Molecular expression of SLC4-derived Na+-dependent anion transporters in selected human tissues

NaHCO(3) transporters are involved in maintenance of intracellular pH and transepithelial HCO(3)(-) movement in many rodent tissues. To establish the human relevance of the many investigations on rodents, this study aimed to map these transporters and a related polypeptide, NaBC1 [solute carrier 4 (SLC4)A11], to several human tissues by using PCR on reverse transcribed human mRNA and immunoperoxidase histochemistry. The mRNA encoding the electroneutral Na(+):HCO(3)(-) cotransporter (NBCe1; SLC4A4), was expressed in renal cortex, renal medulla, stomach, duodenum, jejunum, ileum, colon, pancreas, choroid plexus, cerebellum, cerebrum, and hippocampus. NBCe2 (SLC4A5) and NBCn1 (SLC4A7) mRNAs were mainly found in kidney and brain tissues, as was mRNA encoding the Na(+)-dependent anion exchangers NCBE (SLC4A10) and NDCBE1 (SLC4A8). In addition to previous findings, NBCn1 protein was localized to human renal medullary thick ascending limbs and duodenal epithelial villus cells and NBCe2 protein to renal collecting ducts. Finally, the message encoding NaBC1 was found in kidney, stomach, duodenum, pancreas, and brain, and the corresponding protein in the anterior and posterior corneal epithelia, renal corpuscules, proximal tubules, collecting ducts, pancreatic ducts, and the choroid plexus epithelium. In conclusion, the selected human tissues display distinct expression patterns of HCO(3)(-) transporters, which closely resemble that of rodent tissues.     

Euryhaline pufferfish NBCe1 differs from nonmarine species NBCe1 physiology

Marine fish drink seawater and eliminate excess salt by active salt transport across gill and gut epithelia. Euryhaline pufferfish (Takifugu obscurus, mefugu) forms a CaCO(3) precipitate on the luminal gut surface after transitioning to seawater. NBCe1 (Slc4a4) at the basolateral membrane of intestinal epithelial cell plays a major role in transepithelial intestinal HCO(3)(-) secretion and is critical for mefugu acclimation to seawater. We assayed fugu-NBCe1 (fNBCe1) activity in the Xenopus oocyte expression system. Similar to NBCe1 found in other species, fNBCe1 is an electrogenic Na(+)/HCO(3)(-) cotransporter and sensitive to the stilbene inhibitor DIDS. However, our experiments revealed several unique and distinguishable fNBCe1 transport characteristics not found in mammalian or other teleost NBCe1-orthologs: electrogenic Li(+)/nHCO(3)(-) cotransport; HCO(3)(-) independent, DIDS-insensitive transport; and increased basal intracellular Na(+) accumulation. fNBCe1 is a voltage-dependent Na(+)/nHCO(3)(-) cotransporter that rectifies, independently from the extracellular Na(+) or HCO(3)(-) concentration, around -60 mV. Na(+) removal (0Na(+) prepulse) is necessary to produce the true HCO(3)(-)-elicited current. HCO(3)(-) addition results in huge outward currents with quick current decay. Kinetic analysis of HCO(3)(-) currents reveals that fNBCe1 has a much higher transport capacity (higher maximum current) and lower affinity (higher K(m)) than human kidney NBCe1 (hkNBCe1) does in the physiological range (membrane potential = -80 mV; [HCO(3)(-)] = 10 mM). In this state, fNBCe1 is in favor of operating as transepithelial HCO(3)(-) secretion, opposite of hkNBCe1, from blood to the luminal side. Thus, fugu-NBCe1 represents the first ortholog-based tool to study amino acid substitutions in NBCe1 and how those change ion and voltage dependence.     

