Cloning, Tissue Distribution, Genomic Organization, and Functional Characterization of NBC3, a New Member of the Sodium Bicarbonate Cotransporter Family*

Alexander Pushkin, Natalia AbuladzeDagger , Ivan Lee, Debra Newman, James Hwang, and Ira Kurtz§

From the Division of Nephrology, Center for Health Sciences, UCLA School of Medicine, Los Angeles, California 90095-1698

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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Previous functional studies have demonstrated that muscle intracellular pH regulation is mediated by sodium-coupled bicarbonate transport, Na+/H+ exchange, and Cl-/bicarbonate exchange. We report the cloning, sequence analysis, tissue distribution, genomic organization, and functional analysis of a new member of the sodium bicarbonate cotransporter (NBC) family, NBC3, from human skeletal muscle. mNBC3 encodes a 1214-residue polypeptide with 12 putative membrane-spanning domains. The ~ 7.8-kilobase transcript is expressed uniquely in skeletal muscle and heart. The NBC3 gene (SLC4A7) spans ~80 kb and is composed of 25 coding exons and 24 introns that are flanked by typical splice donor and acceptor sequences. Expression of mNBC3 cRNA in Xenopus laevis oocytes demonstrated that the protein encodes a novel stilbene-insensitive 5-(N-ethyl-N-isopropyl)-amiloride-inhibitable sodium bicarbonate cotransporter.

    INTRODUCTION
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INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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Intracellular pH regulatory mechanisms are critically important for the maintenance of many cellular processes in skeletal muscle, smooth muscle, and myocardial cells (1-8). In muscle cells, contractile processes, metabolic reactions, and membrane transport processes are influenced by pH. Importantly, during periods of increased energy demands and ischemia, muscle cells produce large amounts of lactic acid (3). In these circumstances, intracellular pH (pHi)1 regulatory processes prevent the acidification of the sarcoplasm due to lactic acid accumulation.

Several different transport mechanisms have been described in muscle cells, which maintain a relatively constant intracellular pH during changes in metabolic proton production or an elevation in ambient CO2. The relative contribution of each process varies with cell type, the metabolic requirements of the cell, and local environmental conditions. Intracellular pH regulatory processes that have been characterized functionally in skeletal, smooth muscle, and cardiac cells include: Na+/H+ exchange (1-3, 9-22), Na(HCO3)n cotransport (12, 13, 16, 17, 21, 23-29), Na+-dependent (12, 18, 19, 26, 30, 31) and -independent (19, 24, 26, 29, 32, 33) Cl-/base exchange, and lactate/proton cotransport (3).

With the exception of guinea pig femoral artery, when present, Na+-dependent HCO3- transport plays a significant role in regulating pHi in the tissues where it has been investigated. However, the characteristics of Na+-dependent HCO3- transport vary between different muscle cell preparations. Specifically, the Na(HCO3)n cotransporter in cardiac Purkinje fibers and ventricular myocytes (17, 24) and in certain smooth muscle cells (11, 26-29) appears to be electroneutral in contrast to the electrogenicity reported in other cell types (13, 25, 34). Furthermore, stilbene-insensitive Na+-dependent HCO3- transport has been reported in various smooth muscle cells (27, 28). Finally, EIPA-inhibitable stilbene-insensitive Na+-dependent HCO3- transport has been described in vascular smooth muscle cells (12, 15, 21). The functional differences that have been reported in various skeletal muscle, smooth muscle, and cardiac myocytes preparations suggest that several transporters may exist that differ in their functional properties and sensitivity to inhibitors. However, the molecular mechanisms mediating Na+-dependent HCO3- transport in muscle cells have not previously been investigated. An electrogenic sodium bicarbonate cotransporter (NBC1) has recently been cloned from salamander and rat kidney (35-37). Furthermore, a sodium bicarbonate cotransporter has also been cloned from human kidney (kNBC1) and human pancreas (pNBC1) (38, 39). The kidney uniquely expresses the kNBC1 isoform, predominantly in the proximal tubule, whereas expression of pNBC1 is more widespread (39, 40). pNBC1 was only weakly expressed in human skeletal muscle on Northern blots. Ishibashi et al. (41) have recently cloned an additional member of the NBC family (NBC2) from human retina whose function is unknown.

