Cloning and functional expression of an SGLT-1-like protein from the Xenopus laevisintestine

1999 ◽  
Vol 276 (5) ◽  
pp. G1251-G1259 ◽  
Author(s):  
Katsumi Nagata ◽  
Naohiro Hori ◽  
Kenzo Sato ◽  
Kunimasa Ohta ◽  
Hideaki Tanaka ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Functional Expression ◽  
Hill Coefficient ◽  
Xenopus Laevis Oocytes ◽  
Membrane Hyperpolarization ◽  
Apparent Concentration ◽  
Inward Currents ◽  
Rapid Amplification ◽  
The Hill

A cDNA encoding an Na+-glucose cotransporter type 1 (SGLT-1)-like protein was cloned from the Xenopus laevis intestine by the 5′- and 3′-rapid amplification of cDNA ends method. The deduced amino acid sequence was 673 residues long, with a predicted mass of 74.1 kDa and 52–53% identity to mammalian SGLT-1s. This gene was expressed in the small intestine and kidney, reflecting a tissue distribution similar to that of SGLT-1. The function of the protein was studied using the two-microelectrode voltage-clamp technique after injection of cRNA into Xenopus laevis oocytes. Perfusion with myo-inositol elicited about twofold larger inward currents than perfusion withd-glucose. The order of the substrate specificity was myo-inositol > d-glucose >d-galactose ≥ α-methyl-d-glucoside. The current induced by myo-inositol increased with membrane hyperpolarization and depended on external myo-inositol and Na+: the apparent Michaelis-Menten constant was 0.25 ± 0.07 (SD) mM with myo-inositol, whereas the apparent concentration for half-maximal activation was 12.5 ± 1.0 mM and the Hill coefficient was 1.6 ± 0.1 with Na+. In conclusion, the cloned protein shares features with both SGLT-1 and the Na+- myo-inositol cotransporter.

scholarly journals The transport properties of the human renal Na+- dicarboxylate cotransporter under voltage-clamp conditions

2000 ◽  
Vol 279 (1) ◽  
pp. F54-F64 ◽  
Author(s):  
Xiaozhou Yao ◽  
Ana M. Pajor
Keyword(s):  
Transport Properties ◽  
Voltage Clamp ◽  
Transport Model ◽  
Hill Coefficient ◽  
Xenopus Laevis Oocytes ◽  
Sodium Ions ◽  
Cation Selectivity ◽  
Half Saturation Constant ◽  
Inward Currents ◽  
The Hill

The transport properties of the human Na+-dicarboxylate cotransporter, (hNaDC-1), expressed in Xenopus laevis oocytes were characterized using the two-electrode voltage clamp technique. Steady-state succinate-evoked inward currents in hNaDC-1 were dependent on the concentrations of succinate and sodium, and on the membrane potential. At −50 mV, the half-saturation constant for succinate ( K 0.5 succinate) was 1.1 mM and the half-saturation constant for sodium ( K 0.5 sodium) was 65 mM. The Hill coefficient was 2.3, which is consistent with a transport stoichiometry of 3 Na+:1 divalent anion substrate. The hNaDC-1 exhibits a high-cation selectivity. Sodium is the preferred cation and other cations, such as lithium, were not able to support transport of succinate. The preferred substrates of hNaDC-1 are fumarate ( K 0.5 1.8 mM) and succinate, followed by methylsuccinate ( K 0.5 2.8 mM), citrate ( K 0.5 6.8 mM) and α-ketoglutarate ( K 0.5 16 mM). The hNaDC-1 may also transport sodium ions through an uncoupled leak pathway, which is sensitive to phloretin inhibition. We propose a transport model for hNaDC-1 in which the binding of three sodium ions is followed by substrate binding.

