New paradigms on the transport functions of maturation-stage ameloblasts.

Fully matured dental enamel is an architecturally and mechanically complex hydroxyapatite-based bioceramic devoid of most of the organic material that was essential in its making. Enamel formation is a staged process principally involving secretory and maturation stages, each associated with major changes in gene expression and cellular function. Cellular activities that define the maturation stage of amelogenesis include ion (e.g., calcium and phosphate) transport and storage, control of intracellular and extracellular pH (e.g., bicarbonate and hydrogen ion movements), and endocytosis. Recent studies on rodent amelogenesis have identified a multitude of gene products that appear to be linked to these cellular activities. This review describes the main cellular activities of these genes during the maturation stage of amelogenesis.

Role of NBCe1 and AE2 in secretory ameloblasts.

The H(+)/base transport processes that control the pH of the microenvironment adjacent to ameloblasts are not currently well-understood. Mice null for the AE2 anion exchanger have abnormal enamel. In addition, persons with mutations in the electrogenic sodium bicarbonate co-transporter NBCe1 and mice lacking NBCe1 have enamel abnormalities. These observations suggest that AE2 and NBCe1 play important roles in amelogenesis. In the present study, we aimed to understand the roles of AE2 and NBCe1 in ameloblasts. Analysis of the data showed that NBCe1 is expressed at the basolateral membrane of secretory ameloblasts, whereas AE2 is expressed at the apical membrane. Transcripts for AE2a and NBCe1-B were detected in RNA isolated from cultured ameloblast-like LS8 cells. Our data are the first evidence that AE2 and NBCe1 are expressed in ameloblasts in vivo in a polarized fashion, thereby providing a mechanism for ameloblast transcellular bicarbonate secretion in the process of enamel formation and maturation.

Molecular mechanisms of electrogenic sodium bicarbonate cotransport: structural and equilibrium thermodynamic considerations.

The electrogenic Na(+)-HCO(3)(-) cotransporters play an essential role in regulating intracellular pH and extracellular acid-base homeostasis. Of the known members of the bicarbonate transporter superfamily (BTS), NBC1 and NBC4 proteins have been shown to be electrogenic. The electrogenic nature of these transporters results from the unequal coupling of anionic and cationic fluxes during each transport cycle. This unique property distinguishes NBC1 and NBC4 proteins from other sodium bicarbonate cotransporters and members of the bicarbonate transporter superfamily that are known to be electroneutral. Structure-function studies have played an essential role in revealing the basis for the modulation of the coupling ratio of NBC1 proteins. In addition, the recent transmembrane topographic analysis of pNBC1 has shed light on the potential structural determinants that are responsible for ion permeation through the cotransporter. The experimentally difficult problem of determining the nature of anionic species being transported by these proteins (HCO(3)(-) versus CO(3)(2-)) is analyzed using a theoretical equilibrium thermodynamics approach. Finally, our current understanding of the molecular mechanisms responsible for the regulation of ion coupling and flux through electrogenic sodium bicarbonate cotransporters is reviewed in detail.

Phosphorylation-induced modulation of pNBC1 function: distinct roles for the amino- and carboxy-termini.