Entry to "formula tunnel" revealed by SLC4A4 human mutation and structural model

Glaucoma, cataracts, and proximal renal tubular acidosis are diseases caused by point mutations in the human electrogenic Na(+) bicarbonate cotransporter (NBCe1/SLC4A4) (1, 2). One such mutation, R298S, is located in the cytoplasmic N-terminal domain of NBCe1 and has only moderate (75%) function. As SLC transporters have high similarity in their membrane and N-terminal primary sequences, we homology-modeled NBCe1 onto the crystal structure coordinates of Band 3(AE1) (3). Arg-298 is predicted to be located in a solvent-inaccessible subsurface pocket and to associate with Glu-91 or Glu-295 via H-bonding and charge-charge interactions. We perturbed these putative interactions between Glu-91 and Arg-298 by site-directed mutagenesis and used expression in Xenopus oocyte to test our structural model. Mutagenesis of either residue resulted in reduced transport function. Function was "repaired" by charge reversal (E91R/R298E), implying that these two residues are interchangeable and interdependent. These results contrast the current understanding of the AE1 N terminus as protein-binding sites and propose that hkNBCe1 (and other SLC4) cytoplasmic N termini play roles in controlling HCO(3)(-) permeation.     

The human NBCe1-A mutant R881C, associated with proximal renal tubular acidosis, retains function but is mistargeted in polarized renal epithelia

The human electrogenic renal Na-HCO₃ cotransporter (NBCe1-A; SLC4A4) is localized to the basolateral membrane of proximal tubule cells. Mutations in the SLC4A4 gene cause an autosomal recessive proximal renal tubular acidosis (pRTA), a disease characterized by impaired ability of the proximal tubule to reabsorb HCO₃^– from the glomerular filtrate. Other symptoms can include mental retardation and ocular abnormalities. Recently, a novel homozygous missense mutant (R881C) of NBCe1-A was reported from a patient with a severe pRTA phenotype. The mutant protein was described as having a lower than normal activity when expressed in Xenopus oocytes, despite having normal Na⁺ affinity. However, without trafficking data, it is impossible to determine the molecular basis for the phenotype. In the present study, we expressed wild-type NBCe1-A (WT) and mutant NBCe1-A (R881C), tagged at the COOH terminus with enhanced green fluorescent protein (EGFP). This approach permitted semiquantification of surface expression in individual Xenopus oocytes before assay by two-electrode voltage clamp or measurements of intracellular pH. These data show that the mutation reduces the surface expression rather than the activity of the individual protein molecules. Confocal microscopy on polarized mammalian epithelial kidney cells [Madin-Darby canine kidney (MDCK)I] expressing nontagged WT or R881C demonstrates that WT is expressed at the basolateral membrane of these cells, whereas R881C is retained in the endoplasmic reticulum. In summary, the pathophysiology of pRTA caused by the R881C mutation is likely due to a deficit of NBCe1-A at the proximal tubule basolateral membrane, rather than a defect in the transport activity of individual molecules. bicarbonate; intracellular pH; acidbase; SLC4A4; Na⁺-HCO₃ cotransporter 1     

Electrogenic Na/HCO3 cotransporter (NBCe1) variants expressed in Xenopus oocytes: functional comparison and roles of the amino and carboxy termini

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 CO(2)/HCO(3)(-) solution displayed all the hallmarks of an electrogenic Na(+)/HCO(3)(-) cotransporter: (a) a DIDS-sensitive pH(i) recovery following the initial CO(2)-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 HCO(3)(-) 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 HCO(3)(-)-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 HCO(3)(-)-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.     

Topology of NBCe1 Protein Transmembrane Segment 1 and Structural Effect of Proximal Renal Tubular Acidosis (pRTA) S427L Mutation

