The disulfonic stilbene DIDS and the marine poison maitotoxin activate the same two types of endogenous cation conductance in the cell membrane of Xenopus laevis oocytes

2001 ◽  
Vol 442 (5) ◽  
pp. 700-708 ◽  
Author(s):  
Alexey Diakov ◽  
Jan-Peter Koch ◽  
Olivier Ducoudret ◽  
Suzanne Müller-Berger ◽  
Eberhard Frömter
Keyword(s):  
Cell Membrane ◽  
Xenopus Laevis ◽  
Xenopus Laevis Oocytes ◽  
Cation Conductance ◽  
Disulfonic Stilbene

Effect of reactive oxygen species on NH 4 + permeation in Xenopus laevis oocytes

2002 ◽  
Vol 282 (6) ◽  
pp. C1445-C1453 ◽  
Author(s):  
Marc Cougnon ◽  
Samia Benammou ◽  
Franck Brouillard ◽  
Philippe Hulin ◽  
Gabrielle Planelles
Keyword(s):  
Reactive Oxygen Species ◽  
Xenopus Laevis ◽  
Reactive Oxygen ◽  
Membrane Conductance ◽  
Xenopus Laevis Oocytes ◽  
Oxygen Species ◽  
Cation Conductance ◽  
Β Amyloid ◽  
Oocyte Membrane ◽  
Ros Scavengers

To investigate the effects of reactive oxygen species (ROS) on NH[Formula: see text]permeation in Xenopus laevis oocytes, we used intracellular double-barreled microelectrodes to monitor the changes in membrane potential ( V m) and intracellular pH (pHi) induced by a 20 mM NH4Cl-containing solution. Under control conditions, NH4Cl exposure induced a large membrane depolarization (to V m = 4.0 ± 1.5 mV; n = 21) and intracellular acidification [reaching a change in pHi(ΔpHi) of 0.59 ± 0.06 pH units in 12 min]; the initial rate of cell acidification (dpHi/d t) was 0.06 ± 0.01 pH units/min. Incubation of the oocytes in the presence of H2O2 or β-amyloid protein had no marked effect on the NH4Cl-induced ΔpHi. By contrast, in the presence of photoactivated rose bengal (RB), tert-butyl-hydroxyperoxide ( t-BHP), or xanthine/xanthine oxidase (X/XO), the same experimental maneuver induced significantly greater ΔpHi and dpHi/d t. These increases in ΔpHiand dpHi/d t were prevented by the ROS scavengers histidine and desferrioxamine, suggesting involvement of the reactive species 1ΔgO2 and ·OH. Using the voltage-clamp technique to identify the mechanism underlying the ROS-measured effects, we found that RB induced a large increase in the oocyte membrane conductance ( G m). This RB-induced G m increase was prevented by 1 mM diphenylamine-2-carboxylate (DPC) and by a low Na+concentration in the bath. We conclude that RB, t-BHP, and X/XO enhance NH[Formula: see text] influx into the oocyte via activation of a DPC-sensitive nonselective cation conductance pathway.

Proton transport mechanism in the cell membrane of Xenopus laevis oocytes

1992 ◽  
Vol 420 (1) ◽  
pp. 78-82 ◽  
Author(s):  
Birgitta-Christina Burckhardt ◽  
Beate Kroll ◽  
Eberhard Fr�mter
Keyword(s):  
Cell Membrane ◽  
Xenopus Laevis ◽  
Proton Transport ◽  
Transport Mechanism ◽  
Xenopus Laevis Oocytes

scholarly journals Monovalent cation conductance in Xenopus laevis oocytes expressing hCAT-3

2005 ◽  
Vol 1668 (2) ◽  
pp. 234-239 ◽  
Author(s):  
Wolfgang Gilles ◽  
Sebastian D. Vulcu ◽  
Jana F. Liewald ◽  
Alice Habermeier ◽  
Nicole Vékony ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Monovalent Cation ◽  
Xenopus Laevis Oocytes ◽  
Cation Conductance

Cation transport activity of anion exchanger 1 mutations found in inherited distal renal tubular acidosis