The human NBC1 (SLC4A4) gene encodes the electrogenic sodium bicarbonate cotransporters kNBC1 and pNBC1, which are highly expressed in the kidney and pancreas, respectively. The HCO3-:Na+ stoichiometry of these cotransporters is an important determinant of the direction of ion flux. Recently we showed in a mouse proximal tubule (mPCT) cell line expressing kNBC1, that 8-Br-cAMP shifts the stoichiometry of the cotransporter from 3:1 to 2:1 via protein kinase A (PKA)-dependent phosphorylation of Ser982. pNBC1 has the identical carboxy-terminal consensus phosphorylation PKA site (KKGS1026), and an additional site in its amino-terminus (KRKT49). In this study we determined the potential role of these sites in regulating the function of pNBC1. The results demonstrated that in mPCT cells expressing pNBC1, PKA-dependent phosphorylation of Ser1026 following 8-Br-cAMP treatment shifted the stoichiometry from 3:1 to 2:1. The effect was electrostatic in nature as replacing Ser1026 with Asp resulted in a similar stoichiometry shift. In addition to shifting the stoichiometry, 8-Br-cAMP caused a significant increase in the 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)-sensitive basolateral membrane conductance (GDS) of cells expressing pNBC1, but not kNBC1. Although, the effect did not involve phosphorylation of Thr49, which was endogenously phosphorylated, replacing this residue with Asp or Ala abolished the 8-Br-cAMP-induced increase in GDS. In the mPEC pancreatic duct cell line, where endogenous pNBC1 functions with a HCO3-:Na+ stoichiometry of 2:1, 8-Br-cAMP increased GDS by ~90 % without altering the stoichiometry or inducing phosphorylation of the cotransporter. The results demonstrate that phosphorylation of Ser1026 mediates the cAMP-dependent shift in the stoichiometry of pNBC1, whereas Thr49 plays an essential role in the cAMP-induced increase in GDS.

Immunolocalization of electroneutral Na(+)-HCO cotransporters in human and rat salivary glands.

Patterns of salivary HCO secretion vary widely among species and among individual glands. In particular, virtually nothing is known about the molecular identity of the HCO transporters involved in human salivary secretion. We have therefore examined the distribution of several known members of the Na(+)-HCO cotransporter (NBC) family in the parotid and submandibular glands. By use of a combination of RT-PCR and immunoblotting analyses, the electroneutral cotransporters NBC3 and NBCn1 mRNA and protein expression were detected in both human and rat tissues. Immunohistochemistry demonstrated that NBC3 was present at the apical membranes of acinar and duct cells in both human and rat parotid and submandibular glands. NBCn1 was strongly expressed at the basolateral membrane of striated duct cells but not in the acinar cells in the human salivary glands, whereas little or no NBCn1 labeling was observed in the rat salivary glands. The presence of NBCn1 at the basolateral membrane of human striated duct cells suggests that it may contribute to ductal HCO secretion. In contrast, the expression of NBC3 at the apical membranes of acinar and duct cells in both human and rat salivary glands indicates a possible role of this isoform in HCO salvage under resting conditions.

Cellular bicarbonate protects rat duodenal mucosa from acid-induced injury.

Secretion of bicarbonate from epithelial cells is considered to be the primary mechanism by which the duodenal mucosa is protected from acid-related injury. Against this view is the finding that patients with cystic fibrosis, who have impaired duodenal bicarbonate secretion, are paradoxically protected from developing duodenal ulcers. Therefore, we hypothesized that epithelial cell intracellular pH regulation, rather than secreted extracellular bicarbonate, was the principal means by which duodenal epithelial cells are protected from acidification and injury. Using a novel in vivo microscopic method, we have measured bicarbonate secretion and epithelial cell intracellular pH (pH(i)), and we have followed cell injury in the presence of the anion transport inhibitor DIDS and the Cl(-) channel inhibitor, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). DIDS and NPPB abolished the increase of duodenal bicarbonate secretion following luminal acid perfusion. DIDS decreased basal pH(i), whereas NPPB increased pH(i); DIDS further decreased pH(i) during acid challenge and abolished the pH(i) overshoot over baseline observed after acid challenge, whereas NPPB attenuated the fall of pH(i) and exaggerated the overshoot. Finally, acid-induced epithelial injury was enhanced by DIDS and decreased by NPPB. The results support the role of intracellular bicarbonate in the protection of duodenal epithelial cells from luminal gastric acid.

Phosphorylation of Ser(982) in the sodium bicarbonate cotransporter kNBC1 shifts the HCO(3)(-) : Na(+) stoichiometry from 3 : 1 to 2 : 1 in murine proximal tubule cells.

