at progressively lower concentrations of Na⁺ (b vs. d vs. f vs. h). Using an approach similar to that described above for our bicarbonate-dependence experiments, each low-Na⁺, HCO₃^– exposure was flanked by full-Na⁺ (98.5 mM) HCO₃^– exposures. A similar experiment on an H₂O-injected oocyte subjected to the same experimental protocol is shown in Fig. 7 B. The CO₂/HCO₃^– solutions of different Na⁺ concentrations elicited only small outward currents.

View larger version (10K): [in this window] [in a new window]   Figure 7. External Na+ dependencies of NBCe1-A and -C. (A) A voltage-clamped oocyte (Vh = –60 mV) expressing NBCe1-C was exposed to 5% CO2/33 mM HCO3– solutions containing different [Na+]s. (B) The experimental protocol used in A was performed on an H2O-injected oocyte. (C) Normalized HCO3–-induced currents as a function of external [Na+] for oocytes expressing NBCe1-A (r2 = 0.98). HCO3–-induced outward currents at different Na+ concentrations were normalized to the mean currents elicited by the flanking exposures to the 33 mM HCO3– solution containing "full" 98.5 mM Na+. (D) Normalized HCO3–-induced currents as a function of external [Na+] for oocytes expressing NBCe1-C (r2 = 0.97).

We used data from experiments similar to that shown in Fig. 7 A to determine the Na⁺ dependencies of NBCe1-A, -B, and -C. For each experiment, we normalized the current elicited by 5% CO₂/33 mM HCO₃^– solutions containing different [Na⁺]s to the mean current elicited by a flanking CO₂/HCO₃^– solution containing full, 98.5 mM Na⁺. HCO₃^– currents obtained in the absence of Na⁺ were subtracted before normalization. We plot the external Na⁺ dependencies for the two most dissimilar variants: NBCe1-A in Fig. 7 C and NBCe1-C in Fig. 7 D. The apparent K_m and normalized V_max values are similar for both the A and C variants. The apparent K_M values are 20.6 ± 1.7 mM for NBCe1-A (n = 33 from five oocytes) and 31.7 ± 2.6 mM for NBCe1-C (n = 71 from 12 oocytes). Sciortino and Romero (1999) reported a similar apparent K_M of 30 mM for NBCe1-A. Our corresponding normalized V_max values are 1.21 ± 0.03 for NBCe1-A and 1.31 ± 0.04 for NBCe1-C. In additional studies, we also examined the external Na⁺ dependence of NBCe1-B and obtained an apparent K_M of 35.5 ± 3.7 mM and normalized V_max of 1.35 ± 0.05 (n = 23 from five oocytes). In conclusion, all three NBCe1 variants have similar external Na⁺ dependencies.

Role of the Amino Termini on the Function and Expression of NBCe1 Variants in Oocytes
According to the aforementioned HCO₃^– and Na⁺ dependence data, the higher NBC current seen with NBCe1-A vs. -B and -C (Figs. 4 and 5) is not due to higher substrate affinities. Therefore, the A variant must have a higher transport velocity compared to the B and C variants. As shown in Fig. 1, the differences in amino acid sequence among these three variants reside only at the amino and/or carboxy termini. The amino terminus is likely responsible for differences in NBC activity because the B and C variants (with low activity) share the same amino terminus, which is different than the unique amino terminus of the A variant (with high activity). Furthermore, the A/C chimera containing the unique carboxy terminus of C attached to the A variant displays NBC currents similar to those of the wild-type A variant.

Activity and Surface Expression of NBCe1 Variants Truncated before the First Transmembrane Domain.
We used the HCO₃^–-induced, outward-current assay shown in Fig. 5 A to examine the function of NBCe1 constructs with amino-terminal truncations. The three experimental traces shown in Fig. 8 A are from voltage-clamped oocytes injected with cRNA encoding wild-type NBCe1-C (C_WT), NBCe1-C missing the first 426 residues before the first predicted transmembrane domain (C_N426), or NBCe1-C missing the first 213 residues (C_N213). Oocytes exposed to a solution containing 5% CO₂/33 mM HCO₃^– elicited an outward current of 250 nA in the oocyte expressing C_WT, but little/no current in the oocyte expressing either C_N426 or C_N213.

