scholarly journals Boric acid transport activity of human aquaporins expressed in Xenopus oocytes

2022 ◽  
Vol 10 (1) ◽  
Kazutaka Ushio ◽  
Erika Watanabe ◽  
Takehiro Kamiya ◽  
Ayumi Nagashima ◽  
Tadaomi Furuta ◽  
2002 ◽  
Vol 364 (3) ◽  
pp. 767-775 ◽  
Sabine WOLF ◽  
Annette JANZEN ◽  
Nicole VÉKONY ◽  
Ursula MARTINÉ ◽  
Dennis STRAND ◽  

Member 4 of human solute carrier family 7 (SLC7A4) exhibits significant sequence homology with the SLC7 subfamily of human cationic amino acid transporters (hCATs) [Sperandeo, Borsani, Incerti, Zollo, Rossi, Zuffardi, Castaldo, Taglialatela, Andria and Sebastio (1998) Genomics 49, 230–236]. It is therefore often referred to as hCAT-4 even though no convincing transport activity has been shown for this protein. We expressed SLC7A4 in Xenopus laevis oocytes, but could not detect any transport activity for cationic, neutral or anionic amino acids or for the polyamine putrescine. In addition, human glioblastoma cells stably overexpressing a fusion protein between SLC7A4 and the enhanced green fluorescent protein (EGFP) did not exhibit an increased transport activity for l-arginine. The lack of transport activity was not due to a lack of SLC7A4 protein expression in the plasma membrane, as in both cell types SLC7A4-EGFP exhibited a similar subcellular localization and level of protein expression as functional hCAT-EGFP proteins. The expression of SLC7A4 can be induced in NT2 teratocarcinoma cells by treatment with retinoic acid. However, also for this endogenously expressed SLC7A4, we could not detect any transport activity for l-arginine. Our data demonstrate that the expression of SLC7A4 in the plasma membrane is not sufficient to induce an amino acid transport activity in X. laevis oocytes or human cells. Therefore, SLC7A4 is either not an amino acid transporter or it needs additional (protein) factor(s) to be functional.

1988 ◽  
Vol 255 (3) ◽  
pp. 963-969 ◽  
A R Quesada ◽  
J D McGivan

A rapid method for the functional reconstruction of amino acid transport from liver plasma-membrane vesicles using the neutral detergent decanoyl-N-glucamide (‘MEGA-10’) is described. The method is a modification of that previously employed in this laboratory for reconstitution of amino acid transport systems from kidney brush-border membranes [Lynch & McGivan (1987) Biochem. J. 244, 503-508]. The transport activities termed ‘System A’, ‘System N’, and ‘System L’ are all reconstituted. The reconstitution procedure is rapid and efficient and is suitable as an assay for transport activity in studies involving membrane fractionation. By using this reconstitution procedure, System A transport activity was partially purified by lectin-affinity chromatography.

1998 ◽  
Vol 275 (5) ◽  
pp. G1045-G1055 ◽  
An-Qiang Sun ◽  
Meenakshisundaram Ananthanarayanan ◽  
Carol J. Soroka ◽  
Sundararajah Thevananther ◽  
Benjamin L. Shneider ◽  