1. Adenosine 3',5'-cyclic monophosphate (cAMP) modulates proximal tubule sodium and bicarbonate absorption by decreasing the rate of apical Na(+)-H(+) exchange and basolateral sodium bicarbonate efflux, through activation of protein kinase A (PKA). The electrogenic sodium bicarbonate cotransporter kNBC1 mediates basolateral sodium and bicarbonate efflux in the proximal tubule by coupling the transport of 1 Na(+) cation to that of 3 HCO(3)(-) anions. In this work we studied the effects of cAMP on the function of kNBC1 expressed heterologously in a proximal tubule cell line. 2. A mouse renal proximal tubule cell line, deficient in electrogenic sodium bicarbonate cotransport function, was transfected with kNBC1. Cells were grown on a permeable support to confluence, mounted in an Ussing chamber and permeabilized apically with amphotericin B. Current through the cotransporter was isolated as the difference current due to the reversible inhibitor dinitrostilbene disulfonate. The HCO(3)(-) : Na(+) stoichiometry of kNBC1 was calculated from its reversal potential by measuring the current-voltage relationships of the cotransporter at different Na(+) concentration gradients. 3. Addition of the potent cAMP agonist 8-Br-cAMP caused the stoichiometry of kNBC1 to shift from 3 HCO(3)(-) : 1 Na(+) to 2 HCO(3)(-) : 1 Na(+). Pretreatment of the cells with the PKA inhibitor H-89 abolished the effect of the agonist on the stoichiometry change. Replacing Ser(982) at the C-terminus consensus PKA phosphorylation site with alanine resulted in a failure of PKA to phosphorylate the transporter and induce a stoichiometry shift. 4. Our data indicate that cAMP modulates the stoichiometry of kNBC1 through activation of PKA. The change in stoichiometry from 3 : 1 to 2 : 1 is predicted to cause a shift in the direction of basolateral membrane sodium bicarbonate transport from efflux to influx. Ser(982) in the C-terminus of kNBC1 is a target for PKA phosphorylation. This is the first example of modulation of the stoichiometry of a membrane transporter by phosphorylation.

Immunolocalization of electrogenic sodium-bicarbonate cotransporters pNBC1 and kNBC1 in the rat eye.

The human NBC1 gene encodes two electrogenic sodium-bicarbonate cotransport proteins, pNBC1 and kNBC1, which are candidate proteins for mediating electrogenic sodium-bicarbonate cotransport in ocular cells. Mutations in the coding region of the human NBC1 gene in exons common to both pNBC1 and kNBC1 result in a syndrome with a severe ocular and renal phenotype (blindness, band keratopathy, glaucoma, cataracts, and proximal renal tubular acidosis). In the present study, we determined the pattern of electrogenic sodium-bicarbonate cotransporter protein expression in rat eye. For this purpose, pNBC1- and kNBC1-specific antibodies were generated and used to detect these NBC1 protein variants by immunoblotting and immunocytochemistry. pNBC1 is expressed in cornea, conjunctiva, lens, ciliary body, and retina, whereas the expression of kNBC1 is restricted to the conjunctiva. These results provide the first evidence for extrarenal kNBC1 protein expression. The data in this study will serve as a basis for understanding the molecular mechanisms responsible for abnormalities in ocular electrogenic sodium-bicarbonate cotransport in patients with mutations in the NBC1 gene.

Use of base in the treatment of severe acidemic states.

Severe acidemia (blood pH < 7.1 to 7.2) suppresses myocardial contractility, predisposes to cardiac arrhythmias, causes venoconstriction, and can decrease total peripheral vascular resistance and blood pressure, reduce hepatic blood flow, and impair oxygen delivery. These alterations in organ function can contribute to increased morbidity and mortality. Although it seemed logical to administer sodium bicarbonate to attenuate acidemia and therefore lessen the impact on cardiac function, the routine use of bicarbonate in the treatment of the most common causes of severe acidemia, diabetic ketoacidosis, lactic acidosis, and cardiac arrest, has been an issue of great controversy. Studies of animals and patients with these disorders have reported conflicting data on the benefits of bicarbonate, showing both beneficial and detrimental effects. Alternative alkalinizing agents, tris-hydroxymethyl aminomethane and Carbicarb, have shown some promise in studies of animals and humans, and reevaluation of these buffers in the treatment of severe acidemic states seems warranted. The potential value of base therapy in the treatment of severe acidemia remains an important issue, and further studies are required to determine which patients should be administered base therapy and what base should be used.