Electrophysiological characteristics of the proton-coupled peptide transporter PEPT2 cloned from rat brain

1998 ◽  
Vol 275 (4) ◽  
pp. C967-C975 ◽  
Author(s):  
Hong Wang ◽  
You-Jun Fei ◽  
Vadivel Ganapathy ◽  
Frederick H. Leibach
Keyword(s):  
Rat Brain ◽  
Rat Kidney ◽  
Proton Gradient ◽  
Hill Coefficient ◽  
Peptide Transporter ◽  
Xenopus Laevis Oocytes ◽  
Electrophysiological Properties ◽  
Inward Currents ◽  
Charged Peptides ◽  
The Hill

We have cloned a peptide transporter from rat brain and found it to be identical to rat kidney PEPT2. In the present study we characterize the transport function of the rat brain PEPT2, with special emphasis on electrophysiological properties and interaction with N-acetyl-l-aspartyl-l-glutamate (NAAG). When heterologously expressed in HeLa cells and in SK-N-SH cells, PEPT2 transports several dipeptides but not free amino acids in the presence of a proton gradient. NAAG competes with other peptides for the PEPT2-mediated transport process. When PEPT2 is expressed in Xenopus laevis oocytes, substrate-induced inward currents are detectable with dipeptides of differing charge in the presence of a proton gradient. Proton activation kinetics are similar for differently charged peptides. NAAG is a transportable substrate for PEPT2, as evidenced by NAAG-induced currents. The Hill coefficient for protons for the activation of the transport of differently charged peptides, including NAAG, is 1. Although the peptide-to-proton stoichiometry for negatively charged peptides is 1, the transport nonetheless is associated with transfer of positive charge into the oocyte, as indicated by peptide-induced inward currents.

scholarly journals Mode of Action of Neonicotinoid Insecticides Imidacloprid and Thiacloprid to the Cockroach Pameα7 Nicotinic Acetylcholine Receptor

2021 ◽  
Vol 22 (18) ◽  
pp. 9880
Author(s):  
Alison Cartereau ◽  
Emiliane Taillebois ◽  
Jean-Yves Le Questel ◽  
Steeve H. Thany
Keyword(s):  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Partial Agonist ◽  
Functional Expression ◽  
Binding Pocket ◽  
Xenopus Laevis Oocytes ◽  
Nicotinic Acetylcholine ◽  
Neonicotinoid Insecticides ◽  
Neonicotinoid Insecticide ◽  
Inward Currents

The functional expression of the cockroach Pameα7 nicotinic acetylcholine receptor subunit has been previously studied, and was found to be able to form a homomeric receptor when expressed in Xenopus laevis oocytes. In this study, we found that the neonicotinoid insecticide imidacloprid is unable to activate the cockroach Pameα7 receptor, although thiacloprid induces low inward currents, suggesting that it is a partial agonist. In addition, the co-application or 5 min pretreatment with 10 µM imidacloprid increased nicotine current amplitudes, while the co-application or 5 min pretreatment with 10 µM thiacloprid decreased nicotine-evoked current amplitudes by 54% and 28%, respectively. This suggesting that these two representatives of neonicotinoid insecticides bind differently to the cockroach Pameα7 receptor. Interestingly, the docking models demonstrate that the orientation and interactions of the two insecticides in the cockroach Pameα7 nAChR binding pocket are very similar. Electrophysiological results have provided evidence to suggest that imidacloprid and thiacloprid could act as modulators of the cockroach Pameα7 receptors.

Selective enhancement of bovine papillomavirus type 1 DNA replication in Xenopus laevis eggs by the E6 gene product

1989 ◽  
Vol 9 (2) ◽  
pp. 406-414
Author(s):  
H Romanczuk ◽  
W M Wormington
Keyword(s):  
Dna Replication ◽  
Xenopus Laevis ◽  
Gene Product ◽  
Mammalian Cells ◽  
Xenopus Laevis Oocytes ◽  
Bovine Papillomavirus ◽  
In Vitro Synthesis ◽  
E6 Gene ◽  
E6 Protein