In the kidney proximal tubule, NBCe1-A plays a critical role in absorbing HCO₃⁻ from cell to blood. NBCe1-A transmembrane segment 1 (TM1) is involved in forming part of the ion permeation pathway, and a missense mutation S427L in TM1 impairs ion transport, causing proximal renal tubular acidosis. In the present study, we examined the topology of NBCe1-A-TM1 in detail and its structural perturbation induced by S427L. We analyzed the N-terminal cytoplasmic region (Cys-389–Gln-424) of NBCe1-A-TM1 using the substituted cysteine scanning accessibility method combined with extensive chemical stripping, in situ chemical probing, and functional transport assays. NBCe1-A-TM1 was previously modeled on the anion exchanger 1 TM1 (AE1-TM1); however, our data demonstrated that the topology of AE1-TM1 differs significantly from NBCe1-A-TM1. Our findings revealed that NBCe1-A-TM1 is unusually long, consisting of 31 membrane-embedded amino acids (Phe-412 to Thr-442). The linker region (Arg-394–Pro-411) between the N terminus of TM1 and the cytoplasmic domain is minimally exposed to aqueous and is potentially folded in a helical structure that intimately interacts with the NBCe1-A cytoplasmic domain. In contrast, AE1-TM1 contains 25 amino acids connected to an aqueous-exposed cytoplasmic region. Based on our new NBCe1-A-TM1 model, Ser-427 resides in the middle of TM1. Leucine substitution at Ser-427 blocks the normal aqueous access to Thr-442, Ala-435, and Lys-404, implying a significant alteration of NBCe1-TM1 orientation. Our study provides novel structural insights into the pathogenic mechanism of S427L in mediating proximal renal tubular acidosis.     

Inhibition of the Na/Bicarbonate Cotransporter NBCe1-A by diBAC Oxonol Dyes Relative to Niflumic Acid and a Stilbene

Na/HCO(3) cotransporters (NBCs) are important regulators of intracellular pH (pH(i) in a variety of organ systems where acid-base status is critical for tissue function. To characterize the pharmacology of NBCs in more detail, we used the two-electrode voltage-clamp technique to examine the effect of previously identified inhibitors of anion exchanger 1 (AE1) on the activity of rat NBCe1-A expressed in Xenopus laevis oocytes. NBC-expressing oocytes voltage-clamped at -60 mV and exposed to a 5% CO(2)/33 mM HCO(3)(-) solution displayed NBC-mediated outward currents that were inhibited by either niflumic acid or one of the two bis-oxonol dyes diBA(3)C4 and diBA(5)C4. NBCe1-A was less sensitive to niflumic acid (apparent K(i) of 100 microM) than 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, apparent K(i) of 36 microM) but more sensitive to the diBAC dyes (apparent K(i) of approximately 10 microM). Based on current-voltage relationships, the diBAC dyes inhibited HCO(3)(-) -induced NBCe1-mediated inward currents more so than outward currents. NBCe1 sensitivity to the dyes was (1) lower in the presence of 40 microM DIDS, (2) unaffected by changes in external HCO(3)(-) concentration and (3) only modestly higher at an external Na(+) concentration of 5, but not 15 or 33, mM. Therefore, the diBAC dyes compete with DIDS but not appreciably with Na(+) or HCO(3)(-) for binding. The mechanism of diBAC inhibition of NBCe1 appears similar to that previously reported for AE1.     

The electrogenic Na+-HCO3- cotransporter NBCe1-B is regulated by intracellular Mg2+

NBCe1-B, a major splice variant of the electrogenic Na+--HCO3- cotransporter (NBCe1) fulfills basic cellular functions including regulation of intracellular pH and epithelial HCO3- secretion. However, its cellular regulatory mechanism still remains elusive. Here, we provide evidence for the first time that NBCe1-B activity can be controlled by intracellular Mg2+ (Mg2+(i)), the physiologically most abundant intracellular divalent cation. Using the whole-cell patch-clamp technique, we found that recombinant NBCe1-B currents expressed in HEK293 and NIH3T3 cells were inhibited voltage-independently by Mg2+(i) in a concentration-dependent manner (K(i) approximately 0.01 mM). The Mg2+(i) inhibition was partially relieved by truncation of the NBCe1-B specific N-terminal region (K(i) approximately 0.3 mM), and was also observed for native electrogenic Na+--HCO3- cotransporter current in bovine parotid acinar cells that endogenously express NBCe1-B (K(i) approximately 1 mM). These results suggest that Mg2+ may be a cytosolic factor that limits intrinsic cotransport activity of NBCe1-B in mammalian cells.     

