Upregulation of the Na+-Coupled Phosphate Cotransporters NaPi-IIa and NaPi-IIb by B-RAF

B-RAF, a serine/threonine protein kinase, contributes to signaling of insulin-like growth factor IGF1. Effects of IGF1 include stimulation of proximal renal tubular phosphate transport, accomplished in large part by Na⁺-coupled phosphate cotransporter NaPi-IIa. The related Na⁺-coupled phosphate cotransporter NaPi-IIb accomplishes phosphate transport in intestine and tumor cells. The present study explored whether B-RAF influences protein abundance and/or activity of type II Na⁺-coupled phosphate cotransporters NaPi-IIa and NaPi-IIb. cRNA encoding wild-type NaPi-IIa and wild-type NaPi-IIb was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type B-RAF, and electrogenic phosphate transport determined by dual-electrode voltage clamp. NaPi-IIa protein abundance in Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified by chemiluminescence. Moreover, in HEK293 cells, the effect of B-RAF inhibitor PLX-4720 on NaPi-IIa cell surface protein abundance was quantified utilizing biotinylation of cell surface proteins and western blotting. In NaPi-IIa-expressing Xenopus oocytes, but not in oocytes injected with water, addition of phosphate to extracellular bath generated a current (I _P), which was significantly increased following coexpression of B-RAF. According to kinetic analysis, coexpression of B-RAF enhanced the maximal I_P. Coexpression of B-RAF further enhanced NaPi-IIa protein abundance in the Xenopus oocyte cell membrane. Treatment of HEK293 cells for 24 h with PLX-4720 significantly decreased NaPi-IIa cell membrane protein abundance. Coexpression of B-RAF, further significantly increased I_P in NaPi-IIb-expressing Xenopus oocytes. Again, B-RAF coexpression enhanced the maximal I_P. In conclusion, B-RAF is a powerful stimulator of the renal and intestinal type II Na⁺-coupled phosphate cotransporters NaPi-IIa and NaPi-IIb, respectively.     

Functional characterization of the human intestinal NaPi-IIb cotransporter in hamster fibroblasts and Xenopus oocytes

The recently cloned NaPi-IIb cotransporter is an apical membrane protein that is involved in the absorption of phosphate in the intestine. To expedite functional and structural studies, the human intestinal NaPi-IIb cotransporter was stably expressed in hamster fibroblast (PS120) cells. The hNaPi-IIb cDNA stably transfected cells exhibited a 1.8-fold higher sodium-dependent phosphate uptake than vector DNA transfected cells, and had a K(m) for Pi of approximately 106 microM and a K(m) for Na(+) of approximately 34 mM. The hNaPi-IIb cotransporter was also expressed in Xenopus oocytes and it exhibited a K(m) for Pi of approximately 113 microM and a K(m) for Na(+) of approximately 65 mM. The hNaPi-IIb cotransporter expressed in both PS120 cells and oocytes was inhibited by high external pH. Furthermore, the phosphate uptake mediated by the hNaPi-IIb cotransporter was inhibited by 5 mM phosphonoformic acid (PFA), 1 mM arsenate and 100 nM phorbol myristate acetate (PMA). These results demonstrate that the human intestinal NaPi-IIb cotransporter is functional when expressed in hamster fibroblasts, and that this model system may be useful in the future to identify NaPi-IIb cotransporter-specific inhibitors.     

Klotho Sensitivity of the Neuronal Excitatory Amino Acid Transporters EAAT3 and EAAT4

Klotho, a transmembrane protein, which can be cleaved off as β-glucuronidase and hormone, is released in both, kidney and choroid plexus and encountered in blood and cerebrospinal fluid. Klotho deficiency leads to early appearance of age-related disorders and premature death. Klotho may modify transport by inhibiting 1,25(OH)₂D₃ formation or by directly affecting channel and carrier proteins. The present study explored whether Klotho influences the activity of the Na⁺-coupled excitatory amino acid transporters EAAT3 and EAAT4, which are expressed in kidney (EAAT3), intestine (EAAT3) and brain (EAAT3 and EAAT4). To this end, cRNA encoding EAAT3 or EAAT4 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho. EAAT expressing Xenopus oocytes were further treated with recombinant human β-Klotho protein with or without β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL). Electrogenic excitatory amino acid transport was determined as L-glutamate-induced current (I_glu) in two electrode voltage clamp experiments. EAAT3 and EAAT4 protein abundance in the Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified utilizing chemiluminescence. As a result, coexpression of Klotho cRNA significantly increased I_glu in both, EAAT3 or EAAT4-expressing Xenopus oocytes. Klotho cRNA coexpression significantly increased the maximal current and cell membrane protein abundance of both EAAT3 and EAAT4. The effect of Klotho coexpression on EAAT3 and EAAT4 activity was mimicked by treating EAAT3 or EAAT4-expressing Xenopus oocytes with recombinant human β-Klotho protein. The effects of Klotho coexpression and of treatment with recombinant human β-Klotho protein were both abrogated in the presence of DSAL (10 µM). In conclusion, Klotho is a novel, powerful regulator of the excitatory amino acid transporters EAAT3 and EAAT4.     