View larger version (16K): [in this window] [in a new window]   Figure 8. Inhibited NBCe1 activity elicited by removing regions of the cytosolic amino terminus. (A) Exposing oocytes to a solution containing 5% CO2/33 mM HCO3– elicited an outward current in the oocyte expressing wild-type NBCe1-C (CWT), but little/no current in the oocyte expressing either CN426 or CN213. (B) Summary of HCO3–-induced outward currents from experiments similar to those shown in A. For each bar, n 3 from two batches of oocytes. SEM values for the CN426 or CN213 data are small. (C) Compared to the mean normalized luminescence (Norm. Lum.) for CWT, mean Norm. Lum. was 1.5-fold greater for CN426, and similar for CN213 in oocytes from the same two batches in B. n 10 for each bar.

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SOC analysis was performed on the same two oocyte batches, and luminescence for each oocyte was normalized to the mean luminescence of oocytes expressing wild-type C_WT. Compared with the mean Norm. Lum. for C_WT, the mean values were actually 50% higher (P = 0.002) for C_N426 and similar (P = 0.14) for C_N213 (Fig. 8 C). In summary, the lost NBC activity of C_N426 and C_N213 is not due to the absence of expression at the plasma membrane.

In further structure–function studies, we examined the effects on transporter activity and plasma membrane expression of removing the NH₂-terminal 43 residues of NBCe1-A (A_N43) and 87 residues of NBCe1-C (C_N87). Because the amino terminus of the B and C variants is identical, studies on C_N87 provide NH₂-terminal information on the B variant.

Activity and Surface Expression of NBCe1-A Lacking its Unique Amino Terminus.
At a holding potential of –60 mV, an oocyte expressing A_N43 (Fig. 9 A , left) displayed an HCO₃^–-induced outward current that was 50% smaller than the current seen in the oocyte expressing wild-type NBCe1-A (A_WT). Therefore, removing the amino-terminal 43 residues of the A variant decreases transporter activity. The summary data of HCO₃^–-mediated outward currents are shown in Fig. 9 B (left). From five batches of oocytes, the mean HCO₃^–-induced current for A_N43 (443 ± 31 nA, n = 18) was 55% smaller (P < 0.001) than the mean current for A_WT (990 ± 81 nA, n = 15). The mean HCO₃^–-induced current from batch-matched, H₂O-injected oocytes was only 0.8% of the mean current from A_WT-expressing oocytes.

View larger version (16K): [in this window] [in a new window]   Figure 9. Changes in NBCe1 activity elicited by removing the different amino termini. (A, left) An oocyte expressing mutant AN43 displayed a peak HCO3–-induced outward current that was 50% smaller than seen in the oocyte expressing AWT. (A, right) An oocyte expressing mutant CN87 displayed a peak HCO3–-induced outward current that was approximately threefold larger than seen in the oocyte expressing CWT. (B) Summary of HCO3–-induced outward currents from experiments similar to those shown in A. n 15 for each bar. (C, left) The mean normalized luminescence (Norm. Lum.) was 10% lower for AN43 compared with AWT. n 19 for each bar. (C, right) The mean Norm. Lum. was 33% higher for CN87 compared with CWT. n 23 for each bar.

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Activity and Surface Expression of NBCe1-C Lacking the Different Amino Terminus.
Different results were obtained with the homologous NH₂-terminal truncation of NBCe1-C (C_N87). At a holding potential of –60 mV, an oocyte expressing C_N87 (Fig. 9 A, right) displayed an HCO₃^–-induced outward current that was 3.4-fold larger than the current seen in the oocyte expressing C_WT. Therefore, removing the amino-terminal 87 residues of C_WT increases transporter activity. The summary data of HCO₃^–-mediated outward currents from experiments similar to those shown in Fig. 9 A (right) are shown in B (right). From seven batches of oocytes, the mean HCO₃^–-induced current for C_N87 (654 ± 48 nA, n = 23) was 2.7-fold larger (P < 0.001) than the mean current for C_WT (242 ± 12 nA, n = 19). As expected from Fig. 5 data, the mean current for C_WT was 25% of the mean current for A_WT.