The rat ileal apical Na+-dependent bile acid transporter (ASBT) and the liver Na+-taurocholate cotransporting polypeptide (Ntcp) are members of a new family of anion transporters. These transport proteins share limited sequence homology and almost identical predicted secondary structures but are localized to the apical surface of ileal enterocytes and the sinusoidal surface of hepatocytes, respectively. Stably transfected Madin-Darby canine kidney (MDCK) cells appropriately localized wild-type ASBT and Ntcp apically and basolaterally as assessed by functional activity and immunocytochemical localization studies. Truncated and chimeric transporters were used to determine the functional importance of the cytoplasmic tail in bile acid transport activity and membrane localization. Two cDNAs were created encoding a truncated transporter in which the 56-amino-acid COOH-terminal tail of Ntcp was removed or substituted with an eight-amino-acid epitope FLAG. For both mutants there was some loss of fidelity in basolateral sorting in that ∼75% of each protein was delivered to the basolateral surface compared with ∼90% of the wild-type Ntcp protein. In contrast, deletion of the cytoplasmic tail of ASBT led to complete loss of transport activity and sorting to the apical membrane. An Ntcp chimera in which the 56-amino-acid COOH-terminal tail of Ntcp was replaced with the 40-amino-acid cytoplasmic tail of ASBT was largely redirected (82.4 ± 3.9%) to the apical domain of stably transfected MDCK cells, based on polarity of bile acid transport activity and localization by confocal immunofluorescence microscopy. These results indicate that a predominant signal for sorting of the Ntcp protein to the basolateral domain is located in a region outside of the cytoplasmic tail. These studies have further shown that a novel apical sorting signal is localized to the cytoplasmic tail of ASBT and that it is transferable and capable of redirecting a protein normally sorted to the basolateral surface to the apical domain of MDCK cells.

1995 ◽  
Vol 268 (6) ◽  
pp. C1430-C1439 ◽  
R. T. Franceschi ◽  
J. X. Wilson ◽  
S. J. Dixon

Ascorbic acid is necessary for expression of the osteoblast phenotype. We examined whether Na(+)-dependent transport is required for MC3T3-E1 preosteoblast cells to respond to vitamin C and investigated the role of membrane transport in the intracellular accumulation and function of ascorbate. MC3T3-E1 cells were found to possess a saturable, stereoselective, Na(+)-dependent ascorbic acid transport activity that is sensitive to the transport inhibitors sulfinpyrazone, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and phloretin. Transport activity showed no competition with glucose or 2-deoxyglucose and was not inhibited by cytochalasin B, indicating that it is distinct from known hexose transporters. On addition of 100 microM ascorbic acid to the extracellular medium, intracellular concentrations of 10 mM were reached within 5-10 h and remained constant for up to 24 h. A good correlation was observed between intracellular ascorbic acid concentration and rate of hydroxyproline synthesis. Although ascorbic acid was transported preferentially compared with D-isoascorbic acid, both isomers had equivalent activity in stimulating hydroxyproline formation once they entered cells. Marked stereoselectivity for extracellular L-ascorbic acid relative to D-isoascorbic acid was also seen when alkaline phosphatase and total hydroxyproline were measured after 6 days in culture. Moreover, ascorbic acid transport inhibitors that prevented intracellular accumulation of vitamin blocked the synthesis of hydroxyproline. Thus Na(+)-dependent ascorbic acid transport is required for MC3T3-E1 cells to achieve the millimolar intracellular vitamin C concentrations necessary for maximal prolyl hydroxylase activity and expression of the osteoblast phenotype.

2001 ◽  
Vol 280 (3) ◽  
pp. R612-R622
Virtudes Medina ◽  
Antonio Lorenzo ◽  
Mario Dı́az

l-Alanine transport across the isolated duodenal mucosa of the lizard Gallotia galloti has been studied in Ussing chambers under short-circuit conditions. Net l-alanine fluxes, transepithelial potential difference (PD), and short-circuit current ( Isc) showed concentration-dependent relationships. Na+-dependent l-alanine transport was substantially inhibited by the analog α-methyl aminoisobutyric acid (MeAIB). Likewise, MeAIB fluxes were completely inhibited byl-alanine, indicating the presence of system A for neutral amino acid transport. System A transport activity was electrogenic and exhibited hyperbolic relationships for net MeAIB fluxes, PD, and Isc, which displayed similar apparent K m values. Na+-dependentl-alanine transport, but not MeAIB transport, was partially inhibited by l-serine and l-cysteine, indicating the participation of system ASC. This transport activity represents the major pathway for l-alanine absorption and seemed to operate in an electroneutral mode with a negligible contribution to the l-alanine-induced electrogenicity. It is concluded from the present study that the active Na+-dependent l-alanine transport across the isolated duodenal mucosa of Gallotia galloti results from the independent activity of systems A and ASC for neutral amino acid transport.

Sign in / Sign up

Export Citation Format

Share Document