The renal electrogenic Na+:HCO-3 cotransporter.

1997 ◽  
Vol 200 (2) ◽  
pp. 263-268 ◽  
Author(s):  
V F Boron ◽  
M A Hediger ◽  
E L Boulpaep ◽  
M F Romero
Keyword(s):  
Amino Acids ◽  
Cdna Library ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Full Length ◽  
Xenopus Laevis Oocytes ◽  
New Family ◽  
All Solutions ◽  
Single Clone ◽  
Membrane Spanning

The electrogenic Na+:HCO3- cotransporter (symporter) is the major transporter for HCO3- reabsorption across the basolateral membrane of the renal proximal tubule and also contributes significantly to Na+ reabsorption. We expression-cloned the salamander renal electrogenic Na+:Bicarbonate Cotransporter (NBC) in Xenopus laevis oocytes. After injecting poly(A)+ RNA, fractionated poly(A)+ RNA or cRNA, we used microelectrodes to monitor membrane potential (Vm) and intracellular pH (pHi) All solutions contained ouabain to block the Na+/K+ pump (P-ATPase). After applying 1.5% CO2/10 mmol l-1 HCO3- (pH 7.5) and allowing pHi to stabilize from the CO2-induced acidification, we removed Na+. In native oocytes or water-injected controls, removing Na+ hyperpolarized the cell by -5 mV and had no effect on pHi. In oocytes injected with poly(A)+ RNA, removing Na+ transiently depolarized the cell by -10 mV and caused pHi to decrease; both effects were blocked by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) and required HCO3-. We enriched the signal by electrophoretic fractionation of the poly(A)+ RNA, and constructed a size-selected cDNA library in pSPORT1 using the optimal fraction. Screening the Ambystoma library yielded a single clone (aNBC). Expression was first obvious 3 days after injection of NBC cRNA. Adding CO2/HCO3- induced a large (> 50 mV) and rapid hyperpolarization, followed by a partial relaxation as pHi stabilized. Subsequent Na+ removal depolarized the cell by more than 40 mV and decreased pHi. aNBC is a full-length clone with a start Met and a poly(A)+ tail; it encodes a protein with 1025 amino acids and several putative membrane-spanning domains. aNBC is the first member of a new family of Na(+)-linked HCO3- transporters. We used aNBC to screen a rat kidney cDNA library, and identified a full-length cDNA clone (rNBC) that encodes a protein of 1035 amino acids. rNBC is 86% identical to aNBC and can be functionally expressed in oocytes.

scholarly journals Immunolocalization of the electrogenic Na+- HCO 3 − cotransporter in mammalian and amphibian kidney

1999 ◽  
Vol 276 (1) ◽  
pp. F27-F38 ◽  
Author(s):  
Bernhard M. Schmitt ◽  
Daniel Biemesderfer ◽  
Michael F. Romero ◽  
Emile L. Boulpaep ◽  
Walter F. Boron
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Indirect Immunofluorescence ◽  
Immunofluorescence Microscopy ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Distal Tubule ◽  
Xenopus Laevis Oocytes ◽  
Major Band ◽  
Indirect Immunofluorescence Microscopy