Identification of a sodium-dependent organic anion transporter from rat adrenal gland

In this study, a novel sodium-dependent organic anion transporter (Soat) was identified. Soat is expressed in rat brain, heart, kidney, lung, muscle, spleen, testis, adrenal gland, small intestine, and colon. The Soat protein consists of 370 amino acids and shows 42% and 31% overall amino acid sequence identity to the ileal sodium-dependent bile acid transporter (Isbt) and the Na(+)/taurocholate cotransporting polypeptide (Ntcp), respectively. Soat is predicted to have nine transmembrane domains, with an N-terminus outside the cell and an intracellular C-terminus. The Soat gene is localized on chromosome 14 and is coded by six exons mapped in region 14p22. When expressed in Xenopus laevis oocytes, Soat shows transport function for estrone-3-sulfate (Km = 31 microM, Vmax = 5557 fmol/oocyte/30 min) and dehydroepiandrosterone sulfate (Km = 30 microM, Vmax = 5682 fmol/oocyte/30 min). Soat does not transport taurocholate, estradiol-17beta-glucuronide, nor ouabain.     

Characterisation and cloning of a Na-dependent broad-specificity neutral amino acid transporter from NBL-1 cells: a novel member of the ASC/B transporter family

Na⁺-dependent neutral amino acid transport into the bovine renal epithelial cell line NBL-1 is catalysed by a broad-specificity transporter originally termed System B⁰. This transporter is shown to differ in specificity from the B⁰ transporter cloned from JAR cells [J. Biol. Chem. 271 (1996) 18657] in that it interacts much more strongly with phenylalanine. Using probes designed to conserved transmembrane regions of the ASC/B⁰ transporter family we have isolated a cDNA encoding the NBL-1 cell System B⁰ transporter. When expressed in Xenopus oocytes the clone catalysed Na⁺-dependent alanine uptake which was inhibited by glutamine, leucine and phenylalanine. However, the clone did not catalyse Na⁺-dependent phenylalanine transport, again as in NBL-1 cells. The clone encoded a protein of 539 amino acids; the predicted transmembrane domains were almost identical in sequence to those of the other members of the B⁰/ASC transporter family. Comparison of the sequences of NBL-1 and JAR cell transporters showed some differences near the N-terminus, C-terminus and in the loop between helices 3 and 4. The NBL-1 B⁰ transporter is not the same as the renal brush border membrane transporter since it does not transport phenylalanine. Differences in specificity in this protein family arise from relatively small differences in amino acid sequence.     

The 4F2 antigen heavy chain induces uptake of neutral and dibasic amino acids in Xenopus oocytes.

The 4F2 cell surface antigen is a disulfide-linked heterodimer induced during the process of cellular activation and expressed widely in mammalian tissues (Parmacek, M. S., Karpinski, B. A., Gottesdiener, K. M., Thompson, C. B., and Leiden, J. M. (1989) Nucleic Acids Res. 17, 1915-1931). The human heavy chain component, a type II membrane glycoprotein, has 29% identity to the amino acid transport-related protein encoded by the recently cloned rat D2 cDNA. We have demonstrated that Xenopus oocytes injected with in vitro transcribed cRNA from D2 take up cystine and dibasic and neutral amino acids (Wells, R. G., and Hediger, M. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5596-5600). In the present study, we examine the role of the human 4F2 heavy chain in amino acid transport. In vitro transcribed 4F2 cRNA was injected into Xenopus oocytes which were assayed for the uptake of radiolabeled amino acids. Our results show that cRNA from 4F2 stimulates the uptake of dibasic and neutral amino acids into oocytes at levels up to 3-fold higher than for water-injected control oocytes. There is no demonstrable uptake of cystine. Uptake is saturable, with characteristics of high affinity transport, and inhibition data suggest that uptake occurs via a single transporter. Dibasic amino acids are taken up by both 4F2 and D2 cRNA-injected oocytes in a sodium-independent manner. In contrast, 4F2-induced but not D2-induced neutral amino acid uptake has a significant component of sodium dependence. Likewise, neutral amino acids in excess inhibit the 4F2-induced uptake of radiolabeled arginine but not leucine in a sodium-dependent manner. The 4F2-induced uptake we observe most likely represents the activity of a single transport system with some characteristics of systems y+, b0,+, and B0,+. We suggest that 4F2 and D2 represent a new family of proteins which induce amino acid transport with distinct characteristics, possibly functioning as transport activators or regulators.     