The Sodium Bicarbonate Cotransporter (NBCe1) Is Essential for Normal Development of Mouse Dentition

Proximal renal tubular acidosis (pRTA) is a syndrome caused by abnormal proximal tubule reabsorption of bicarbonate resulting in metabolic acidosis. Patients with mutations to the SLC4A4 gene (coding for the sodium bicarbonate cotransporter NBCe1), have pRTA, growth delay, ocular defects, and enamel abnormalities. In an earlier report, we provided the first evidence that enamel cells, the ameloblasts, express NBCe1 in a polarized fashion, thereby contributing to trans-cellular bicarbonate transport. To determine whether NBCe1 plays a critical role in enamel development, we studied the expression of NBCe1 at various stages of enamel formation in wild-type mice and characterized the biophysical properties of enamel in NBCe1^−/− animals. The enamel of NBCe1^−/− animals was extremely hypomineralized and weak with an abnormal prismatic architecture. The expression profile of amelogenin, a known enamel-specific gene, was not altered in NBCe1^−/− animals. Our results show for the first time that NBCe1 expression is required for the development of normal enamel. This study provides a mechanistic model to account for enamel abnormalities in certain patients with pRTA.     

Physiological relevance of cell-specific distribution patterns of CFTR, NKCC1, NBCe1, and NHE3 along the crypt-villus axis in the intestine

We examined the cell-specific subcellular expression patterns for sodium- and potassium-coupled chloride (NaK2Cl) cotransporter 1 (NKCC1), Na(+) bicarbonate cotransporter (NBCe1), cystic fibrosis transmembrane conductance regulator (CFTR), and Na(+)/H(+) exchanger 3 (NHE3) to understand the functional plasticity and synchronization of ion transport functions along the crypt-villus axis and its relevance to intestinal disease. In the unstimulated intestine, all small intestinal villus enterocytes coexpressed apical CFTR and NHE3, basolateral NBCe1, and mostly intracellular NKCC1. All (crypt and villus) goblet cells strongly expressed basolateral NKCC1 (at approximately three-fold higher levels than villus enterocytes), but no CFTR, NBCe1, or NHE3. Lower crypt cells coexpressed apical CFTR and basolateral NKCC1, but no NHE3 or NBCe1 (except NBCe1-expressing proximal colonic crypts). CFTR, NBCe1, and NKCC1 colocalized with markers of early and recycling endosomes, implicating endocytic recycling in cell-specific anion transport. Brunner's glands of the proximal duodenum coexpressed high levels of apical/subapical CFTR and basolateral NKCC1, but very low levels of NBCe1, consistent with secretion of Cl(-)-enriched fluid into the crypt. The cholinergic agonist carbachol rapidly (within 10 min) reduced cell volume along the entire crypt/villus axis and promoted NHE3 internalization into early endosomes. In contrast, carbachol induced membrane recruitment of NKCC1 and CFTR in all crypt and villus enterocytes, NKCC1 in all goblet cells, and NBCe1 in all villus enterocytes. These observations support regulated vesicle traffic in Cl(-) secretion by goblet cells and Cl(-) and HCO(3)(-) secretion by villus enterocytes during the transient phase of cholinergic stimulation. Overall, the carbachol-induced membrane trafficking profile of the four ion transporters supports functional plasticity of the small intestinal villus epithelium that enables it to conduct both absorptive and secretory functions.     