Electrogenic cotransport of Na+ and[Formula: see text] is a crucial element of[Formula: see text] reabsorption in the renal proximal tubule (PT). An electrogenic Na+-[Formula: see text]cotransporter (NBC) has recently been cloned from salamander and rat kidney. In the present study, we generated polyclonal antibodies (pAbs) to NBC and used them to characterize NBC on the protein level by immunochemical methods. We generated pAbs in guinea pigs and rabbits by immunizing with a fusion protein containing the carboxy-terminal 108 amino acids (amino acids 928–1035) of rat kidney NBC (rkNBC). By indirect immunofluorescence microscopy, the pAbs strongly labeled HEK-293 cells transiently expressing NBC, but not in untransfected cells. By immunoblotting, the pAbs recognized a ∼130-kDa band in Xenopus laevis oocytes expressing rkNBC, but not in control oocytes injected with water or cRNA for the Cl−/[Formula: see text]exchanger AE2. In immunoblotting experiments on renal microsomes, the pAbs specifically labeled a major band at ∼130 kDa in both rat and rabbit, as well as a single ∼160-kDa band in salamander kidney. By indirect immunofluorescence microscopy on 0.5-μm cryosections of rat and rabbit kidneys fixed in paraformaldehyde-lysine-periodate (PLP), the pAbs produced a strong and exclusively basolateral staining of the PT. In the salamander kidney, the pAbs labeled only weakly the basolateral membrane of the PT. In contrast, we observed strong basolateral labeling in the late distal tubule, but not in the early distal tubule. The specificity of the pAbs for both immunoblotting and immunohistochemistry was confirmed in antibody preabsorption experiments using either the fusion protein used for immunization or similarly prepared control fusion proteins. In summary, we have developed antibodies specific for NBC, determined the apparent molecular weights of rat, rabbit, and salamander kidney NBC proteins, and described the localization of NBC within the kidney of these mammalian and amphibian species.

Interaction of the Ca2+-sensing receptor with the inwardly rectifying potassium channels Kir4.1 and Kir4.2 results in inhibition of channel function

2007 ◽  
Vol 292 (3) ◽  
pp. F1073-F1081 ◽  
Author(s):  
Chunfa Huang ◽  
Aleksandra Sindic ◽  
Ceredwyn E. Hill ◽  
Kristine M. Hujer ◽  
Kim W. Chan ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Basolateral Membrane ◽  
Xenopus Laevis Oocytes ◽  
Hek 293 ◽  
Salt Wasting ◽  
Novel Proteins ◽  
293 Cells ◽  
Adult Kidney ◽  
Cell Current ◽  
Inwardly Rectifying

The Ca2+-sensing receptor (CaR), a G protein-coupled receptor, is expressed in many epithelial tissues including the parathyroid glands, kidney, and GI tract. Although its role in regulating PTH levels and Ca2+ metabolism are best characterized, it may also regulate salt and water transport in the kidney as demonstrated by recent reports showing association of potent gain-of-function mutations in the CaR with a Bartter-like, salt-wasting phenotype. To determine whether this receptor interacts with novel proteins that control ion transport, we screened a human adult kidney cDNA library with the COOH-terminal 219 amino acid cytoplasmic tail of the CaR as bait using the yeast two-hybrid system. We identified two independent clones coding for ∼125 aa from the COOH terminus of the inwardly rectifying K+ channel, Kir4.2. The CaR and Kir4.2 as well as Kir4.1 (another member of Kir4 subfamily) were reciprocally coimmunoprecipitated from HEK-293 cells in which they were expressed, but the receptor did not coimmunoprecipitate with Kir5.1 or Kir1.1. Both Kir4.1 and Kir4.2 were immunoprecipitated from rat kidney extracts with the CaR. In Xenopus laevis oocytes, expression of the CaR with either Kir4.1 or Kir4.2 channels resulted in inactivation of whole cell current as measured by two-electrode voltage clamp, but the nonfunctional CaR mutant CaRR796W, and that does not coimmunoprecipitate with the channels, had no effect. Kir4.1 and the CaR were colocalized in the basolateral membrane of the distal nephron. The CaR interacts directly with Kir4.1 and Kir4.2 and can decrease their currents, which in turn could reduce recycling of K+ for the basolateral Na+-K+-ATPase and thereby contribute to inhibition of Na+ reabsorption.

scholarly journals The heterodimeric amino acid transporter 4F2hc/y+LAT2 mediates arginine efflux in exchange with glutamine

2000 ◽  
Vol 349 (3) ◽  
pp. 787-795 ◽  
Author(s):  
Angelika BRÖER ◽  
Carsten A. WAGNER ◽  
Florian LANG ◽  
Stefan BRÖER
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Mammalian Cells ◽  
Substrate Inhibition ◽  
Cell Types ◽  
Xenopus Laevis Oocytes ◽  
Cationic Amino Acid ◽  
New Family ◽  
Arginine Uptake ◽  
Intracellular Binding