In five of the seven batches above in which SOC data were obtained, the mean Norm. Lum. was 33% higher (P < 0.001) for oocytes injected with C_N87 compared to those injected with C_WT (Fig. 9 C, right). A 33% increase in surface expression would not explain the 170% increase in transporter activity of C_N87 compared with C_WT. Moreover, in two of these five batches in which the surface expression of C_N87 (n = 9) was 102 ± 4% of C_WT (n = 10), the mean NBC-mediated current for C_N87 (713 ± 137 nA, n = 6) was also 2.7- fold larger (P = 0.01) than for C_WT (264 ± 20 nA, n = 5). Therefore, the higher activity of C_N87 compared to C_WT cannot be explained by a difference in surface expression. Data from Fig. 9 are consistent with the amino-terminal residues of NBCe1-A stimulating transporter activity, and the amino-terminal residues of NBCe1-B and C inhibiting transporter activity.

If the amino termini of the NBCe1 variants are solely responsible for the higher transporter activity seen with A compared to B and C, then the activities of A_N43 and C_N87 should be identical. However, as shown in Fig. 9, the mean C_N87 current (654 nA) is 50% larger (P < 0.001) than the mean A_N43 current (443 nA). The larger mean C_N87 current could be explained by the slightly higher surface expression of C_N87 vs. A_N43 as revealed in the SOC analysis shown in Fig. 9 C. However, a difference in surface expression does not appear to be the only explanation according to current–voltage (I-V) analyses (Fig. 10 ).

View larger version (16K): [in this window] [in a new window]   Figure 10. Current–voltage (I-V) relationships from oocytes expressing wild-type and amino-terminal truncations of NBCe1 variants. (A) Representative experiment on an oocyte expressing CN87 in which I-V plots were obtained with the oocyte initially bathed in ND96 (diamonds), followed by 1 min in 5% CO2/33 mM HCO3– (circles) and then 10 min in the physiological buffer (squares), and finally in the HCO3– solution containing 200 µM DIDS (triangles). (B) HCO3–-dependent I-V plots for NBCe1-A (closed diamonds), AN43 (open diamonds), NBCe1-C (closed triangles), and CN87 (open triangles). Data were obtained from experiments similar to that shown in A. n = 3 for each data point from a single batch of oocytes. SEM bars smaller than symbol sizes are not shown. Similar results were obtained from a second batch of oocytes.

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From experiments similar to that shown in Fig. 10 A, mean HCO₃^–-dependent I-V plots were obtained from oocytes injected with wild-type NBCe1-A and -C, and the truncated constructs A_N43 and C_N87 (Fig. 10 B). The data were from day-matched experiments, and mean I-V data from H₂O-injected oocytes were subtracted. Similar to the Fig. 9 results obtained at a fixed potential of –60 mV, the voltage-dependent A_N43 currents (Fig. 10 B, open diamonds) were smaller than the corresponding wild-type A currents (closed diamonds), and the C_N87 currents (open triangles) were larger than the corresponding wild-type C currents (closed triangles) at all holding potentials from –200 to +20 mV (except at –80 mV). For each construct, the NBC conductance (g_NBC) was determined from the slope of a linear fit to either the mean inward currents (V_h from –200 to –100 mV), or the mean outward currents (V_h from –80 to +20 mV) plotted in Fig. 10 B. For the inward currents, the g_NBC of A_N43 was 64% lower than that of A_WT, and the g_NBC of C_N87 was 2.3-fold higher than that of C_WT. Expectedly, the g_NBC of C_WT was only 28% of the g_NBC of A_WT. Similar g_NBC results were obtained for the outward currents.

C_N87 currents were also found to be consistently larger than the A_N43 currents. The mean C_N87 current was approximately twofold larger than the mean A_N43 current at –200 and +20 mV. Furthermore, g_NBC of C_N87 was 1.8-fold higher than the g_NBC of A_N43. These data are consistent with the unique carboxy terminus of the C variant vs. the carboxy terminus of the A/B variant contributing to higher NBC activity in the absence of the amino terminus.