Binding of carbonic anhydrase IX to extracellular loop 4 of the NBCe1 Na+/HCO3- cotransporter enhances NBCe1-mediated HCO3- influx in the rat heart

Na(+)/HCO(3)(-) cotransporter (NBC)e1 catalyze the electrogenic movement of 1 Na(+):2 HCO(3)(-) into cardiomyocytes cytosol. NBC proteins associate with carbonic anhydrases (CA), CAII, and CAIV, forming a HCO(3)(-) transport metabolon. Herein, we examined the physical/functional interaction of NBCe1 and transmembrane CAIX in cardiac muscle. NBCe1 and CAIX physical association was examined by coimmunoprecipitation, using rat ventricular lysates. NBCe1 coimmunoprecipitated with anti-CAIX antibody, indicating NBCe1 and CAIX interaction in the myocardium. Glutathione-S-transferase (GST) pull-down assays with predicted extracellular loops (EC) of NBCe1 revealed that NBCe1-EC4 mediated interaction with CAIX. Functional NBCe1/CAIX interaction was examined using fluorescence measurements of BCECF in rat cardiomyocytes to monitor cytosolic pH. NBCe1 transport activity was evaluated after membrane depolarization with high extracellular K(+) in the presence or absence of the CA inhibitors, benzolamide (BZ; 100 μM) or 6-ethoxyzolamide (ETZ; 100 μM) (*P < 0.05). This depolarization protocol produced an intracellular pH (pH(i)) increase of 0.17 ± 0.01 (n = 11), which was inhibited by BZ (0.11 ± 0.02; n = 7) or ETZ (0.06 ± 0.01; n = 6). NBCe1 activity was also measured by changes of pH(i) in NBCe1-transfected human embryonic kidney 293 cells subjected to acid loads. Cotransfection of CAIX with NBCe1 increased the rate of pH(i) recovery (in mM/min) by about fourfold (12.1 ± 0.8; n = 9) compared with cells expressing NBCe1 alone (3.1 ± 0.5; n = 7), which was inhibited by BZ (7.5 ± 0.3; n = 9). We demonstrated that CAIX forms a complex with EC4 of NBCe1, which activates NBCe1-mediated HCO(3)(-) influx in the myocardium. CAIX and NBCe1 have been linked to tumorigenesis and cardiac cell growth, respectively. Thus inhibition of CA activity might be useful to prevent activation of NBCe1 under these pathological conditions.     

Electrogenic NBCe1 (SLC4A4), but not electroneutral NBCn1 (SLC4A7), cotransporter undergoes cholinergic-stimulated endocytosis in salivary ParC5 cells

Cholinergic agonists are major stimuli for fluid secretion in parotid acinar cells. Saliva bicarbonate is essential for maintaining oral health. Electrogenic and electroneutral Na(+)-HCO(3)(-) cotransporters (NBCe1 and NBCn1) are abundant in parotid glands. We previously reported that angiotensin regulates NBCe1 by endocytosis in Xenopus oocytes. Here, we studied cholinergic regulation of NBCe1 and NBCn1 membrane trafficking by confocal fluorescent microscopy and surface biotinylation in parotid epithelial cells. NBCe1 and NBCn1 colocalized with E-cadherin monoclonal antibody at the basolateral membrane (BLM) in polarized ParC5 cells. Inhibition of constitutive recycling with the carboxylic ionophore monensin or the calmodulin antagonist W-13 caused NBCe1 to accumulate in early endosomes with a parallel loss from the BLM, suggesting that NBCe1 is constitutively endocytosed. Carbachol and PMA likewise caused redistribution of NBCe1 from BLM to early endosomes. The PKC inhibitor, GF-109203X, blocked this redistribution, indicating a role for PKC. In contrast, BLM NBCn1 was not downregulated in parotid acinar cells treated with constitutive recycling inhibitors, cholinergic stimulators, or PMA. We likewise demonstrate striking differences in regulation of membrane trafficking of NBCe1 vs. NBCn1 in resting and stimulated cells. We speculate that endocytosis of NBCe1, which coincides with the transition to a steady-state phase of stimulated fluid secretion, could be a part of acinar cell adjustment to a continuous secretory response. Stable association of NBCn1 at the membrane may facilitate constitutive uptake of HCO(3)(-) across the BLM, thus supporting HCO(3)(-) luminal secretion and/or maintaining acid-base homeostasis in stimulated cells.     