To gain additional insight into the reason for the functional variability in Na+-dependent HCO3- transport among various muscle cell preparations, we screened a human skeletal muscle cell library and obtained a unique clone that is expressed specifically in skeletal muscle and heart. In this paper, we report the cloning, sequence analysis, tissue distribution, genomic organization, and functional analysis of this new member of the NBC family, NBC3.

    MATERIALS AND METHODS
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Cloning and Sequencing of mNBC3-- Human expressed sequence tag clones were identified by a homology search of the expressed sequence tag data base for pNBC1-related sequences. The clones were used as a template for sequencing and 5' and 3' RACE products were generated. 5' and 3' oligonucleotide probes based on the RACE products were synthesized, random primer labeled with 32P, and used to screen a human muscle lambda gt11 cDNA library (CLONTECH, Palo Alto, CA). Positive clones were verified by sequencing. Two overlapping clones were obtained that contained the entire coding region. To obtain a full-length clone containing the complete open reading frame, these two clones were spliced together using a common BamHI restriction site and subcloned into pPCR-Script SK+ (Stratagene, La Jolla, CA). The 5' end of the coding sequence for mNBC3 was confirmed by 5' RACE PCR amplification. Furthermore, to confirm that the mNBC3 amino acid sequence was derived from a bona fide transcript, we amplified the entire open reading frame of human mNBC3 by reverse transcription-PCR using Marathon Ready cDNA prepared from human muscle (CLONTECH) as a template. Nucleotide sequences were determined bidirectionally by automated sequencing (ABI 310, Perkin-Elmer, Foster City, CA) using Taq Polymerase (Ampli-Taq FS, Perkin-Elmer). Sequence assembly and analysis was carried out using Geneworks software (Oxford, UK). Secondary structure and hydropathy analyses were performed using the Tmpred program (www.isrec.isb-sib.ch/software/TMPRED_form.html).

Northern Analysis and Tissue Distribution-- Northern blots with various human tissues were obtained from CLONTECH. The probe was primed with [32P]dCTP and [32P]dATP to a specific activity of about 1.5 × 109 dpm/µg. The filters were prehybridized at 42 °C for 2 h using 50% formamide, 6× SSPE, 0.5% SDS, Denhardt's solution, and 0.1 mg/ml of sheared herring testes-denatured DNA. Following the prehybridization, the filters were incubated with the 32P probe using 25 ml of hybridization buffer. The probes were denatured and added to the hybridization solution at 107 dpm/ml. The filters were probed at 42 °C for 18 h and washed in 1× SSC,0.1% SDS at 45 °C for 60 min (three changes, 350 ml/wash); after exposure for 9.5 h, the filters were rewashed in 1× SSC, 0.1% SDS at 65 °C for 30 min and 0.1× SSC, 0.1% SDS at 65 °C for 1 h. A labeled synthetic 95-bp oligonucleotide probe (nucleotides 1002-1096) specific for the unique N-terminal region of mNBC3 was randomly primed and labeled with [32P]dCTP and [32P]dATP.

Genomic Organization-- To isolate the NBC3 gene, we screened a BAC human genomic DNA library (Genome Systems, St.Louis, MO) using a PCR approach. The PCR primers designed for screening the BAC human library were: 5'-CTGCAACAGTGTCAATAAGTCATG-3' (2995) and 5'-ACTGAAGTCATGAACACAGAGAGGC-3' (3130). The PCR product was 160 bp. BAC clone 15 was isolated, and sequence analysis revealed that this clone encompassed the entire human NBC3 gene and was therefore used for all subsequent analysis. A series of primers were designed based on the known mNBC3 cDNA sequence (AF047033) to determine the structure of the NBC3 gene. Sequences at the intron-exon boundaries of the NBC3 gene were determined by aligning the cDNA sequence of mNBC3 with the genomic sequence.