The stoichiometry of the electrogenic sodium bicarbonate cotransporter NBC1 is cell-type dependent.

1. The pancreatic variant of the sodium bicarbonate cotransporter, pNBC1, mediates basolateral bicarbonate influx in the exocrine pancreas by coupling the transport of bicarbonate to that of sodium, with a 2 HCO3-:1 Na+ stoichiometry. The kidney variant, kNBC1, mediates basolateral bicarbonate efflux in the proximal tubule by coupling the transport of 3 HCO3- to 1 Na+. The molecular basis underlying the different stoichiometries is not known. 2. pNBC1 and kNBC1 are 93 % identical to each other with 41 N-terminal amino acids of kNBC1 replaced by 85 distinct amino acids in pNBC1. In this study we tested the hypothesis that the differences in stoichiometry are related to the difference between the N-termini of the two proteins. 3. Mouse renal proximal tubule and collecting duct cells, deficient in both pNBC1- and kNBC1-mediated electrogenic sodium bicarbonate cotransport function were transfected with either pNBC1 or kNBC1. Cells were grown on a permeable support to confluence, mounted in an Ussing chamber and permeabilized apically with amphotericin B. Current through the cotransporter was isolated as the difference current due to the reversible inhibitor dinitrostilbene disulfonate. The stoichiometry was calculated from the reversal potential by measuring the current-voltage relationships of the cotransporter at different Na+ concentration gradients. 4. Our data indicate that both kNBC1 and pNBC1 can exhibit either a 2:1 or 3:1 stoichiometry depending on the cell type in which each is expressed. In proximal tubule cells, both pNBC1 and kNBC1 exhibit a 3 HCO3-:1 Na+ stoichiometry, whereas in collecting duct cells, they have a 2:1 stoichiometry. These data argue against the hypothesis that the stoichiometric differences are related to the difference between the N-termini of the two proteins. Moreover, the results suggest that as yet unidentified cellular factor(s) may modify the stoichiometry of these cotransporters.

The stoichiometry of the electrogenic sodium bicarbonate cotransporter pNBC1 in mouse pancreatic duct cells is 2 HCO(3)(-):1 Na(+).

The electrogenic sodium bicarbonate cotransporter pNBC1 is believed to play a major role in the secretion of bicarbonate by pancreatic duct cells, by transporting bicarbonate into the cell across the basolateral membrane. Thermodynamics predict that this function can be achieved only if the reversal potential of the cotransporter is negative to the cell's membrane potential, or equivalently that the HCO3-:Na+ stoichiometry is not larger then 2:However, there are no data available on either the reversal potential or the HCO3-:Na+ stoichiometry of pNBC1 in pancreatic cells. We studied pNBC1 function in mouse pancreatic duct cells. RT-PCR analysis of total RNA revealed that these cells contain the message for pNBC1, but not for kNBC1, NBC2 or NBC3. To measure cotransporter activity, mouse pancreatic duct cells were grown to confluence on a porous substrate, mounted in an Ussing chamber, and the apical plasma membrane permeabilized with amphotericin B. Ion flux through pNBC1 was achieved by applying Na+ concentration gradients across the basolateral plasma membrane. The current through the cotransporter was isolated as the difference current due to the reversible inhibitor dinitrostilbene disulfonate (DNDS). Current-voltage relationships for the cotransporter, measured at three different Na+ concentration gradients, were linear over a range of about 100 mV. The reversal potential data, obtained from these current-voltage relationships, all corresponded to a 2 HCO3-:1 Na+ stoichiometry. The data indicate that pNBC1 is functionally expressed in mouse pancreatic duct cells. The cotransporter operates with a 2 HCO3-:1 Na+ stoichiometry in these cells, and mediates the transport of bicarbonate into the cell across the basolateral membrane.

HCO3- salvage mechanisms in the submandibular gland acinar and duct cells.