Carbonic anhydrase II increases the activity of the human electrogenic Na+/HCO3- cotransporter

Several acid/base-coupled membrane transporters, such as the electrogenic sodium-bicarbonate cotransporter (NBCe1), have been shown to bind to different carbonic anhydrase isoforms to create a "transport metabolon." We have expressed NBCe1 derived from human kidney in oocytes of Xenopus leavis and determined its transport activity by recording the membrane current in voltage clamp, and the cytosolic H(+) and Na(+) concentrations using ion-selective microelectrodes. When carbonic anhydrase isoform II (CAII) had been injected into oocytes, the membrane current and the rate of cytosolic Na(+) rise, indicative for NBCe1 activity, increased significantly with the amount of injected CAII (2-200 ng). The CAII inhibitor ethoxyzolamide reversed the effects of CAII on the NBCe1 activity. Co-expressing wild-type CAII or NH(2)-terminal mutant CAII together with NBCe1 provided similar results, whereas co-expressing the catalytically inactive CAII mutant V143Y had no effect on NBCe1 activity. Mass spectrometric analysis and the rate of cytosolic H(+) change following addition of CO(2)/HCO(3)(-) confirmed the catalytic activity of injected and expressed CAII in oocytes. Our results show that the transport capacity of NBCe1 is enhanced by the catalytic activity of CAII, in line with the notion that CAII forms a transport metabolon with NBCe1.     

Effect of chronic inhibition of converting enzyme on proximal tubule acidification

The acute effect of angiotensin-converting enzyme inhibition (ACEi) on proximal convoluted tubule (PCT) function is well documented. However, the effect of chronic treatment is less known. The aim of this work was to evaluate the effect of chronic ACEi on PCT acidification (J(HCO(3)(-))). Rats received enalapril (10 mg.kg(-1).day(-1), added to the drinking water) during 3 mo. Micropuncture experiments were performed to measure the effect of chronic ACEi on J(HCO(3)(-)). Nitric oxide (NO.) synthesis in kidney cortex homogenates was assessed by quantifying the conversion of [(14)C]-L-arginine to [(14)C]-L-citrulline. Western blot analysis was performed to determine the abundances of V-H(+)ATPase and NHE3 isoform of the Na(+)/H(+) exchanger in proximal brush-border membrane vesicles (BBMV). Enalapril treatment induced an approximately 50% increase in J(HCO(3)(-)). Luminal perfusion with ethyl-isopropyl amiloride (EIPA) 10(-4)M or bafilomycin 10(-6)M decreased J(HCO(3)(-)) by approximately 60% and approximately 30%, respectively, in both control and enalapril-treated rats. The effect of EIPA and bafilomycin on absolute J(HCO(3)(-)) was larger in enalapril-treated than in control rats. Acute inhibition of NO. synthesis with N(G)-nitro-L-arginine methyl ester abolished the enalapril-induced increase in J(HCO(3)(-)). Cortex homogenates from enalapril-treated rats displayed a 46% increase in nitric oxide synthase (NOS) activity compared with those from untreated animals. Enalapril treatment did not affect the abundances of NHE3 and V-H(+)ATPase in BBMV. Our results suggest that PCT acidification is increased during chronic ACEi probably due to an increase in NO. synthesis, which would stimulate Na(+)/H(+) exchange and electrogenic proton transport.     