Role of the Carboxy Termini on the Function and Expression of NBCe1 Variants in Oocytes
Activity and Surface Expression of NBCe1 Truncated after the Last Transmembrane Domain.
To determine the importance of the cytosolic carboxy terminus of NBCe1, we removed the 97 residues after the last predicted transmembrane domain of NBCe1-C (C_C97). This construct is identical to the homologous truncation of NBCe1-B. After expressing C_C97 in oocytes, we used pH-sensitive microelectrodes and the voltage-clamp technique to measure simultaneously pH_i and membrane current of oocytes voltage clamped at –60 mV.

Records from simultaneous pH_i and voltage-clamp experiments on oocytes expressing wild-type NBCe1-C (C_WT) and C_C97 are shown in Fig. 11 . After stable pH_i and V_m values were obtained, oocytes were voltage clamped at –60 mV and exposed to 5% CO₂/33 mM HCO₃^–. As expected from Fig. 2 and Fig. 5 data, the C_WT-expressing oocyte (panel A) exhibited an instantaneous HCO₃^–-stimulated outward current (point a', bottom trace), as well as a pH_i recovery following the initial CO₂-induced acidification (abc, top trace). Removing external Na⁺ reversed the outward current (c'), and blocked the pH_i recovery (cd). In contrast, the C_C97-expressing oocyte (panel B) exposed to CO₂/HCO₃^– exhibited neither a HCO₃^–-stimulated outward current (point a', bottom trace), nor any appreciable pH_i recovery after the CO₂-induced acidification (abc, upper trace). Stars in the voltage traces represent time points during the experiment when I-V plots were obtained (unpublished data).

View larger version (12K): [in this window] [in a new window]   Figure 11. Lost activity of CC97 expressed in oocytes. (A) pHi (top trace), voltage (middle trace), and current (bottom trace) were measured simultaneously in an NBCe1-C–injected oocyte that was initially bathed in ND96. The oocyte was then voltage clamped and exposed to a solution containing 5% CO2/33 mM HCO3– during segment ae. Electrogenic NBC activity was evident from the instantaneous outward current (a'), and the pHi recovery (bc) following the initial CO2-induced acidification (ab). Removing Na+ blocked the pHi recovery (cd), and reversed the hyperpolarization (c'). (B) The same experimental protocol in A was performed on a CC97-injected oocyte. Exposing the oocyte to CO2/HCO3– elicited only a small outward current (a'), and minimal pHi recovery (bc) following the initial decrease in pHi (ab).

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View larger version (19K): [in this window] [in a new window]   Figure 12. Lost activity of CC97 expressed in oocytes due to poor expression at the plasma membrane. (A) Summary data of the segment-bc pHi recovery rates from experiments similar to those shown in Fig. 11, top traces. n 4 for each bar. (B) Summary of H2O-subtraced, HCO3–-induced currents from experiments similar to those shown in Fig. 11, bottom traces. For each bar, n 6 from two batches. (C) Immunoblot analysis of total microsomal protein from single oocytes injected with H2O, CC97 cRNA, and CWT cRNA. An NBCe1 antibody labeled bands of the expected sizes: 130 kD for CWT and 118 kD for CC97. No labeling was observed in protein from an H2O-injected oocyte. (D) The mean normalized luminescence (Norm. Lum.) for oocytes injected with CC97 and H2O are similar. n 10 from two batches of oocyte.

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Activity and Surface Expression of NBCe1 Variants Truncated at the Different Carboxy Termini.
To explore the contribution of the different carboxy termini of the NBCe1 variants to plasma membrane expression, and possibly function, we performed additional studies comparing the expression and function of wild-type and truncated variants. More specifically, we created two additional constructs: NBCe1-A truncated 46 residues from the carboxy terminus (A_C46) and NBCe1-C truncated 61 residues from the carboxy terminus (C_C61). The C_C61 construct is identical to the homologous truncation of NBCe1-B.