Xenopus Oocyte Expression-- The mNBC3 insert in pPCR-Script SK+ was subcloned into the Xenopus expression vector ppT7TS (obtained from Dr. Paul Krieg), which contains the 5'- and 3'-untranslated regions of Xenopus beta -globin mRNA inserted into pGEM4Z. The plasmid containing the complete coding sequence of mNBC3 was linearized by digestion with SalI, and capped cRNA was prepared with T7 RNA polymerase using a T7 mMessage mMachine kit RNA capping kit (Ambion, Austin, TX) as recommended by the manufacturer. This cRNA was used for the Xenopus oocyte expression studies. An aliquot of the synthesized cRNA was run on a denaturing gel to verify the expected size prior to oocyte injection. Defolliculated oocytes were injected with 50 nl of sterile water or a solution containing 0.5-1 ng/nl of capped mNBC3 cRNA (prepared as described above). They were then bathed in Barth's medium at 18 °C and studied 3-14 days post-injection.

22Na+ Influx Measurements-- Defolliculated oocytes were injected with mNBC3 cRNA (50 nl, 0.5-1 µg/ul) or water and incubated in Barth's medium at 18 °C prior to study. In uptake studies performed in the presence of chloride, the oocytes were preincubated for 1 h in 10 ml of a Na+-free solution containing 108 mM tetramethylammonium Cl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 8 mM Hepes, pH 7.4. The oocytes were then transferred into 1.4 ml of the following Na+-containing solution: 22 mM NaCl, 78 mM tetramethylammonium Cl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 8 mM NaHCO3 bubbled with 1.5% CO2, pH 7.4 with 2 µCi of 22Na+. A 10-µl aliquot was removed from the influx solution for later determination of 22Na+ specific activity. 22Na+ influx was measured after 15 min and terminated with three washes of ice-cold Na+-free stop solution. The influx experiments were repeated in the presence of DIDS (1 mM). In the DIDS-containing experiments, the oocytes were exposed to 1 mM DIDS for 1 h prior to and throughout the influx period. The effect of EIPA (50 µM) on 22Na+ uptake was also studied. In uptake studies performed in the absence of chloride, the oocytes were preincubated for 1 h in 10 ml of a Na+-free Cl--free solution containing 108 mM tetramethylammonium hydroxide, 108 mM gluconic acid lactone, 2 mM potassium gluconate, 7 mM calcium gluconate, 2 mM magnesium gluconate, and 8 mM Hepes, pH 7.4. The oocytes were then transferred into 1.4 ml of the following Na+-containing solution: 22 mM sodium gluconate, 78 mM tetramethylammonium hydroxide, 78 mM gluconic acid lactone, 2 mM potassium gluconate, 7 mM calcium gluconate, 2 mM magnesium gluconate, and 8 mM NaHCO3 bubbled with 1.5% CO2, pH 7.4, with 2 µCi of 22Na+.

86Rb Influx Measurements-- The oocytes were preincubated in 10 ml for 1 h in a K+-free solution containing 98 mM NaCl, 10 mM RbCl, 1 mM CaCl2, 1 mM MgCl2, and 8 mM Hepes, pH 7.4, containing 0.1 mM ouabain. The oocytes were then transferred into 1.4 ml of the following solution: 90 mM NaCl, 10 mM RbCl, 1 mM CaCl2, 1 mM MgCl2, and 8 mM NaHCO3 bubbled with 1.5% CO2, pH 7.4, containing 0.1 mM ouabain with 2 µCi of 86Rb. A 10-µl aliquot was removed from the influx solution for later determination of 86Rb specific activity. 86Rb influx was measured after 15 min and terminated with three washes of ice-cold stop solution.