Electrogenic Kinetics of a Mammalian Intestinal Type IIb Na+/Pi Cotransporter

The kinetics of a type IIb Na(+)-coupled inorganic phosphate (Pi) cotransporter (NaPi-IIb) cloned from mouse small intestine were studied using the two-electrode voltage clamp applied to Xenopus oocytes. In the steady state, mouse NaPi-IIb showed a curvilinear I-V relationship, with rate-limiting behavior only for depolarizing potentials. The Pi dose dependence was Michaelian, with an apparent affinity constant for Pi (Km(pi)) of 10 +/- 1 microM: at -60 mV. Unlike for rat NaPi-IIa, (Km(pi)) increased with membrane hyperpolarization, as reported for human NaPi-IIa, flounder NaPi-IIb and zebrafish NaPi-IIb2. The apparent affinity constant for Na(+) (Km(na)) was 23 +/- 1 mM: at -60 mV, and the Na(+) activation was cooperative with a Hill coefficient of approximately 2. Pre-steady-state currents were documented in the absence of Pi and showed a strong dependence on external Na(+). The hyperpolarizing shift of the charge distribution midpoint potential was 65 mV/log[Na]. Approximately half the moveable charge was attributable to the empty carrier. A comparison of the voltage dependence of steady-state Pi-induced current and pre-steady-state charge movement indicated that for -120 mV 相似文献   

Cloning and functional characterization of a cocaine-sensitive dopamine transporter.

We report the cloning of a rat cDNA encoding a functional dopamine transporter. This cDNA, derived from an intron-containing gene, encodes a protein of 620 amino acids. Hydropathicity analysis of the protein sequence suggests the presence of 12 putative transmembrane domains. The protein displays considerable identity with transporters for noradrenaline and GABA (64 and 30%, respectively). Transient expression of the cDNA in COS7 cells directs the expression of dopamine uptake activity with appropriate pharmacology and in a sodium-dependent fashion. In situ hybridization reveals that the mRNA for this transporter is expressed in the substantia nigra and ventral tegmental area, regions that contain dopaminergic cell bodies.     

Functional expression of the rabbit intestinal Na+/L-proline cotransporter (IMINO system) in Xenopus laevis oocytes

Proline absorption across small intestine takes place mainly through a Na+-dependent cotransporter localized at the brush border membrane of the enterocyte named IMINO system. It transports L-proline and 4-OH-proline but not L-alanine, neither cationic nor anionic amino acids. The present work demonstrates the functional expression of this transporter in Xenopus laevis oocytes by mRNA microinjection and radiotracer uptake techniques. Poly (A)+-RNA was isolated from rabbit jejunal mucosa and injected into oocytes. Five days after the injection, results showed 1.5 fold stimulation of 50 microM 3H-proline uptake by the injected oocytes when compared to the non injected oocytes uptake. Poly (A)+-RNA was sized fractionated and fractions were injected again. Increase on Na+-dependent L-proline uptake was obtained with a mRNA fraction between 2,4 and 4,4 kb, which was used to construct a cDNA library. The library was sequentially divided and cRNAs injected into oocytes in order to screen for an increment on the signal. A subdivision containing around 2,000 colonies was found to augment L-proline uptake 25 fold over the non injected oocytes uptake. This cRNA pool was used to further characterize the transporter. Results showed that in the absence of Na+ there was no L-proline uptake, 2 mM 4-OH-L-proline completely inhibited 50 microM proline uptake and there was no 50 microM alanine uptake. In summary, these results demonstrate the expression of the rabbit small intestine IMINO transporter in Xenopus laevis oocytes and support the next steps in the isolation of the clone.     

BcNRT1, a plasma membrane-localized nitrate transporter from non-heading Chinese cabbage

A nitrate transporter, BcNRT1, was isolated from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) cultivar 'Suzhouqing'. The full-length cDNA was obtained using the rapid amplification of cDNA ends technique and contains an open reading frame of 1,770?bp that predicts a protein of 589 acid residues that possesses 12 putative transmembrane domains. Using the GUS marker gene driven by the BcNRT1 promoter, we found BcNRT1 expression to be concentrated in primary and lateral root tips and in shoots of transgenic Arabidopsis plants. The YFP fused to BcNRT1 and transformed into cabbage protoplasts indicated that BcNRT1 was localized to the plasma membrane. The expression of BcNRT1 in roots was induced by exposure to 25?mM nitrate, and the BcNRT1 cRNA heterologously expressed in Xenopus laevis oocytes showed nitrate conductance when nitrate was included in the medium. Moreover, mutant chl1-5 plants harboring 35S::BcNRT1 showed sensitivity to chlorate treatment and exhibited restored nitrate uptake. In conclusion, the results indicate that BcNRT1 functions as a low affinity nitrate transporter in non-heading Chinese cabbage.     

