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Homozygous mutations in SLC4A4 , which encodes the electrogenic Na + /[Formula: see text] cotransporter (NBCe1), cause proximal renal tubular acidosis associated with extrarenal symptoms. Although 17` mutated sites in SLC4A4 have thus far been identified among patients with proximal renal tubular acidosis, the physiological significance of other nonsynonymous single-nucleotide variants (SNVs) remains largely undetermined. Here, we investigated the functional properties of SNVs in NBCe1. From the National Center for Biotechnology Information dbSNP database, we identified 13 SNVs that have not previously been characterized in the highly conserved, transmembrane domains of NBCe1-A. Immunocytochemical analysis revealed that the I551F variant was present predominantly in the cytoplasm in human embryonic kidney (HEK)-293 cells, whereas all other SNVs did not show as dramatic a change in subcellular distribution. Western blot analysis in HEK-293 cells demonstrated that the I551F variant showed impaired glycosylation and a 69% reduction in cell surface levels. To determine the role of I551 in more detail, we examined the significance of various artificial mutants in both nonpolarized HEK-293 cells and polarized Madin-Darby canine kidney cells, which indicated that only I551F substitution resulted in cytoplasmic retention. Moreover, functional analysis using Xenopus oocytes demonstrated that the I551F variant had a significantly reduced activity corresponding to 39% of that of the wild-type, whereas any other SNVs and artificial I551 mutants did not show significant changes in activity. Finally, immunofluorescence experiments in HEK-293 cells indicated that the I551F variant retained wild-type NBCe1-A in the cytoplasm. These data demonstrate that the I551F variant of NBCe1-A shows impaired transport activity predominantly through cytoplasmic retention and suggest that the variant can have a dominant negative effect by forming complexes with wild-type NBCe1-A. NEW & NOTEWORTHY Electrogenic Na + /[Formula: see text] cotransporter 1-A (NBCe1-A) in the proximal tubule regulates the acid/base balance and fluid volume homeostasis. From the National Center for Biotechnology Information dbSNP database, we identified the I551F variant of NBCe1-A, which showed reduced glycosylation, cell surface expression, and transport activity. We also found that the I551F variant can exert a dominant negative effect on wild-type NBCe1-A, suggesting its physiological significance.

Mutations in Na(+)-HCO(3)(-) cotransporter (NBC-1) cause proximal renal tubular acidosis (pRTA) associated with ocular abnormalities. One pRTA patient had increased serum amylase, suggesting possible evidence of pancreatitis. To further delineate a link between NBC-1 inactivation and pancreatic dysfunction, immunohistochemical analysis was performed on rat and human pancreas using antibodies against kidney-type (kNBC-1) and pancreatic-type (pNBC-1) transporters. In rat pancreas, the anti-pNBC-1 antibody labeled acinar cells and both apical and basolateral membranes of medium and large duct cells. In human pancreas, on the other hand, the anti-pNBC-1 antibody did not label acinar cells, although it did label the basolateral membranes of the entire duct system. The labeling by anti-kNBC-1 antibody was detected in only a limited number of rat pancreatic duct cells. To examine the effects of pRTA-related mutations, R342S and R554H, on pNBC-1 function, we performed functional analysis and found that both mutants had reduced transport activities compared with the wild-type pNBC-1. These results indicate that pNBC-1 is the predominant variant that mediates basolateral HCO(3)(-) uptake into duct cells in both rat and human pancreas. The loss of pNBC-1 function is predicted to have significant impact on overall ductal HCO(3)(-) secretion, which could potentially lead to pancreatic dysfunction.