Measurement of Oocyte pHi-- Optical recordings were made at 21 °C. Intracellular pH was monitored using the fluorescent probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and a microflourometer coupled to the microscope (42). Individual defolliculated oocytes were held in place with pipettes attached to low suction with vegetal pole surface closest to the 40× objective. Prior to loading with BCECF, the background intensity from each oocyte was digitized at 500 and 440 nm (530 nm emission). The oocytes were loaded with 32 µM BCECF-AM for 30-60 min prior to experimentation in the following solution: 108 mM sodium gluconate, 2 mM potassium gluconate, 7 mM calcium gluconate, 2 mM magnesium gluconate, and 8 mM Hepes, pH 7.4. Calibration of intracellular BCECF in the oocytes was performed at the end of each experiment by monitoring the 500/440 nm fluorescence excitation ratio at various values of pHi in the presence of high K+ nigericin standards as described previously (43). The following experimental protocols were performed: 1) For Na+ removal/addition in Cl--free bicarbonate-buffered solutions, the oocytes were bathed in the following Na+-containing Cl--free solution for 1-8 h prior to loading with BCECF: 108 mM sodium gluconate, 2 mM potassium gluconate, 7 mM calcium gluconate, 2 mM magnesium gluconate, and 8 mM Hepes, pH 7.4. After a steady state was reached, the oocytes were exposed to a bicarbonate-buffered Cl--free solution containing 100 mM sodium gluconate, 2 mM potassium gluconate, 7 mM calcium gluconate, 2 mM magnesium gluconate, 8 mM NaHCO3- bubbled with 1.5% CO2, pH 7.4. After a new steady state was achieved, Na+ was removed by exposing the oocytes to a solution containing 100 mM tetramethylammonium hydroxide, 100 mM D-gluconic acid lactone, 2 mM potassium gluconate, 7 mM calcium gluconate, 2 mM magnesium gluconate, 8 mM tetramethylammonium HCO3 bubbled with 1.5% CO2, pH 7.4. Additional experiments were performed in oocytes bathed in 1 mM DIDS or 50 µM EIPA. 2) For Na+ removal/addition in Cl--free Hepes-buffered solutions bubbled continuously with 100% O2, the oocytes were bathed in the following Na+-containing Cl--free solution for 1-8 h prior to loading with BCECF: 108 mM sodium gluconate, 2 mM potassium gluconate, 7 mM calcium gluconate, 2 mM magnesium gluconate, and 8 mM Hepes, pH 7.4. After a steady state was reached in the same solution, Na+ was removed by bathing the oocytes in the following Hepes-buffered Na+-free solution: 108 mM tetramethylammonium hydroxide, 108 mM D-gluconic acid lactone, 2 mM potassium gluconate, 7 mM calcium gluconate, 2 mM magnesium gluconate, and 8 mM Hepes, pH 7.4, bubbled continuously with 100% O2. 3) For K+ addition/removal in Cl--free bicarbonate-buffered solutions, the oocytes were bathed in the following Na+-containing Cl--free solution for 1-8 h prior to loading with BCECF: 108 mM sodium gluconate, 2 mM potassium gluconate, 7 mM calcium gluconate, 2 mM magnesium gluconate, and 8 mM Hepes, pH 7.4. After a steady state was reached, the oocytes were bathed in a solution containing 50 mM sodium gluconate, 2 mM potassium gluconate, 48 mM tetramethylammonium hydroxide, 48 mM gluconic acid lactone, 7 mM calcium gluconate, 2 mM magnesium gluconate, and 8 mM NaHCO3- bubbled with 1.5% CO2, pH 7.4. The K+ concentration was acutely increased by exposing the oocytes to a solution containing the identical Na+ concentration and a K+ concentration of 50 mM: 50 mM sodium gluconate, 50 mM potassium gluconate, 7 mM calcium gluconate, 2 mM magnesium gluconate, and 8 mM NaHCO3- bubbled with 1.5% CO2, pH 7.4.

    RESULTS AND DISCUSSION
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We report the cloning, sequence analysis, tissue distribution, genomic organization, and functional characterization of a new member of the NBC family, NBC3, which is uniquely expressed in skeletal muscle and heart muscle. Partial sequence information obtained from human expressed sequence tag clones and 5' and 3' RACE products were used for generating probes to screen a human muscle lambda gt11 cDNA library to obtain full-length mNBC3. The nucleotide sequence of mNBC3 is 7785 nucleotides in length, with a predicted open reading frame of 3642 nucleotides, as 5'-untranslated region of 71 nucleotides and a 3'-untranslated region of 4072 nucleotides (GenBankTM/EMBL Data Bank with the accession number AF047033). mNBC3 is homologous to several expressed sequence tag sequences (human: AA205898, AA535856 AA569234, AA188932, AA852668, AA243166, AA476540, AA227419, AA216661, AA243228, AA218565, AA101282, Z19373, R57438, AA494043, AA630595, H66521, AF00103, and Z20394; mouse: AA166155, AA689098, and AA592390). Beginning with methionine at position 1, an open reading frame codes for a protein of 1214 amino acids. Fig. 1 demonstrates the protein alignment of mNBC3 with other members of the NBC family and the anion exchanger bAE3. mNBC3 is 78% homologous to NBC2; 39 and 46% homologous to pNBC1 and kNBC1, respectively, and 29% homologous to bAE3.