2008 ◽  
Vol 295 (2) ◽  
pp. F343-F350 ◽  
Author(s):  
Stephen Walsh ◽  
Franck Borgese ◽  
Nicole Gabillat ◽  
Robert Unwin ◽  
Helene Guizouarn
Keyword(s):  
Cell Membrane ◽  
Anion Exchange ◽  
Renal Tubular Acidosis ◽  
Anion Exchanger ◽  
Distal Renal Tubular Acidosis ◽  
Xenopus Laevis Oocytes ◽  
Red Cell ◽  
Anion Exchanger 1 ◽  
Cation Conductance ◽  
Renal Tubular

Anion exchanger 1 (AE1) is encoded by SLC4A1 and mediates electroneutral anion exchange across cell membranes. It is the most abundant protein in the red cell membrane, but it is also found in the basolateral membrane of renal α-intercalated cells, where it is required for normal urinary acidification. Recently, four point mutations in red cell AE1 have been described that convert the anion exchanger to a cation conductance. SLC4A1 mutations can also cause type 1 hypokalemic distal renal tubular acidosis (dRTA). We investigated the properties of four dRTA-associated AE1 mutations (R589H, G609R, S613F, and G701D) by heterologous expression in Xenopus laevis oocytes. Although these AE1 mutants are functional anion exchangers, unlike the red cell disease mutants, we found that they also demonstrated a cation leak. We found a large cation leak in the G701D mutant. This mutant normally requires coexpression with glycophorin A for surface membrane expression in red blood cells and oocytes. However, we found that coexpressing wild-type kidney AE1 with G701D in oocytes still caused a cation leak, consistent with heterodimerized G701D reaching the cell membrane and retaining its cation conductance property. These findings have potential structural and functional implications for AE1, and they indicate that while anion exchange and cation conductance properties are distinct, they can coexist.

scholarly journals A new system to evaluate characteristics of Niemann-Pick C1 Like 1-mediated cholesterol transport using Xenopus laevis oocytes

2021 ◽  
Vol 1863 (2) ◽  
pp. 183508
Author(s):  
Shunsuke Nashimoto ◽  
Saori Yagi ◽  
Naoki Takeda ◽  
Miku Nonaka ◽  
Yoh Takekuma ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Cholesterol Transport ◽  
Xenopus Laevis Oocytes ◽  
Niemann Pick ◽  
New System

Characterization of Transport Activity of SLC11 Transporters in Xenopus laevis Oocytes by Fluorophore Quenching

2021 ◽  
pp. 247255522110041
Author(s):  
Raffaella Cinquetti ◽  
Francesca Guia Imperiali ◽  
Salvatore Bozzaro ◽  
Daniele Zanella ◽  
Francesca Vacca ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Voltage Clamp ◽  
Expression System ◽  
Peptide Transporter ◽  
Xenopus Laevis Oocytes ◽  
Substrate Uptake ◽  
Transport Activity ◽  
Experimental Conditions ◽  
Metal Transporters ◽  
Electrode Voltage

Membrane proteins are involved in different physiological functions and are the target of pharmaceutical and abuse drugs. Xenopus laevis oocytes provide a powerful heterologous expression system for functional studies of these proteins. Typical experiments investigate transport using electrophysiology and radiolabeled uptake. A two-electrode voltage clamp is suitable only for electrogenic proteins, and uptake measurements require the existence of radiolabeled substrates and adequate laboratory facilities. Recently, Dictyostelium discoideum Nramp1 and NrampB were characterized using multidisciplinary approaches. NrampB showed no measurable electrogenic activity, and it was investigated in Xenopus oocytes by acquiring confocal images of the quenching of injected fluorophore calcein. This method is adequate to measure the variation in emitted fluorescence, and thus transporter activity indirectly, but requires long experimental procedures to collect statistically consistent data. Considering that optimal expression of heterologous proteins lasts for 48–72 h, a slow acquiring process requires the use of more than one batch of oocytes to complete the experiments. Here, a novel approach to measure substrate uptake is reported. Upon injection of a fluorophore, oocytes were incubated with the substrate and the transport activity measured, evaluating fluorescence quenching in a microplate reader. The technique permits the testing of tens of oocytes in different experimental conditions simultaneously, and thus the collection of significant statistical data for each batch, saving time and animals. The method was tested with different metal transporters (SLC11), DMT1, DdNramp1, and DdNrampB, and verified with the peptide transporter PepT1 (SLC15). Comparison with traditional methods (uptake, two-electrode voltage clamp) and with quenching images acquired by fluorescence microscopy confirmed its efficacy.

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