Genetic analyses of bovine papillomavirus type 1 (BPV-1) DNA in transformed mammalian cells have indicated that the E6 gene product is essential for the establishment and maintenance of a high plasmid copy number. In order to analyze the direct effect of the E6 protein on the replication of a BPV-1-derived plasmid, a cDNA containing the BPV-1 E6 open reading frame was subcloned into an SP6 vector for the in vitro synthesis of the corresponding mRNA. The SP6 E6 mRNA was injected into Xenopus laevis oocytes to determine the subcellular localization of the E6 gene product and to analyze the effect of the protein on BPV-1 DNA replication. SP6 E6 mRNA microinjected into stage VI oocytes was translated into a 15.5-kilodalton protein that was specifically immunoprecipitated by antibodies directed against the E6 gene product. The E6 protein preferentially accumulated in oocyte nuclei, a localization which is consistent with the replicative functions in which it has been implicated. The expression of E6 in replication-competent mature oocytes selectively enhanced the replication of a BPV-derived plasmid, indicating a direct role for this gene product in the control of BPV-1 DNA replication.

scholarly journals Functional expression of H1-histaminergic receptors in Xenopus laevis oocytes injected with bovine adrenal medullary mRNA.

1991 ◽  
Vol 55 (2) ◽  
pp. 287-290 ◽  
Author(s):  
Kazushige Sugama ◽  
Masakatsu Yamashita ◽  
Hiroyuki Fukui ◽  
Seiji Ito ◽  
Hiroshi Wada
Keyword(s):  
Xenopus Laevis ◽  
Functional Expression ◽  
Xenopus Laevis Oocytes

scholarly journals Neurotoxin Merging: A Strategy Deployed by the Venom of the Spider Cupiennius salei to Potentiate Toxicity on Insects

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 250 ◽  
Author(s):  
Benjamin Clémençon ◽  
Lucia Kuhn-Nentwig ◽  
Nicolas Langenegger ◽  
Lukas Kopp ◽  
Steve Peigneur ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Direct Interaction ◽  
Structural Type ◽  
Structure Type ◽  
Open Loop ◽  
Cytolytic Activity ◽  
Xenopus Laevis Oocytes ◽  
Cupiennius Salei

The venom of Cupiennius salei is composed of dozens of neurotoxins, with most of them supposed to act on ion channels. Some insecticidal monomeric neurotoxins contain an α-helical part besides their inhibitor cystine knot (ICK) motif (type 1). Other neurotoxins have, besides the ICK motif, an α-helical part of an open loop, resulting in a heterodimeric structure (type 2). Due to their low toxicity, it is difficult to understand the existence of type 2 peptides. Here, we show with the voltage clamp technique in oocytes of Xenopus laevis that a combined application of structural type 1 and type 2 neurotoxins has a much more pronounced cytolytic effect than each of the toxins alone. In biotests with Drosophila melanogaster, the combined effect of both neurotoxins was enhanced by 2 to 3 log units when compared to the components alone. Electrophysiological measurements of a type 2 peptide at 18 ion channel types, expressed in Xenopus laevis oocytes, showed no effect. Microscale thermophoresis data indicate a monomeric/heterodimeric peptide complex formation, thus a direct interaction between type 1 and type 2 peptides, leading to cell death. In conclusion, peptide mergers between both neurotoxins are the main cause for the high cytolytic activity of Cupiennius salei venom.

scholarly journals Functional Expression of Hi-Histaminergic Receptors in Xenopus Laevis Oocytes Injected with Bovine Adrenal Medullary mRNA

1991 ◽  
Vol 55 (2) ◽  
pp. 287-290
Author(s):  
Kazushige Sugama ◽  
Masakatsu Yamashita ◽  
Hiroyuki Fukui ◽  
Seiji Ito ◽  
Hiroshi Wada
Keyword(s):  
Xenopus Laevis ◽  
Functional Expression ◽  
Xenopus Laevis Oocytes
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