Apical electrogenic NaHCO3 cotransport. A mechanism for HCO3 absorption across the retinal pigment epithelium

Intracellular microelectrode techniques and intracellular pH (pHi) measurements using the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) were employed to characterize an electrogenic bicarbonate transport mechanism at the apical membrane of the frog retinal pigment epithelium (RPE). Reductions in apical concentrations of both [HCO3]o (at constant Pco2 or pHo) or [Na]o caused rapid depolarization of the apical membrane potential (Vap). Both of these voltage responses were inhibited when the concentration of the other ion was reduced or when 1 mM diisothiocyano-2-2 disulfonic acid stilbene (DIDS) was present in the apical bath. Reductions in apical [HCO3]o or [Na]o also produced a rapid acidification of the cell interior that was inhibited by apical DIDS. Elevating pHi at constant Pco2 (and consequently [HCO3]i) by the addition of apical NH4 (20 mM) produced an immediate depolarization of Vap. This response was much smaller when either apical [HCO3]o or [Na]o was reduced or when DIDS was added apically. These results strongly suggest the presence of an electrogenic NaHCO3 cotransporter at the apical membrane. Apical DIDS rapidly depolarized Vap by 2-3 mV and decreased pHi (and [HCO3]i), indicating that the transporter moves NaHCO3 and net negative charge into the cell. The voltage dependence of the transporter was assessed by altering Vap with transepithelial current and then measuring the DIDS-induced change in Vap. Depolarization of Vap increased the magnitude of the DIDS-induced depolarization, whereas hyperpolarization decreased it. Hyperpolarizing Vap beyond -114 mV caused the DIDS-induced voltage change to reverse direction. Based on this reversal potential, we calculate that the stoichiometry of the transporter is 1.6-2.4 (HCO3/Na).     

A kinetically defined Na+/H+ antiporter within a mathematical model of the rat proximal tubule

The luminal membrane antiporter of the proximal tubule has been represented using the kinetic formulation of E. Heinz (1978. Mechanics and Engergetics of Biological Transport. Springer-Verlag, Berlin) with the assumption of equilibrium binding and 1:1 stoichiometry. Competitive binding and transport of NH+4 is included within this model. Ion affinities and permeation velocities were selected in a least-squares fit to the kinetic parameters determined experimentally in renal membrane vesicles (Aronson, P.S., M.A. Suhm, and J. Nee. 1983. Journal of Biological Chemistry. 258:6767-6771). The modifier role of internal H+ to enhance transport beyond the expected kinetics (Aronson, P.S., J. Nee, and M. A. Suhm. 1982. Nature. 299:161-163) is represented as a velocity effect of H+ binding to a single site. This kinetic formulation of the Na+/H+ antiporter was incorporated within a model of the rat proximal tubule (Weinstein, A. M. 1994. American Journal of Physiology. 267:F237-F248) as a replacement for the representation by linear nonequilibrium thermodynamics (NET). The membrane density of the antiporter was selected to yield agreement with the rate of tubular Na+ reabsorption. Simulation of 0.5 cm of tubule predicts that the activity of the Na+/H+ antiporter is the most important force for active secretion of ammonia. Model calculations of metabolic acid-base disturbances are performed and comparison is made among antiporter representations (kinetic model, kinetic model without internal modifier, and NET formulation). It is found that the ability to sharply turn off Na+/H+ exchange in cellular alkalosis substantially eliminates the cell volume increase associated with high HCO3- conditions. In the tubule model, diminished Na+/H+ exchange in alkalosis blunts the axial decrease in luminal HCO3- and thus diminishes paracellular reabsorption of Cl-. In this way, the kinetics of the Na+/H+ antiporter could act to enhance distal delivery of Na+, Cl-, and HCO3- in acute metabolic alkalosis.     

Genetic ablation of Slc4a10 alters the expression pattern of transporters involved in solute movement in the mouse choroid plexus