In the present work, we characterized H(+) and HCO3- transport mechanisms in the submandibular salivary gland (SMG) ducts of wild type, NHE2-/-, NHE3-/-, and NHE2-/-;NHE3-/- double knock-out mice. The bulk of recovery from an acid load across the luminal membrane (LM) of the duct was mediated by a Na(+)-dependent HOE and ethyl-isopropyl-amiloride (EIPA)-inhibitable and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-insensitive mechanism. HCO3- increased the rate of luminal Na(+)-dependent pH(i) recovery but did not change inhibition by HOE and EIPA or the insensitivity to DIDS. Despite expression of NHE2 and NHE3 in the LM of the duct, the same activity was observed in ducts from wild type and all mutant mice. Measurements of Na(+)-dependent OH(-) and/or HCO3- cotransport (NBC) activities in SMG acinar and duct cells showed separate DIDS-sensitive/EIPA-insensitive and DIDS-insensitive/EIPA-sensitive NBC activities in both cell types. Functional and immunocytochemical localization of these activities in the perfused duct indicated that pNBC1 probably mediates the DIDS-sensitive/EIPA-insensitive transport in the basolateral membrane, and splice variants of NBC3 probably mediate the DIDS-insensitive/EIPA-sensitive NBC activity in the LM of duct and acinar cells. Notably, the acinar cell NBC3 variants transported HCO3- but not OH(-). By contrast, duct cell NBC3 transported both OH(-) and HCO3-. Accordingly, reverse transcription-polymerase chain reaction analysis revealed that both cell types expressed mRNA for pNBC1. However, the acini expressed mRNA for the NBC3 splice variants NBCn1C and NBCn1D, whereas the ducts expressed mRNA for NCBn1B. Based on these findings we propose that the luminal NBCs in the HCO3- secreting SMG acinar and duct cells function as HCO3- salvage mechanisms at the resting state. These studies emphasize the complexity but also begin to clarify the mechanism of HCO3- homeostasis in secretory epithelia.

Genomic organization of the DCTN1-SLC4A5 locus encoding both NBC4 and p150(Glued).

In eukaryotes, it is rare for a single gene to encode two functionally unrelated proteins. p150(Glued) is a component of the dynactin heteromultimeric complex of proteins which is required for dynein-mediated vesicle and organelle transport by microtubules. NBC4 is an electrogenic sodium bicarbonate cotransporter, which regulates intracellular pH. Here we report that NBC4 and p150(Glued) are encoded by the same locus, DCTN1-SLC4A5. We have characterized the genomic organization of the human DCTN1-SLC4A5 locus which spans approximately 230 kilobases on chromosome 2p13 and contains 66 exons. This information should allow the study of potential genomic alterations of DCTN1-SLC4A5 in patients with diseases mapping to this genomic region.

Two C-terminal variants of NBC4, a new member of the sodium bicarbonate cotransporter family: cloning, characterization, and localization.

We report the cloning, characterization, and chromosomal assignment of a new member of the sodium bicarbonate cotransporter (NBC) family, NBC4. The NBC4 gene was mapped to chromosome 2p13 and is a new candidate gene for Alstrom syndrome. Two variants of the transporter have been isolated from human testis and heart, which differ in their C termini. NBC4a encodes a 1137-residue polypeptide and is widely expressed in various tissues, including liver, testis, and spleen. NBC4b is identical to NBC4a except that it has a 16-nucleotide insert, creating a C-terminal frame shift. NBC4b encodes a 1074-residue polypeptide and is highly expressed in heart. Amino acids 1-1046 are common to both NBC4 variants. NBC4a has two protein-interacting domains that are lacking in NBC4b: a proline-rich sequence, PPPSVIKIP (amino acids 1102-1110), and a consensus PDZ-interacting domain, SYSL (1134-1137). NBC4b lacks the stretch of charged residues present in the C terminus of NBC4a and other members of the NBC family. Unlike other members of the NBC family, both NBC4a and NBC4b have a unique glycine-rich region (amino acids 440-469). In comparison with other members of the bicarbonate transport superfamily, NBC4a and NBC4b are most similar structurally to the electrogenic sodium bicarbonate cotransporters (NBC1).

