We report the cloning, sequence analysis, tissue distribution, functional expression, and chromosomal localization of the human pancreatic sodium bicarbonate cotransport protein (pancreatic NBC (pNBC)). The transporter was identified by searching the human expressed sequence tag data base. An I.M.A.G.E. clone W39298 was identified, and a polymerase chain reaction probe was generated to screen a human pancreas cDNA library. pNBC encodes a 1079-residue polypeptide that differs at the N terminus from the recently cloned human sodium bicarbonate cotransporter isolated from kidney (kNBC) (Burnham, C. E., Amlal, H., Wang, Z., Shull, G. E., and Soleimani, M. (1997) J. Biol. Chem. 272, 19111-19114). Northern blot analysis using a probe specific for the N terminus of pNBC revealed an ϳ7.7-kilobase transcript expressed predominantly in pancreas, with less expression in kidney, brain, liver, prostate, colon, stomach, thyroid, and spinal chord. In contrast, a probe to the unique 5 region of kNBC detected an ϳ7.6-kilobase transcript only in the kidney. In situ hybridization studies in pancreas revealed expression in the acini and ductal cells. The gene was mapped to chromosome 4q21 using fluorescent in situ hybridization. Expression of pNBC in Xenopus laevis oocytes induced sodium bicarbonate cotransport. These data demonstrate that pNBC encodes the sodium bicarbonate cotransporter in the mammalian pancreas. pNBC is also expressed at a lower level in several other organs, whereas kNBC is expressed uniquely in kidney.

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 ϳ 7.8-kilobase transcript is expressed uniquely in skeletal muscle and heart. The NBC3 gene (SLC4A7) spans ϳ80 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.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 (pH i ) 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 CO 2 . 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 ϩ /H ϩ exchange (1-3, 9

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Na+-coupled acid–base transporters play essential roles in human biology. Their dysfunction has been linked to cancer, heart, and brain disease. High-resolution structures of mammalian Na+-coupled acid–base transporters are not available. The sodium-bicarbonate cotransporter NBCe1 functions in multiple organs and its mutations cause blindness, abnormal growth and blood chemistry, migraines, and impaired cognitive function. Here, we have determined the structure of the membrane domain dimer of human NBCe1 at 3.9 Å resolution by cryo electron microscopy. Our atomic model and functional mutagenesis revealed the ion accessibility pathway and the ion coordination site, the latter containing residues involved in human disease-causing mutations. We identified a small number of residues within the ion coordination site whose modification transformed NBCe1 into an anion exchanger. Our data suggest that symporters and exchangers utilize comparable transport machinery and that subtle differences in their substrate-binding regions have very significant effects on their transport mode.

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.

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. 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. 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. 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 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.

Normal sensory transduction requires the efficient disposal of acid (H+) generated by neuronal and sensory receptor activity. Multiple highly sensitive transport mechanisms have evolved in prokaryotic and eukaryotic organisms to maintain acidity within strict limits. It is currently assumed that the multiplicity of these processes provides a biological robustness. Here we report that the visual and auditory systems have a specific requirement for H+ disposal mediated by the sodium bicarbonate cotransporter NBC3 (refs. 7,8). Mice lacking NBC3 develop blindness and auditory impairment because of degeneration of sensory receptors in the eye and inner ear as in Usher syndrome. Our results indicate that in certain sensory organs, in which the requirement to transduce specific environmental signals with speed, sensitivity and reliability is paramount, the choice of the H+ disposal mechanism used is limited.