Isolation and characterization of a sodium-dependent phosphate transporter gene in Dunaliella viridis

A sodium-dependent phosphate transporter gene, DvSPT1, was isolated from a cDNA library using a probe derived from a subtracted cDNA library of Dunaliella viridis. Sequencing analyses revealed a cDNA sequence of 2649 bp long and encoded an open-reading frame consisting of 672 amino acids. The deduced amino acid sequence of DvSPT1 exhibited 31.2% identity to that of TcPHO from Tetraselmis chui. Hydrophobicity and secondary structure prediction revealed 11 conserved transmembrane domains similar to those found in PHO89 from Saccharomyces cerevisiae and PHO4 from Neurospora crassa. Northern blot analysis indicated that the DvSPT1 expression was induced upon NaCl hyperosmotic stress or phosphate depletion. Functional characterization in yeast Na+ export pump mutant G19 suggested that DvSPT1 encoded a Na+ transporter protein. The gene sequence of GDvSPT1 (7922 bp) was isolated from a genomic library of D. viridis. Southern blot analysis indicated that there exist at least two homologous genes in D. viridis.     

Cloning and characterization of a type III Na-dependent phosphate cotransporter from mouse intestine

Intestinal and renalabsorption of inorganic phosphate (P_i) is critical forphosphate homeostasis in mammals. We have isolated a cDNA that encodesa type III Na-dependent phosphate cotransporter from mouse smallintestine (mPit-2). The nucleotide sequence of mPit-2 predicts aprotein of 653 amino acids with at least 10 putative transmembranedomains. Kinetic studies, carried out in Xenopus oocytes,showed that mPit-2 cRNA induces significant Na-dependent P_iuptake with an apparent Michaelis constant (K_m)for phosphate of 38 µM. The transport of phosphate by mPit-2 isinhibited at high pH. Northern blot analysis demonstrated the presenceof mPit-2 mRNA in various tissues, including intestine, kidney, heart,liver, brain, testis, and skin. The highest expression of mPit-2 in the intestine was found in the jejunum. In situ hybridization revealed thatmPit-2 mRNA is expressed throughout the vertical crypt-villus axis ofthe intestinal epithelium. The presence of mPit-2 in the mouseintestine and its unique transport characteristics suggest thatmultiple Na-dependent cotransporters may contribute to phosphate absorption in the mammalian small intestine.

     

Cloning and functional characterization of the human sodium-dependent vitamin C transporters hSVCT1 and hSVCT2

Two sodium-dependent vitamin C transporters, hSVCT1 and hSVCT2, were cloned from a human kidney cDNA library. hSVCT1 had a 1797 bp open reading frame encoding a 598 amino acid polypeptide. The 1953 bp open reading frame of hSVCT2 encoded a 650 amino acid polypeptide. Using a Xenopus laevis oocyte expression system, both transporters were functionally expressed. By Eadie-Hofstee transformation the apparent K(m) of hSVCT1 for ascorbate was 252.0 microM and of hSVCT2 for ascorbate was 21.3 microM. Both transporters were sodium-dependent and did not transport dehydroascorbic acid. Incubation of oocytes expressing either transporter with phorbol 12-myristate 13-acetate (PMA) inhibited ascorbate transport activity. Availability of the human transporter clones may facilitate new strategies for determining vitamin C intake.     

Insect protein synthesis in frog cells: the translation of honey bee promelittin messenger RNA in Xenopus oocytes

Venom glands of young queen bees (Apis mellifera) synthesize the toxic peptide melittin as their main product. Melittin is formed by proteolytic cleavage of a precursor, promelittin. Unfractionated RNA prepared from venom glands was injected into Xenopus oocytes and was shown to direct the synthesis of a promelittin-like substance. About half of the peptide chain made in oocytes has been sequenced; the 17 amino acid residues identified correspond exactly with sequences found in promelittin from venom gland cells. These results yield final proof that injected messenger RNAs can be read with great fidelity. The translation of a messenger from an insect gland shows that at least some of the translational systems within the oocyte are neither cell-type nor phylum specific. It seems likely that the oocyte can be used to assay any kind of eukaryotic mRNA.The conversion of promelittin to melittin could not be detected in oocytes. Moreover, the promelittin synthesized in oocytes differs at the carboxyl end from the product made in gland cells, for the latter terminates with glutamine amide while the oocyte material probably ends with an amino acid with a free α-carboxyl group. Some of the post-translational modifications characteristic of gland cells thus do not seem to take place in oocytes.