NBC-mediated outward currents in response to CO₂/HCO₃^– are shown in Fig. 13 A for two voltage-clamped oocytes; one expressing A_WT and the other expressing A_C46. The HCO₃^–-induced outward current of 0.43 µA in the A_C46-expressing oocyte was 71% smaller than the current of 1.45 µA in the A_WT-expressing oocyte. The summary data from similar experiments performed on A_wt and A_C46, as well as C_wt and C_C61 are plotted in Fig. 13 B. The mean currents obtained from H₂O-injected oocytes were subtracted from the corresponding currents seen in the NBC-injected oocytes. The mean NBC-mediated currents of the truncated NBCs were 30% of the corresponding wild-type NBCs. As shown in Fig. 13 C, both A_WT and A_C46 are expressed in a microsomal membrane fraction. Similar results were obtained with C_WT and C_C61 (not depicted). However, according to summary SOC data (Fig. 13 D) from the same batches of oocytes, A_C46 and C_C61 are poorly expressed at the plasma membrane compared to the corresponding wild-type NBCs. Therefore, the reduced activity of A_C46 and C_C61 in oocytes is predominantly due to low surface expression.

View larger version (22K): [in this window] [in a new window]   Figure 13. Reduced activity and low surface expression of AC46 and CC61 expressed in oocytes. (A) 5% CO2/33 mM HCO3– elicited an outward current in an oocyte expressing NBCe1-A (AWT) that was markedly larger than the current in the oocyte expressing AC46. (B) Summary of H2O-subtracted, HCO3–-induced currents from experiments similar to those shown in A. n 6 for each bar from two batches of oocytes. (C) Immunoblot analysis of total microsomal protein from single oocytes injected with H2O, AWT cRNA, or AC46 cRNA. An NBCe1 antibody labeled bands of the expected sizes: 130 kD for AWT and 120 kD for AC46. (D) The mean normalized luminescence (Norm. Lum.) for oocytes expressing the truncated NBCs were markedly less than the Norm. Lum. for oocytes expressing the corresponding wild-type NBCs. n 11 for each bar from two batches of oocytes.

N87

N43

C_WT and C_N87.
As shown in Fig. 14 B , there was no appreciable change in current when a macropatch from an H₂O-injected oocyte was exposed first to CO₂/HCO₃^–, and then to the HCO₃^– solution containing 200 µM DIDS. In contrast, exposing a macropatch from a C_WT-expressing oocyte to CO₂/HCO₃^– elicited an inward current of 5 pA (Fig. 14 C). This inward current is consistent with NBC- mediated net-negative charge moving from the bath to the pipette held at +60 mV. Switching back to the ND96 solution caused the current to return to the original level. In many experiments with either the C or A variant, we could reactivate this HCO₃^–-induced inward current by reexposing the patch to the CO₂/HCO₃^– solution (unpublished data). However, the current change elicited by the second HCO₃^– exposure was often smaller—a result consistent with "rundown" of transporter activity. The phenomenon of rundown is observed with channels such as ROMK1 (McNicholas et al., 1994), and reflects events such as protein dephosphorylation (McNicholas et al., 1994) or loss of protein interaction with membrane phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP₂) (Huang et al., 1998; Suh and Hille, 2005).

View larger version (14K): [in this window] [in a new window]   Figure 14. NBCe1 activity from inside-out macropatches excised from oocytes. (A) NBC-mediated inward currents were elicited by exposing patches to a low-Cl–, 5% CO2/33 mM HCO3– solution ± 200 µM DIDS with the patch pipette (–Vp = –60 mV) containing the same low-Cl–, 33 mM HCO3– solution. (B) H2O-injected oocyte. The HCO3– solution without or with DIDS did not elicit any change in current. (C) CWT-injected oocyte. The HCO3– solution elicited a small inward current that was reversible when the patch was returned to ND96. (D) CN87-injected oocyte. The inward current elicited by the HCO3– solution was larger than the current seen in C, and completely inhibited by DIDS. (E) AWT-injected oocyte. The HCO3– solution elicited a small, reversible inward current. (F) AN43-injected oocyte. No change in current was observed when the patch was exposed to HCO3–.