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Fig. 1.   Multiple protein sequence alignment of human mNBC3 with human NBC2, pNBC1, kNBC1, and AE3 (GenBankTM accession numbers AF047033, AB012130, AF011390, AF007216, and HSU05596). mNBC3 encodes a polypeptide of 1214 amino acids.

The secondary structure analyses and hydropathy profile of the protein predict an intrinsic membrane protein with 12 putative transmembrane domains (Fig. 2); N-linked glycosylation sites in exofacial loops 1-2 and 5-6; protein kinases A and C, casein kinase II, and ATP/GTP-binding consensus phosphorylation sites; potential sites for myristylation and amidation; and cytoplasmic N and C termini. A large exofacial loop is present between predicted membrane domains 1 and 2. The first of two putative stilbene-binding motifs (K(M/L)XK) shared by other members of the anion exchanger superfamily (AE1, AE2, AE3, pNBC1, and kNBC1) is lacking in mNBC3, which has the sequence KLFD (amino acids 742-745). The replacement of the highly conserved lysine at position 745 by aspartic acid may block negatively charged stilbene disulfonates from binding and alter the Ki of these compounds for mNBC3 (44, 45). It is of interest that stilbene-insensitive Na+-dependent bicarbonate transport has been reported in cultured smooth muscle cells from human internal mammary artery (15, 21), rat aorta (12), guinea pig femoral artery (27), and guinea pig ureter (28). The second putative stilbene-binding motif KLKK (amino acids 944-947) in mNBC3 is identical in sequence to the homologous stilbene-binding site in human kNBC1, pNBC1, and NBC2 (38, 39, 41).


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Fig. 2.   Putative topology of human mNBC3 in the membrane. Charged amino acids are depicted by plus and minus signs. Potential N-linked glycosylation sites are depicted by psi . mNBC3 also has numerous potential intracellular consensus phosphorylation sites: 1) protein kinase A: Lys222 and Lys239; 2) protein kinase C: Ser39, Ser55, Ser76, Ser91, Thr132, Ser203, The225, Ser260, Ser271, Ser288, Thr320, Ser324, Ser327, Thr847, Thr1127, Ser1141, and Ser1194; 3) casein kinase II: Thr30, Ser76, Thr100, Thr114, Ser161, Ser181, Thr182, Ser203, Ser233, Ser407, Ser412, Thr424, Ser639, Thr858, Thr1127, Ser1132, and Ser1198; and 4) ATP-GTP-binding site motif A: Ala17.

The expression of mNBC3 was examined in various human tissues by Northern blot analysis (Fig. 3). A synthetic 95-bp oligonucleotide probe (nucleotides 1002-1096) specific for the unique region of mNBC3 was randomly primed and labeled with [32P]dCTP and [32P]dATP. This probe hybridized to a ~7.8-kb transcript in mRNA from skeletal muscle and heart. These results indicate the mNBC3 transcript is muscle-specific. We have recently determined the chromosomal localization of the mNBC3 gene by fluorescent in situ hybridization (46). mNBC3 maps to a position that is 19% of the distance from the centromere to the telomere of chromosome arm 3p, an area that corresponds to band 3p22. Recently, Olsen and Keating (47) have studied a family with dilated cardiomyopathy associated with sinus node dysfunction, supraventricular tachyarrythmias, conduction delay, and stroke. The disease gene has been mapped to a 30 cM region of chromosome 3p22-3p25 excluding genes encoding a G-protein (GNA12), calcium channel (CACNL1A2), sodium channel (SCN5A), and inositol triphosphate receptor. The finding that mNBC3 maps to this region suggests that it is a new candidate gene potentially causing familial dilated cardiomyopathy.