The cationic amino acid arginine, due to its positive charge, is usually accumulated in the cytosol. Nevertheless, arginine has to be released by a number of cell types, e.g. kidney cells, which supply other organs with this amino acid, or the endothelial cells of the blood–brain barrier which release arginine into the brain. Arginine release in mammalian cells can be mediated by two different transporters, y+LAT1 and y+LAT2. For insertion into the plasma membrane, these transporters have to be associated with the type-II membrane glycoprotein 4F2hc [Torrents, Estevez, Pineda, Fernandez, Lloberas, Shi, Zorzano and Palacin (1998) J. Biol. Chem. 273, 32437–32445]. The present study elucidates the function and distribution of y+LAT2. In contrast to y+LAT1, which is expressed mainly in kidney epithelial cells, lung and leucocytes, y+LAT2 has a wider tissue distribution, including brain, heart, testis, kidney, small intestine and parotis. When co-expressed with 4F2hc in Xenopus laevis oocytes, y+LAT2 mediated uptake of arginine, leucine and glutamine. Arginine uptake was inhibited strongly by lysine, glutamate, leucine, glutamine, methionine and histidine. Mutual inhibition was observed when leucine or glutamine was used as substrate. Inhibition of arginine uptake by neutral amino acids depended on the presence of Na+, which is a hallmark of y+LAT-type transporters. Although arginine transport was inhibited strongly by glutamate, this anionic amino acid was only weakly transported by 4F2hc/y+LAT2. Amino acid transport via 4F2hc/y+LAT2 followed an antiport mechanism similar to the other members of this new family. Only preloaded arginine could be released in exchange for extracellular amino acids, whereas marginal release of glutamine or leucine was observed under identical conditions. These results indicated that arginine has the highest affinity for the intracellular binding site and that arginine release may be the main physiological function of this transporter.

Molecular characterization of a novel urea transporter from kidney inner medullary collecting ducts

2001 ◽  
Vol 280 (3) ◽  
pp. F487-F494 ◽  
Author(s):  
Chairat Shayakul ◽  
Hiroyasu Tsukaguchi ◽  
Urs V. Berger ◽  
Matthias A. Hediger
Keyword(s):  
Amino Acid ◽  
Molecular Characterization ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Functional Characterization ◽  
Xenopus Laevis Oocytes ◽  
Urea Transporter ◽  
Urea Uptake

In the terminal part of the kidney collecting duct, rapid urea reabsorption is essential to maintaining medullary hypertonicity, allowing maximal urinary concentration to occur. This process is mediated by facilitated urea transporters on both apical and basolateral membranes. Our previous studies have identified three rat urea transporters involved in the urinary concentrating mechanism, UT1, UT2 and UT3 , herein renamed UrT1-A, UrT1-B, and UrT2, which exhibit distinct spatial distribution in the kidney. Here we report the molecular characterization of an additional urea transporter isoform, UrT1-C, from rat kidney that encodes a 460-amino acid residue protein. UrT1-C has 70 and 62% amino acid identity to rat UrT1-B and UrT2 (UT3), respectively, and 99% identity to a recently reported rat isoform (UT-A3; Karakashian A, Timmer RT, Klein JD, Gunn RB, Sands JM, and Bagnasco SM. J Am Soc Nephrol 10: 230–237, 1999). We report the anatomic distribution of UrT1-C in the rat kidney tubule system as well as a detailed functional characterization. UrT1-C m RNA is primarily expressed in the deep part of the inner medulla. When expressed in Xenopus laevis oocytes, UrT1-C induced a 15-fold stimulation of urea uptake, which was inhibited almost completely by phloretin (0.7 mM) and 60–95% by thiourea analogs (150 mM). The characteristics are consistent with those described in perfusion studies with inner medullary collecting duct (IMCD) segments, but, contrary to UrT1-A, UrT1-C-mediated urea uptake was not stimulated by activation of protein kinase A. Our data show that UrT1-C is a phloretin-inhibitable urea transporter expressed in the terminal collecting duct that likely serves as an exit mechanism for urea at the basolateral membrane of IMCD cells.