N87

N87

A_WT and A_N43.
We performed macropatch experiments on A_WT and A_N43 using the aforementioned protocol. Based on the finding that macropatch currents for C_N87 were larger than those of C_WT, an observation that paralleled the whole-cell data, we anticipated A_WT to exhibit the largest macropatch currents of all the NBC constructs in the present study. As shown in Fig. 14 E, an HCO₃^–-induced, NBC-mediated current of 7 pA was indeed obtained in a macropatch from an oocyte expressing A_WT. Unexpectedly however, the mean A_WT current was of similar magnitude to the C_WT current (Fig. 14 C), and considerably less than the C_N87 current (D). In additional studies (not depicted), we confirmed that this A_WT-mediated HCO₃^–-induced current was eliminated with 200 µM DIDS. The inward current elicited by CO₂/HCO₃^– in a macropatch expressing A_N43 was absent (Fig. 14 F). The NBC-mediated current rose much faster for the A_WT (Fig. 14 E) variant than the C_WT variant (C)—an observation also made in whole-cell experiments (see Fig. 5). Therefore, the activation kinetics of A appear faster than those of C, even in the absence of the cytosol.

From experiments similar to those shown in Fig. 14 (B–F), we plotted the mean HCO₃^–-induced currents from a total of 68 inside-out macropatch experiments (Fig. 15 ). Similar to the results from the whole-cell data, the mean macropatch current for C_N87 is 3.2-fold larger than the mean current for C_WT (P = 0.03), whereas the mean current for A_N43 is 71% less than that of A_WT (P < 0.01). In fact, the mean macropatch currents from oocytes injected with A_N43 and H₂O are indistinguishable (P = 0.25). Macropatch data in disagreement with the whole-cell data include the absence of A_N43 current, as well as similar mean currents for A_WT and C_WT (P = 0.32). These disagreements may reflect the absence of a cytosolic component in the macropatch experiments that is necessary for both the A_N43 current and the large A_WT current seen in the whole-cell experiments.

View larger version (12K): [in this window] [in a new window]   Figure 15. Summary macropatch data from experiments similar to those shown in Fig. 14 on oocytes injected with H2O, CWT, CN87, AWT, and AN43. The mean HCO3–-induced macropatch current for CN87 is 3.2-fold larger than the mean current for CWT (, P = 0.03). The mean HCO3–-induced currents for AWT and CWT are similar (, P = 0.32), and both are larger than the mean current seen in H2O-injected oocytes (P < 0.05). The mean current for AN43 is 71% smaller than that of AWT (*, P < 0.01). n values are shown under bars.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Voltage and Ion Dependencies of NBCe1 Variants in Xenopus Oocytes
While all three variants exhibited similar voltage and external ion dependencies, the reversal potential (E_rev) of NBCe1-A was more positive (–85 mV) than the E_rev values of the B and C variants (–160 mV) (Fig. 4 A). Working on rat kidney NBCe1-A expressed in oocytes, Sciortino and Romero (1999) obtained an E_rev similar to ours, and calculated a corresponding 1:2 Na⁺:HCO₃^– stoichiometry for the transporter. As mentioned in Results, the more negative E_rev values for the B and C variants are unlikely to reflect different Na⁺:HCO₃^– stoichiometries; but rather, different substrate and pH gradients established immediately across the plasma membrane by the variants. Indeed, we observed a similar negative shift of E_rev in oocytes displaying smaller NBCe1-A currents in response to less cRNA injected (Fig. 4 B). Furthermore, the I-V relationships for the three variants looked similar when we adjusted expression levels and matched the magnitude of the whole-cell, transporter currents (Fig. 4 C).

For the NBCe1 variants, we calculated transporter stoichiometry using E_rev values and the extracellular Na⁺ and HCO₃^– concentrations used in the study. The equilibrium potential of an electrogenic NBC (E_NBC) is described by the following equation (Boron and Boulpaep, 1983; Deitmer and Schlue, 1989):

where R, T, and F have their usual meanings, and n is the HCO₃^–:Na⁺ stoichiometry. Solving Eq. 1 for n yields the transformed equation:

We determined the mean intracellular HCO₃^– concentration of 8.3 mM from simultaneous pH_i and voltage-clamp experiments on NBCe1-C–expressing oocytes similar to those shown in Fig. 11 A. For the intracellular Na⁺ concentration, we used a measured value of 10.4 mM previously reported by Sciortino and Romero (1999) for unclamped oocytes expressing NBCe1-A. Our calculated Na⁺:HCO₃^– stoichiometries are 1:1.8 for the B variant and 1:1.7 for the C variant. These values are similar to the 1:2 Na⁺:HCO₃^– stoichiometry previously calculated for the A variant (Sciortino and Romero, 1999). Moreover, we used the E_rev of –120/–140 mV from the I-V relationship shown in Fig. 4 (B and C) to calculate a similar Na⁺:HCO₃^– stoichiometry of 1:2 for the A variant. We used the lower-current I-V relationships for the analysis to minimize the complication of a large NBC-mediated substrate/pH gradient across the membrane (see above). In summary, all three variants expressed in oocytes appear to have the same 1:2 Na⁺:HCO₃^– stoichiometry.

