Alexander Pushkin

Molecular Cloning, Chromosomal Localization, Tissue Distribution, and Functional Expression of the Human Pancreatic Sodium Bicarbonate Cotransporter

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 ∼7.7-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 ∼7.6-kilobase transcript only in the kidney. In situhybridization studies in pancreas revealed expression in the acini and ductal cells. The gene was mapped to chromosome 4q21 using fluorescentin 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.

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

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 inXenopus laevis oocytes demonstrated that the protein encodes a novel stilbene-insensitive 5-(N-ethyl-N-isopropyl)-amiloride-inhibitable sodium bicarbonate cotransporter.

Blindness and auditory impairment caused by loss of the sodium bicarbonate cotransporter NBC3

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.

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.

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.

Regulation of the sodium bicarbonate cotransporter knbc1 function: role of Asp986, Asp988 and kNBC1‐carbonic anhydrase II binding

The HCO3− : Na+ cotransport stoichiometry of the electrogenic sodium bicarbonate cotransporter kNBC1 determines the reversal potential (Erev) and thus the net direction of transport of these ions through the cotransporter. Previously, we showed that phosphorylation of kNBC1‐Ser982 in the carboxy‐terminus of kNBC1 (kNBC1‐Ct), by cAMP‐protein kinase A (PKA), shifts the stoichiometry from 3 : 1 to 2 : 1 and that binding of bicarbonate to the cotransporter is electrostaticaly modulated. These results raise the possibility that phosphorylated kNBC1‐Ser982, or other nearby negatively charged residues shift the stoichiometry by blocking a bicarbonate‐binding site. In the current study, we examined the role of the negative charge on Ser982‐phosphate and three aspartate residues in a D986NDD custer in altering the stoichiometry of kNBC1. mPCT cells expressing kNBC1 mutants were grown on filters and mounted in an Ussing chamber for electrophysiological studies. Enhanced green fluorescence protein (EGFP)‐tagged mutant constructs expressed in the same cells were used to determine the phosphorylation status of kNBC1‐Ser982. The data indicate that both kNBC1‐Asp986 and kNBC1‐Asp988, but not kNBC1‐Asp989, are required for the phosphorylation‐induced shift in stoichiometry. A homologous motif (D887ADD) in the carboxy‐terminus of the anion exchanger AE1 binds to carbonic anhydrase II (CAII). In isothermal titration calorimetry experiments, CAII was found to bind to kNBC1‐Ct with a KD of 160 ± 10 nm. Acetazolamide inhibited the short‐circuit current through the cotransporter by 65 % when the latter operated in the 3 : 1 mode, but had no effect on the current in the 2 : 1 mode. Acetazolamide did not affect the cotransport stoichiometry or the ability of 8‐Br‐cAMP to shift the stoichiometry. Although CAII does not affect the transport stoichiometry, it may play an important role in enhancing the flux through the transporter when kNBC1‐Ser982 is unphosphorylated.

Molecular mechanism of kNBC1–carbonic anhydrase II interaction in proximal tubule cells

We have recently shown that carbonic anhydrase II (CAII) binds in vitro to the C‐terminus of the electrogenic sodium bicarbonate cotransporter kNBC1 (kNBC1‐ct). In the present study we determined the molecular mechanisms for the interaction between the two proteins and whether kNBC1 and CAII form a transport metabolon in vivo wherein bicarbonate is transferred from CAII directly to the cotransporter. Various residues in the C‐terminus of kNBC1 were mutated and the effect of these mutations on both the magnitude of CAII binding and the function of kNBC1 expressed in mPCT cells was determined. Two clusters of acidic amino acids, L958DDV and D986NDD in the wild‐type kNBC1‐ct involved in CAII binding were identified. In both acidic clusters, the first aspartate residue played a more important role in CAII binding than others. A significant correlation between the magnitude of CAII binding and kNBC1‐mediated flux was shown. The results indicated that CAII activity enhances flux through the cotransporter when the enzyme is bound to kNBC1. These data are the first direct evidence that a complex of an electrogenic sodium bicarbonate cotransporter with CAII functions as a transport metabolon.

Phosphorylation of Ser982 in the sodium bicarbonate cotransporter kNBC1 shifts the HCO3− : 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 HCO3− 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 HCO3− : 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 agonsit 8‐Br‐cAMP caused the stoichiometry of kNBC1 to shift from 3 HCO3− : 1 Na+ to 2 HCO3− : 1 Na+. Pretreatment of the cells with the PKA inhibitor H‐89 abolished the effect of the agonist on the stoichiometry change. Replacing Ser982 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. Ser982 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.

The stoichiometry of the electrogenic sodium bicarbonate cotransporter pNBC1 in mouse pancreatic duct cells is 2 HCO3−:1 Na+

1 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 cells membrane potential, or equivalently that the HCO3−:Na+ stoichiometry is not larger then 2:1. However, there are no data available on either the reversal potential or the HCO3−:Na+ stoichiometry of pNBC1 in pancreatic cells. 2 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. 3 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). 4 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. 5 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.

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.