scholarly journals The arachidonate-activatable, NADPH oxidase-associated H+ channel is contained within the multi-membrane-spanning N-terminal region of gp91-phox

1997 ◽  
Vol 325 (3) ◽  
pp. 701-705 ◽  
Author(s):  
Lydia M. HENDERSON ◽  
Stephen THOMAS ◽  
George BANTING ◽  
J. Brian CHAPPELL
Keyword(s):  
Amino Acids ◽  
Nadph Oxidase ◽  
Cell Line ◽  
Proton Motive Force ◽  
Transmembrane Domains ◽  
Terminal Region ◽  
Full Length ◽  
Protein Component ◽  
Cho Cell ◽  
Membrane Spanning

The generation of superoxide by the NADPH oxidase of neutrophils is accompanied by the efflux of H+ ions through a H+ channel. gp91-phox, a protein component of the oxidase, has been shown previously to function as a H+ channel [Henderson, Banting and Chappell (1995) J. Biol. Chem. 270, 5909–5916]. We have constructed a CHO cell line (CHO-N) that expresses an N-terminal fragment of gp91-phox containing the predicted multiple transmembrane domains of the protein. These cells exhibit H+ fluxes in response to an imposed proton motive force and in the presence of arachidonate (to open the channel). The H+ fluxes were indistinguishable from those observed in cells expressing full-length gp91-phox. Therefore the N-terminal 230 amino acids of gp91-phox contain all that is required to function as the NADPH oxidase-associated H+ channel.

scholarly journals AMP-activated protein kinase inhibits KCNQ1 channels through regulation of the ubiquitin ligase Nedd4-2 in renal epithelial cells

2010 ◽  
Vol 299 (6) ◽  
pp. F1308-F1319 ◽  
Author(s):  
Rodrigo Alzamora ◽  
Fan Gong ◽  
Christine Rondanino ◽  
Jeffrey K. Lee ◽  
Christy Smolak ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Epithelial Cells ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Xenopus Laevis Oocytes ◽  
Ampk Activation ◽  
Channel Inhibition ◽  
Amp Activated Protein Kinase

The KCNQ1 K+ channel plays a key role in the regulation of several physiological functions, including cardiac excitability, cardiovascular tone, and body electrolyte homeostasis. The metabolic sensor AMP-activated protein kinase (AMPK) has been shown to regulate a growing number of ion transport proteins. To determine whether AMPK regulates KCNQ1, we studied the effects of AMPK activation on KCNQ1 currents in Xenopus laevis oocytes and collecting duct epithelial cells. AMPK activation decreased KCNQ1 currents and channel surface expression in X. laevis oocytes, but AMPK did not phosphorylate KCNQ1 in vitro, suggesting an indirect regulatory mechanism. As it has been recently shown that the ubiquitin-protein ligase Nedd4-2 inhibits KCNQ1 plasma membrane expression and that AMPK regulates epithelial Na+ channels via Nedd4-2, we examined the role of Nedd4-2 in the AMPK-dependent regulation of KCNQ1. Channel inhibition by AMPK was blocked in oocytes coexpressing either a dominant-negative or constitutively active Nedd4-2 mutant, or a Nedd4-2 interaction-deficient KCNQ1 mutant, suggesting that Nedd4-2 participates in the regulation of KCNQ1 by AMPK. KCNQ1 is expressed at the basolateral membrane in mouse polarized kidney cortical collecting duct (mpkCCDc14) cells and in rat kidney. Treatment with the AMPK activators AICAR (2 mM) or metformin (1 mM) reduced basolateral KCNQ1 currents in apically permeabilized polarized mpkCCDc14 cells. Moreover, AICAR treatment of rat kidney slices ex vivo induced AMPK activation and intracellular redistribution of KCNQ1 from the basolateral membrane in collecting duct principal cells. AICAR treatment also induced increased ubiquitination of KCNQ1 immunoprecipitated from kidney slice homogenates. These results indicate that AMPK inhibits KCNQ1 activity by promoting Nedd4-2-dependent channel ubiquitination and retrieval from the plasma membrane.