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Fig. 3.   Northern blot analysis of expression of mNBC3 in human tissues. Lane 1, heart; lane 2, brain; lane 3, placenta; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, pancreas; lane 9, spleen; lane 10, thymus; lane 11, prostate; lane 12, testis; lane 13, ovary; lane 14, small intestine; lane 15, colon; lane 16, peripheral blood leukocytes; lane 17, stomach; lane 18, thyroid; lane 19, spinal chord; lane 20, lymph node; lane 21, trachea; lane 22, adrenal gland; lane 23, bone marrow. Each lane was loaded with ~2 µg of poly(A)+ human RNA. The multiple tissue Northern blots were from CLONTECH. The following 32P-labeled mNBC3 probe was used: a synthetic 95-bp oligonucleotide specific for mNBC3 (nucleotides 1002-1096).

NBC3 (approved symbol SLC4A7) is the first member of the NBC family in which the genomic structure has been analyzed (Fig. 4). The gene coding for mNBC3 is composed of 25 exons and 24 introns and spans ~80 kb. The exon sizes ranged from 82 to 4123 bp. The large 3'-untranslated region is contained in exon 25. Intron sizes were not determined. All intron sequences match known consensus donor and acceptor splice sites. The genomic organization of the related anion exchange gene family differs in several respects. There are 20 exons in the AE1 gene and 23 exons in the AE2 and AE3 genes if alternate exons transcribed from internal promoters are excluded. For all AE family members the first exon consists of the 5'-untranslated region, and the second exon contains translation start site. In the NBC3 gene there are 25 exons, and the translation start site is contained in the first exon. In NBC3 (SLC4A7), the large 3'-end exon (exon 25) codes for the 3'-terminus of the polypeptide and the 3'-untranslated region.


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Fig. 4.   Intron/exon boundaries of the human NBC3 (approved symbol SLC4A7) gene. Nucleotide sequences at the intron/exon boundaries are shown, with exon sequences in uppercase letters and intron sequences in lowercase letters. Exons are numbered from left to right. The length of each exon determined by sequence analysis is shown.

We next examined the functional properties of mNBC3 using measurements of pHi, 22Na, and 86Rb uptake after injecting the corresponding polyadenylated cRNA into Xenopus oocytes. Polyadenylated cRNA prepared as described above was injected into oocytes and allowed to express for at least 72 h. As shown in Fig. 5, augmented 22Na uptake was observed in oocytes injected with mNBC3 cRNA. The uptake was increased by approximately 12 nmol/h/oocyte in cRNA-injected oocytes (3.44 ± 0.34 nmol/h/oocyte in control oocytes (n = 24) versus 15.3 ± 1.44 nmol/h/oocyte (n = 32), p < 0.001). 22Na uptake was not significantly different in oocytes exposed to Cl--free solutions (15.3 ± 1.44 nmol/h/oocyte (n = 32) versus 13.8 ± 1.30 nmol/h/oocyte (n = 16) in oocytes exposed to Cl--free solutions). Uptake was not significantly affected by DIDS (1 mM; 16.5 ± 1.56 nmol/h/oocyte (n = 8)) but was completely inhibited by EIPA (50 µM; 3.28 ± 0.38 nmol/h/oocyte (n = 16), p < 0.02). 86Rb uptake was similar in control oocytes and cRNA-injected oocytes (controls 1.04 ± 0.04 nmol/h/oocyte (n = 16) versus 2.55 ± 0.04 nmol/h/oocyte (n = 16) in cRNA-injected oocytes).


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Fig. 5.   Functional expression of mNBC3 in Xenopus laevis oocytes. 22Na uptake (expressed in nmol/h/oocyte) in control oocytes or with mNBC3 cRNA-injected oocytes 3-6 days after injection was measured in chloride-free solutions and in the presence of DIDS (1 mM) or EIPA (50 µM). Each bar represents the mean ± S.E. of 8-32 oocytes. 22Na uptake was significantly increased by mNBC3 cRNA versus control oocytes (p < 0.001) and inhibited in the presence of 50 µM EIPA (p < 0.02). Furthermore, in cRNA-injected oocytes 22Na uptake was not affected by chloride removal or 1 mM DIDS. 86Rb uptake (expressed as nmol/h/oocyte) was also assessed in control oocytes and in mNBC3 cRNA-injected oocytes. 86Rb uptake was similar in both groups.