Contribution of the Carboxy Termini to NBCe1 Activity
The carboxy-terminal truncations of NBCe1 variants examined in this study express poorly at the plasma membrane of oocytes. Based on our results, one or more regions within the different carboxy termini among the variants is/are required for proper plasma membrane expression in oocytes. Similarly, Cordat et al. (2003) reported that an AE1 construct truncated 11 residues from the carboxy terminus displayed reduced plasma membrane expression when expressed in HEK293 cells. Furthermore, Li et al. (2004) reported that GFP-tagged, human NBCe1-A mutants missing either the carboxy-terminal 26 or 50 residues retargets to the apical membrane of MDCK cells, and the latter construct displays significant intracytoplasmic expression. Our data with both A_C46 and C_C61 are consistent with this finding that the carboxy termini can influence trafficking. However, the effect of truncating the different carboxy termini found in A and C on plasma membrane expression is more dramatic in oocytes. The reduced surface expression could be due to the loss of a residue/region required for protein expression/stability, or an altered conformation that leads to organellar retention.

Contribution of the Amino Terminus to NBCe1 Activity
The following three general models would explain the contribution(s) of the different amino termini to NBC activity: (1) the unique amino terminus of A (A_N41) is stimulatory for an otherwise low-activity transporter, (2) the amino terminus of B/C is inhibitory (C_N85) for an otherwise high-activity transporter, or (3) both termini can influence function of an otherwise intermediate-activity transporter. Our observation that both whole-cell and macropatch NBC currents were inhibited to an intermediate level by A_N43 and stimulated to an intermediate level by C_N87 are consistent with the third possibility. The amino-terminal truncations likely lead to changes in transporter velocity. Indeed, the external Na⁺ and HCO₃^– dependencies for C_N87 were similar to those for C_WT (unpublished data). Therefore, the different amino termini of the variants can influence transporter activity two to threefold even though they only comprise a small fraction of the entire NBCe1 sequence (4% for A and 8% for C). While the different amino termini stimulate or inhibit transporter activity, one or more regions of the cytosolic amino terminus downstream of the variable region are required for NBC function. Removing nearly all of the cytosolic amino terminus of NBCe1-A or -C, or approximately half of this region of NBCe1-C, leads to a complete loss of transporter function without affecting plasma membrane expression.

Potential Mechanism(s) for the Amino-terminal Effects.
There are several possible mechanisms by which the amino terminus of an NBCe1 could affect transporter activity. An amino terminus may bind to a region of the same NBC protein and either elicit a change in protein conformation or directly interact with the ion translocation pathway. However, a specific carboxy terminus does not appear to be essential in light of the observation that the A variant and the A variant containing the carboxy terminus of C (A/C chimera) display a similar level of activity. Interaction with the translocation pathway may be similar to the "ball and chain" mechanism of inactivation of potassium channels (Zagotta et al., 1990; Tseng-Crank et al., 1993). Alternatively, the amino terminus of one NBC may exert its effect by binding to another NBC protein. Evidence for multimerization of NBCe1 has been reported in preliminary form (Espiritu, D.J.D., A.A. Bernardo, and J.A.L. Arruda. 2003. FASEB J. 17:A462; Espiritu, D.J.D., A.A. Bernardo, and J.A.L. Arruda. 2004. J. Am. Soc. Nephrol. 15:F-PO016.). Another mechanism for the amino-terminal effects may involve regulatory proteins/factors that are either cytosolic or membrane bound (see below). Regulatory proteins may serve as intermediate proteins that couple the amino termini to regions of an NBC protein.