Conventional and double-barreled microelectrodes were used to examine the anion selectivity of Cl- conductance in the basolateral membrane of rabbit renal proximal tubule S3 segment. The permeability sequence determined by anion replacements in the presence of K+ channel blocker quinine was SCN- > I- > Br- > Cl- > gluconate in both nonperfused and luminally perfused tubules. The anion-selective microelectrodes with higher sensitivity to I- enabled us to measure intracellular I- activities. With these electrodes, we could compare the conductive movements of Cl- and I- in response to the increase in bath K+ concentrations and confirmed that the conductance sequence was also I- > Cl-. Although the basolateral potential changes generated by Cl- and Br- currents were stimulated by adenosine 3',5'-cyclic monophosphate (cAMP), the potential changes by SCN- and I- currents were somewhat inhibited by cAMP. In addition, the conductive uptake of I- was, in contrast to that of Cl-, inhibited by cAMP These results are consistent with the existence of at least two distinct anion conductances in this membrane, which are differently regulated by cAMP.

Roles of Ca2+ and protein kinase C (PKC) in the regulation of acid/base transport in isolated rabbit proximal tubules were investigated by measuring cytosolic Ca2+ concentrations ([Ca2+]i) and cell pH (pHi) with fluorescent probes. Ionomycin (0.2 microM) increased [Ca2+]i by approximately 200 nM but did not affect the basolateral Na(+)-HCO3- cotransporter. However, the apical Na+/H+ exchanger was inhibited by 50% by ionomycin, and this inhibition was abolished either by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, an intracellular Ca2+ chelator, or by KN-62, an inhibitor of calcium-calmodulin-dependent protein kinase II (CaM kinase II). On the other hand, phorbol 12-myristate 13-acetate (PMA, 0.5 microM) did not affect the apical Na+/H+ exchanger but did stimulate the basolateral Na(+)-HCO3- cotransporter by 60-80%, and this stimulation was prevented by calphostin C, an inhibitor of PKC. Consistent with the cotransporter stimulation, PMA decreased steady-state pHi in the presence of CO2/ HCO3-. These results indicate that 1) the acute increase in [Ca2+]i within physiological ranges inhibits the apical Na+/H+ exchanger, probably through mediation of CaM kinase II; and 2) the short-term PKC activation stimulates the basolateral Na(+)-HCO3- cotransporter.

The effects of extracellular ATP on cytosolic Ca2+ concentrations ([Ca2+]i) and cell membrane potentials (Vb) of rabbit renal proximal tubules were investigated using fura 2 and microelectrodes. ATP transiently increased [Ca2+]i without an apparent sustained phase, and the maximum effect was obtained at 10 microM. ADP, adenosine 5'-O-(3-thiotriphosphate), and 2-methylthioadenosine 5'-triphosphate were equally effective as ATP, whereas UTP, adenosine, and alpha beta-methyleneadenosine 5'-triphosphate were far less effective. The [Ca2+]i responses to ATP were strongly inhibited by reactive blue 2, a P2-purinergic receptor antagonist. The removal of extracellular Ca2+ as well as the addition of thapsigargin also markedly attenuated the responses to ATP. In addition, ATP had virtually no effect on Vb, except for the occasional small depolarization by 300 microM ATP. These results indicate that extracellular ATP increases [Ca2+]i through a P2-purinergic receptor and that this effect of ATP is dependent on both extracellular and intracellular Ca2+.

mRNA of the EP3 prostaglandin E2 (PGE2) receptor was detected and quantitated in microdissected mouse nephron segments by a modified protocol of reverse transcription-polymerase chain reaction (RT-PCR). At the step of RT, a point mutation was introduced in cDNA, which made a new restriction site for the Mbo I enzyme. PCR was performed using a set of primers on the same exon, and genomic DNA was coamplified with cDNA by these primers. PCR products were treated with Mbo I, and signals from genomic DNA and mRNA were separately detected on gel electrophoresis. The relative amount of mRNA per cell was expressed as a ratio of amount of product from mRNA to that from genomic DNA. EP3 PGE2 receptor mRNA expression was abundant in thick ascending limb of Henle, present to a lesser extent in distal convoluted tubules and collecting ducts, and undetectable in glomeruli, proximal tubules, and descending thin limb. The results support the notion that PGE2 modulates water and solute transport through the EP3 receptor in specific structures of the kidney.