Establishment and Identification of a Normalized Full-Length cDNA Library of CCRI 36

2009 ◽  
Vol 35 (4) ◽  
pp. 602-607 ◽  
Author(s):  
Dong WU ◽  
Jun-Jie LIU ◽  
Shu-Xun YU ◽  
Shu-Li FAN ◽  
Mei-Zhen SONG
Keyword(s):  
Cdna Library ◽  
Full Length ◽  
Full Length Cdna

Immunolocalization of sat-1 sulfate/oxalate/bicarbonate anion exchanger in the rat kidney

1998 ◽  
Vol 275 (1) ◽  
pp. F79-F87 ◽  
Author(s):  
Lawrence P. Karniski ◽  
Marius Lötscher ◽  
Monica Fucentese ◽  
Helen Hilfiker ◽  
Jürg Biber ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Anion Exchanger ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Wild Type Virus ◽  
Affinity Column ◽  
Sf9 Cells ◽  
Wild Type ◽  
Metal Affinity ◽  
Sat 1

The rat liver sulfate/bicarbonate/oxalate exchanger (sat-1) transports sulfate across the canalicular membrane in exchange for either bicarbonate or oxalate. Sulfate/oxalate exchange has been detected in the proximal tubule of the kidney, where it is probably involved in the reabsorption of filtered sulfate and the secretion of oxalate and may contribute to oxalate-dependent chloride reabsorption. Screening of a renal cortex cDNA library determined that sat-1 is expressed in the rat kidney. To evaluate this anion exchanger, the sat-1 protein was expressed in Sf9 cells. Sodium-independent sulfate and oxalate uptake was enhanced 7.3-fold and 13.1-fold, respectively, in Sf9 cells expressing the sat-1 protein compared with cells infected with wild-type virus. We determined that sat-1 is glycosylated in the kidney; however, anion exchange via sat-1 is observed despite incomplete glycosylation of sat-1 in Sf9 cells. The sat-1 protein, with an added COOH-terminal 6-histidine tag, was purified on a metal affinity column and used to generate anti-sat-1 monoclonal antibodies. The sat-1 protein was localized to the basolateral membrane, but not the apical membrane, of the proximal tubule by both Western blot analysis and immunohistochemistry. These studies demonstrate that sulfate/oxalate exchange on the apical and basolateral membranes of the proximal tubule represents transport on two different anion exchangers.

scholarly journals Neutral amino acid transporter ASCT2 displays substrate-induced Na+ exchange and a substrate-gated anion conductance

2000 ◽  
Vol 346 (3) ◽  
pp. 705-710 ◽  
Author(s):  
Angelika BRÖER ◽  
Carsten WAGNER ◽  
Florian LANG ◽  
Stefan BRÖER
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Anion Channel ◽  
Amino Acid Transporter ◽  
Neutral Amino Acid ◽  
Xenopus Laevis Oocytes ◽  
Anion Conductance ◽  
Neutral Amino Acid Transporter ◽  
Inward Currents ◽  
Acid Transporter

The neutral amino acid transporter ASCT2 mediates electroneutral obligatory antiport but at the same time requires Na+ for its function. To elucidate the mechanism, ASCT2 was expressed in Xenopus laevis oocytes and transport was analysed by flux studies and two-electrode voltage clamp recordings. Flux studies with 22NaCl indicated that the uptake of one molecule of glutamine or alanine is accompanied by the uptake of four to seven Na+ ions. Similarly to the transport of amino acids, the Na+ uptake was mediated by an obligatory Na+ exchange mechanism that depended on the presence of amino acids but was not stoichiometrically coupled to the amino acid transport. Other cations could not replace Na+ in this transport mechanism. When NaCl was replaced by NaSCN in the transport buffer, the superfusion of oocytes with amino acid substrates resulted in large inward currents, indicating the presence of a substrate-gated anion channel in the ASCT2 transporter. The Km for glutamine derived from these experiments is in good agreement with the Km derived from flux studies; it varied between 40 and 90 μM at holding potentials of -60 and -20 mV respectively. The permeability of the substrate-gated anion conductance decreased in the order SCN- NO3- > I- > Cl- and also required the presence of Na+.

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