Mutational changes of one transporter can have deleterious effects on epithelial function leaving the cells with the options of either compensating for the loss of function or dedifferentiating. Previous studies have shown that the choroid plexus epithelium (CPE) from mice lacking the Na(+)-dependent Cl(-)/HCO(3)(-) exchanger (NCBE) encoded by Slc4a10 leads to retargeting of the Na(+)/H(+) exchanger 1 (NHE1) from the luminal to the basolateral plasma membrane. We hypothesized that disruption of NCBE, the main basolateral Na(+) importer in the CPE, would lead to a compensatory increase in the abundance of other important transport proteins in this tissue. Aquaporin-1 (AQP1) abundance was 42.7% lower and Na,K-ATPase 36.4% lower in the CPE of Slc4a10 knockout mice, respectively. The NHE1 binding ezrin cytoskeleton appeared disrupted in Slc4a10 knockout mice, whereas no changes were observed in cellular polarization with respect to claudin-2 and appearance of luminal surface microvilli. The renal proximal tubule constitutes a leaky epithelium with high transport rate similar to CPE. Here, Slc4a10 knockout did not affect Na,K-ATPase or AQP1 expression. CPE from AQP1 knockout mice has a secretory defect similar to Slc4a10 mice. However, neither NCBE nor Na,K-ATPase expression was affected in CPE from AQP1 knockout mice. By contrast, the abundance of Na,K-ATPase and NBCe1 was decreased by 23 and 31.7%, respectively, in AQP1 knockout proximal tubules, while the NHE3 abundance was unchanged. In conclusion, CPE lacking NCBE seems to spare the molecular machinery involved in CSF secretion rather than compensate for the loss of the Na(+) loader. Slc4a10 knockout seems to be more deleterious to CPE than AQP1 knockout.     

Prevention of the Disrupted Enamel Phenotype in Slc4a4-Null Mice Using Explant Organ Culture Maintained in a Living Host Kidney Capsule

Slc4a4-null mice are a model of proximal renal tubular acidosis (pRTA). Slc4a4 encodes the electrogenic sodium base transporter NBCe1 that is involved in transcellular base transport and pH regulation during amelogenesis. Patients with mutations in the SLC4A4 gene and Slc4a4-null mice present with dysplastic enamel, amongst other pathologies. Loss of NBCe1 function leads to local abnormalities in enamel matrix pH regulation. Loss of NBCe1 function also results in systemic acidemic blood pH. Whether local changes in enamel pH and/or a decrease in systemic pH are the cause of the abnormal enamel phenotype is currently unknown. In the present study we addressed this question by explanting fetal wild-type and Slc4a4-null mandibles into healthy host kidney capsules to study enamel formation in the absence of systemic acidemia. Mandibular E11.5 explants from NBCe1^−/− mice, maintained in host kidney capsules for 70 days, resulted in teeth with enamel and dentin with morphological and mineralization properties similar to cultured NBCe1^+/+ mandibles grown under identical conditions. Ameloblasts express a number of proteins involved in dynamic changes in H⁺/base transport during amelogenesis. Despite the capacity of ameloblasts to dynamically modulate the local pH of the enamel matrix, at least in the NBCe1^−/− mice, the systemic pH also appears to contribute to the enamel phenotype. Extrapolating these data to humans, our findings suggest that in patients with NBCe1 mutations, correction of the systemic metabolic acidosis at a sufficiently early time point may lead to amelioration of enamel abnormalities.     

Functional role of a putative carbonic anhydrase II-binding domain in the electrogenic Na+ -HCO₃- cotransporter NBCe1 expressed in Xenopus oocytes

The electrogenic Na+ -HCO?? cotransporter NBCe1 plays essential roles in the regulation of systemic and/or local pH. Homozygous inactivating mutations in NBCe1 cause proximal renal tubular acidosis associated with ocular abnormalities. We recently showed that defective membrane expression of NBCe1, caused by several mutations such as Delta65bp (S982NfsX4), is also associated with familial migraine. The Delta65bp mutant is quite unique in that it lacks a putative carbonic anhydrase (CA) II-binding domain but still shows an apparently normal transport activity in Xenopus oocytes. In this addendum, we show that the co-expression of CAII together with the wild-type NBCe1 or the Delta65bp mutant does not enhance the NBCe1 activities in oocytes. Moreover, a carbonic anhydrase inhibitor acetazolamide fails to inhibit the wild-type or the Delta65bp activities co-expressed with CAII. These results indicate that a bicarbonate transport metabolon proposed for the interaction between CAII and NBCe1 does not work at least in Xenopus oocytes.