Further studies were done measuring pHi transients in control oocytes and those injected with mNBC3 cRNA (Fig. 6). All studies were performed in the absence of chloride given the lack of dependence of the 22Na uptake results on the presence of chloride. In bicarbonate-buffered solutions, resting pHi was 7.14 ± 0.02 (n = 4) in control oocytes and 7.29 ± 0.04 (n = 5) in cRNA-injected oocytes (p < 0.05). After extracellular Na+ was removed in bicarbonate-buffered solutions, in control oocytes, pHi failed to decrease (Fig. 6a). In contrast, in the cRNA-injected oocytes, at a resting pHi of 7.29 ± 0.04 (n = 5), pHi decreased at a rate of -0.14 ± 0.02 pH/min (n = 5), p < 0.001 (Fig. 6b). Further experiments were done to determine whether K+-induced pHi transients would be present. As shown in a typical experiment in Fig. 6c, in bicarbonate-buffered solutions, increasing the external K+ from 2 to 50 mM in cRNA-injected oocytes had no effect on pHi. These results suggest that 1) mNBC3 is K+-independent as was also shown by the 86Rb flux measurements and 2) depolarizing the oocyte membrane potential using high K+ solutions (48) has no measurable effect on pHi in cRNA-injected oocytes, suggesting that the transport process is electroneutral. In Hepes buffered-solutions, following Na+ removal at a resting pHi of ~7.5, pHi decreased at a rate of -0.17 ± 0.02 pH/min (n = 4) (Fig. 6d). The rate of change of pHi in Hepes-buffered solutions when pHi had decreased to ~7.3 (resting pHi in bicarbonate-buffered solutions), was -0.15 ± 0.01 pH/min (n = 4). Furthermore, at a minimum pHi of ~6.5, following the readdition of Na+ in bicarbonate-buffered solutions pHi increased at a rate of 0.13 ± 0.01 pH/min versus 0.12 ± 0.01 pH/min in Hepes-buffered solutions. Given the difference in intracellular buffer capacities in Hepes and bicarbonate-containing solutions, the results suggest that the rate of equivalent base flux is greater in the presence of bicarbonate in cRNA-injected oocytes. In separate experiments the effect of DIDS and EIPA inhibition was studied. In cRNA-injected oocytes, DIDS (1 mM) did not significantly modify the decrease in pHi induced by Na+ removal (-0.12 ± 0.01 pH/min (n = 3), p = NS; Fig. 6e). In contrast, EIPA (50 µM) completely blocked the Na+-induced pHi transients in cRNA-injected oocytes (0.02 pH/min ± 0.01 pH/min (n = 3), p < 0.001; Fig. 6f). The dependence of mNBC3 transport on external Na+ is shown in Fig. 7a. In these studies, Na+ was repeatedly removed to acidify pHi to a value of ~7.0. Various concentrations of Na+ were added to determine the external Na+ dependence of the transporter. The data indicate that the external Km for external Na+ under these conditions is ~24 mM (Fig. 7b).


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Fig. 6.   Intracellular pH measurements in X. laevis oocytes 5-14 days after injection. Oocytes were bathed initially in a Hepes-buffered chloride-free solution. a and b, following the addition of bicarbonate-buffered chloride-free solutions a steady state was achieved, and Na+ removal/readdition experiments were performed in control oocytes (a) and cRNA-injected oocytes (b). c, increasing the solution K+ from 2 mM to 50 mM had no effect on pHi in cRNA-injected oocytes. d, Na+ removal/readdition in Hepes-buffered chloride-free solutions in cRNA-injected oocytes. e, Na+ removal/readdition in bicarbonate-buffered chloride free solutions in cRNA-injected oocytes in the presence of 1 mM DIDS. f, Na+ removal/readdition in bicarbonate-buffered chloride-free solutions in cRNA-injected oocytes in the presence of 50 µM EIPA.