Immunolocalization of NBC3 and NHE3 in the rat epididymis: colocalization of NBC3 and the vacuolar H+-ATPase.

In the male reproductive tract, the epididymis plays an important role in mediating transepithelial bicarbonate transport and luminal acidification. In the proximal vas deferens, a significant component of luminal acidification is Na+-independent, and mediated by specific cells that possess apical vacuolar proton pumps. In contrast, luminal acidification in the cauda epididymidis is an Na+-dependent process. The specific apical Na+-dependent H+/base transport process(es) responsible for luminal acidification have not been identified. A potential clue as to the identity of these apical Na+-dependent H+/base transporter(s) is provided by similarities between the transport properties of the epididymis and the mammalian nephron. Specifically, the H+/base transport properties of caput epididymidis resemble the mammalian renal proximal tubule, whereas the distal epididymis and vas deferens have characteristics in common with renal collecting duct intercalated cells. Given the known expression of the Na+/H+ antiporter, NHE3, in the proximal tubule, and of the electroneutral sodium bicarbonate cotransporter, NBC3, in renal intercalated cells, we determined the localization of NHE3 and NBC3 in various regions of rat epididymis. NBC3 was highly expressed on the apical membrane of apical (narrow) cells in caput epididymidis, and light (clear) cells in corpus and cauda epididymidis. The number of cells expressing apical NBC3 was highest in cauda epididymidis. The localization of NBC3 in the epididymis was identical to the vacuolar H+-ATPase. The results indicate that colocalization of NBC3 and the vacuolar H+-ATPase is not restricted to kidney intercalated cells. Moreover, the close association of the two transporters appears to be a more generalized phenomenon in cells that express high levels of vacuolar H+-ATPase. Unlike NBC3, NHE3 was most highly expressed on the apical membrane of all epithelial cells in caput epididymidis, with less expression in the corpus, and no expression in the cauda. These results suggest that apical NBC3 and NHE3 potentially play an important role in mediating luminal H+/base transport in epididymis.

Cloning, characterization and chromosomal assignment of NBC4, a new member of the sodium bicarbonate cotransporter family.

We report the cloning, characterization and chromosomal assignment of a new member of the sodium bicarbonate cotransporter (NBC) family, NBC4, from human heart. NBC4 maps to chromosome 2p13 and is a new candidate gene for Alstrom syndrome. NBC4 encodes a 1074-residue polypeptide with 12 putative membrane-spanning domains. Unlike other members of the NBC family, NBC4 has a unique glycine-rich region (amino acids 438-485). In addition, NBC4 lacks the lysine-rich C-terminus of NBC1 with which it is most homologous. The first of two putative stilbene binding motifs (K(M/L)(X)K) is lacking in NBC4 (amino acids 655-658). The approximately 6 kb NBC4 transcript is moderately expressed in heart, with the highest expression in liver, testes and spleen.

Structural organization of the human NBC1 gene: kNBC1 is transcribed from an alternative promoter in intron 3.