NBCe1 Macropatch Currents
NBCe1-mediated HCO₃^– Transport across a patch of Oocyte Membrane.
We used the macropatch technique in the inside-out configuration to examine the potential involvement of a cytosolic component on the activity of NBCe1 constructs expressed in oocytes. The magnitudes of our NBC macropatch currents were similar to those previously reported for rat NBCe1-A (Heyer et al., 1999). In our studies, it was not uncommon to record from patches with little or no NBC-mediated currents. For all NBC groups, there was a broad range of HCO₃^–-mediated currents. For C_WT, current responses ranged from an outward current of 4.2 pA to an inward current of –14 pA. The range of responses may reflect different plasma membrane expression levels of NBCs among patches. Indeed, A_WT currents from three vegetal-pole patches obtained from each of two oocytes also exhibited a range of currents: 0.4, –5.1, and –3.3 pA for one oocyte, and –7.8, –3.3, and –7.4 pA for the other oocyte. NBCe1 expression at the oocyte surface may therefore be somewhat heterogeneous and reflect clustering of transporter proteins.

Influence of the Amino Termini on NBCe1 Activity.
There are three noteworthy observations regarding the macropatch results and the influence of the amino termini on NBC activity. First, the mean macropatch current for C_N87 is 3.2-fold larger than the mean current for C_WT. A similar 2.7-fold larger current for C_N87 was observed in the whole-cell experiments. Second, the mean current for A_N43 is less than that of A_WT. Similar results were obtained in the whole-cell experiments: the mean A_N43 current was 55% less than that of A_WT. Both of these observations corroborate the whole-cell data regarding the functional impact of the different amino termini.

The third observation in the macropatch studies, which deviates from the whole-cell data, is the observation that the mean macropatch current for A_WT is similar to the mean current for C_WT. Furthermore, the mean current for A_N43 is not only lower than for A_WT, but is nonexistent and similar to that seen in patches from H₂O-injected oocytes. These deviations from the whole-cell data may be due to different intracellular ion dependencies or sensitivities among the variants, and the fact that the macropatch experiments were performed with a symmetrical solution containing extracellular ion concentrations. High Na⁺ and HCO₃^– concentrations on the cytosolic side were used to maximize the transporter currents. Another explanation is the involvement of cytosolic factors in the whole-cell experiments that are absent in the macropatch experiments. While such factors could either stimulate A or inhibit C, the A_N43 current observed in whole-cell but not macropatch experiments is consistent with a cytosolic factor stimulating the A variant.

Potential cytosolic factors include cytoskeletal elements such as actin or enzymes. Indeed, the amino-terminal domain of AE1 interacts with actin, protein 4.1, protein 4.2, as well as glycolytic enzymes (Reithmeier et al., 1996). Classic regulatory factors include cyclic AMP and ATP. Heyer et al. (1999) reported that 2 mM ATP increases the activity of rat NBCe1-A from inside-out macropatches by twofold. The effect of ATP on NBCe1 activity may be mediated by ATP-dependent lipid kinases and PIP₂, as previously reported for the cardiac Na-Ca exchanger and K_ATP potassium channels (Hilgemann and Ball, 1996). There is considerable evidence highlighting the regulatory effects of membrane phospholipids such as PIP₂ on ion channels and transporters (Suh and Hille, 2005). Future studies will be necessary to characterize potential regulatory factors such as ATP, PIP₂, and kinases/phosphatases that may differentially modulate the NBCe1 variants.

	ACKNOWLEDGMENTS

 
We thank Mr. Albert Tousson (High Resolution Imaging Center, University of Alabama at Birmingham) for assistance with confocal microscopy used in the immunocytochemical analysis of NBC expression in oocytes. Dr. Peter R. Smith (Physiology and Biophysics, University of Alabama at Birmingham) provided assistance and the luminometer used for single-oocyte chemiluminescence. A special thanks goes to Dr. Chou-Long Huang (University of Texas Southwestern Medical Center at Dallas, Dallas, TX) for providing guidance with the macropatch technique. We thank Mrs. Debeshi Majumdar for her helpful suggestions and comments regarding the manuscript.

This work was supported by the American Heart Association, Southeast Affiliate (0265083B) and National Institutes of Health/National Institute of Neurological Disorders and Stroke (NS046653).

Olaf S. Andersen served as editor.

Submitted: 15 February 2006
Accepted: 24 April 2006

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