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Fig. 7.   a, dependence of mNBC3 transport on extracellular Na+. Na+ was repeatedly removed to acidify pHi to a value of ~7.0. Various concentrations of Na+ were added when pHi decreased to ~7.0. The Na+-dependent rate (at each Na+ concentration) was calculated by subtracting the rate of change in pHi immediately prior to the addition of Na+ (the acidification rate in zero Na+ at pH ~ 7.0) from the initial rate of change in pHi following the addition of a particular concentration of Na+. Once the initial rate of change in pHi was measured for a given Na+ concentration, pHi was returned more rapidly to ~7.5 by the addition of 108 mM Na+ followed by another phase of Na+ removal. b, a Hanes-Woolf analysis of the data revealed that the external Km for Na+ is ~24 mM.

The properties of mNBC3 closely resemble those of Na+-dependent HCO3- transport activity previously described in certain smooth muscle cells. A novel Na+ and HCO3--dependent mechanism, sensitive to EIPA but insensitive to disulfonic stilbenes, has been described in cultured human internal mammary smooth muscle cells (15, 21) and cultured rat aortic smooth muscle cells (12). DIDS-insensitive electroneutral NaHCO3 cotransport has also been described in guinea pig ureter and guinea pig femoral artery (27, 28).) Stilbene-insensitive NaHCO3 cotransport has also been demonstrated in cultured oligodendrocytes from mouse spinal cord (49), rat hippocampal slices (50), and cultured rat cerebellar oligodendrocytes (51). Previous studies have shown that H2DIDS reacts covalently with Lys539 and Lys851 in human AE1 (45). Replacement of the highly conserved lysine at position 745 by aspartic acid in the highly conserved putative stilbene-binding motif (K(M/L)XK) in mNBC3, corresponding to Lys542 in human AE1, may block negatively charged stilbene disulfonates from binding. The negatively charged aspartic acid residue at position 745 in mNBC3 may alter the Ki of these compounds for mNBC3 and account for the lack of DIDS inhibition of 22Na influx and Na+-induced pHi changes that we observed. Electroneutral Na(HCO3)n cotransport has also been identified in sheep cardiac Purkinje fibers (17), guinea pig ventricular myocytes (24), the smooth muscle-like cell line BC3H-1 (26), and rat mesenteric vessels (11, 29). However, in these preparations, the transporter is stilbene-sensitive, suggesting that a different protein is likely involved.

The novel finding that EIPA inhibits the transport of mNBC3 is of interest. Further studies are required to determine which residues react with the EIPA molecule. EIPA has been shown to provide protection against cardiac ischemia and reperfusion and to significantly reduce infarction size in rat and rabbit hearts (52-55). The protective effect of EIPA is not altered by K+ channel blockers or PKC inhibitors and has been attributed to inhibition of Na+/H+ exchange (54). The results of the present study suggest the interesting possibility that inhibition of mNBC3 may also contribute to the protective effect of EIPA.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK46976, the Iris and B. Gerald Cantor Foundation, the Max Factor Family Foundation, the Verna Harrah Foundation, the Richard and Hinda Rosenthal Foundation, and the Fredericka Taubitz Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF047033.

Dagger Supported by Training Grant J891002 from the National Kidney Foundation of Southern California.

§ To whom correspondence should be addressed: UCLA Div. of Nephrology, 10833 Le Conte Ave., Rm. 7-155 Factor Bldg., Los Angeles, CA 90095-1689. Tel.: 310-206-6741; Fax: 310-825-6309; E-mail: IKurtz{at}med1.medsch.ucla.edu.

    ABBREVIATIONS

The abbreviations used are: pHi, intracellular pH; NBC, sodium bicarbonate cotransporter; EIPA, 5-(N-ethyl-N-isopropyl)-amiloride; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; bp, base pair(s); DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; BCECF, 2',7'- biscarboxyethyl-5,6-carboxyfluorescein; mNBC3, muscle sodium bicarbonate cotransporter, 3; AE, anion exchanger.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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