Several electrogenic sodium bicarbonate cotransporters have been cloned from different human organs. In the renal proximal tubule, the electrogenic sodium bicarbonate cotransporter kNBC1 (1035aa) mediates the majority of basolateral sodium bicarbonate absorption. In pancreatic ducts, the electrogenic sodium bicarbonate cotransporter pNBC1 (1079aa) mediates basolateral sodium bicarbonate influx. hNBC1 (hhNBC), cloned from human heart, is identical to pNBC1 at the amino acid level. We have demonstrated that kNBC1 and pNBC1 are highly homologous proteins that have different N-termini. In kNBC1, 41 amino acids replace the initial 85 amino acids of pNBC1. Whether these proteins are coded by one or more genes is unknown. In order to determine the genetic basis for these transcripts, we first characterized the genomic organization of the NBC1 gene (SLC4A4). NBC1 spans approximately 450 kilobases containing 26 exons that are flanked by typical splice donor and acceptor sequences at the intron-exon boundaries. Exon 1 is specific for the pNBC1 transcript. The first alternative exon of the hNBC1 transcript, containing the 5'-untranslated region, is derived from the last 43 nucleotides of intron 1 in the NBC1 gene coupled to exon 2. kNBC1 is transcribed from an alternative promoter in intron 3. In the first alternative exon of kNBC1, the last 313 nucleotides of intron 3 are coupled to exon 4, which is common to pNBC1 and hNBC1. The major transcription initiation site in kNBC1 is located 192 nucleotides upstream from the translation initiation codon. A minor start site is located 182 nucleotides upstream from the translation initiation codon. Structural analysis of the proximal kNBC1 promoter revealed an atypical TATA sequence (-33) and several potentially important transcription factor binding sites. Functional studies showed that the 5'-flanking region of the alternative kNBC1 promoter (-159 to+43) is sufficient for promoter activity. This work is the first demonstration that the three N-terminal transcripts of the human electrogenic sodium bicarbonate cotransporter NBC1 are encoded by the SLC4A4 gene. Furthermore, knowledge of the genomic organization and alternative promoter usage in the NBC1 gene provides a molecular basis for understanding disorders involving electrogenic sodium bicarbonate cotransporters and facilitates the elucidation of transcriptional control of NBC1 expression.

Immunoelectron microscopic localization of NBC3 sodium-bicarbonate cotransporter in rat kidney.

In the present study, we produced a rabbit peptide-derived polyclonal COOH-terminal antibody that selectively recognizes NBC3, to determine the cellular and subcellular localization of NBC3 in rat kidney, using immunocytochemistry and immunoelectron microscopy. Immunocytochemistry with cryostat sections and semithin cryosections revealed specific staining of intercalated cells (ICs) in the connecting tubule and in cortical, outer medullary, and initial inner medullary collecting ducts. In the connecting tubule and in the cortical and medullary collecting duct, the labeling was associated with both type A and type B ICs. In type A ICs, labeling was confined to the apical and subapical domains, whereas in type B ICs, basal domains were exclusively labeled. In contrast, collecting duct principal cells were consistently unlabeled, and this was confirmed using anti-aquaporin-2 antibodies, which labeled principal cells in parallel semithin cryosections. Glomeruli, proximal tubules, descending thin limbs, ascending thin limbs, thick ascending limbs, distal convoluted tubules, and vascular structures were unlabeled. For immunoelectron microscopy, tissue samples were freeze-substituted, and immunolabeling was performed on ultrathin Lowicryl HM20 sections. Immunoelectron microscopy demonstrated that NBC3 labeling was very abundant in the apical plasma membrane, in intracellular vesicles, and in tubulocisternal profiles in the subapical domains of type A ICs. In type B ICs, NBC3 was mainly present in the basolateral plasma membrane. Immunolabeling controls using peptide-absorbed antibody were consistently negative. In conclusion, NBC3 is highly abundant in the apical plasma membrane of type A ICs and in the basolateral plasma membrane of type B ICs. This suggests that NBC3 plays an important role in modulating bicarbonate transport in the connecting tubule and collecting duct.

Oligomeric structure of bAE3 protein.

The "brain" form of the anion exchanger protein 3 (bAE3) has been purified to homogeneity from the rabbit kidney by differential centrifugation and immunoaffinity chromatography. A single protein band of approximately 165 kDa was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Monomers, dimers (a major component), and a higher oligomeric form (apparently tetramers) were found after oxidative cross-linking of purified bAE3. The largest form of bAE3 was separated from dimers and monomers by sucrose gradient centrifugation and was studied by transmission electron microscopy to confirm a tetrameric structure. Two main types of bAE3 images were detected, round (approximately 11-14 nm) and square-shaped (approximately 12 x 12 nm). Image analysis revealed fourfold rotational symmetry of both the round and square-shaped images, indicating that bAE3 consists of multiples of 4 subunits. We conclude that bAE3 in Triton X-100 solution is predominantly a mixture of dimers and tetramers with a smaller amount of monomers.