A hyperpolarization-activated chloride current in xenopus laevis oocytes under voltage-clamp

1983 ◽  
Vol 399 (2) ◽  
pp. 157-159 ◽  
Author(s):  
Antonio Peres ◽  
Giovanni Bernardini
Keyword(s):  
Xenopus Laevis ◽  
Voltage Clamp ◽  
Chloride Current ◽  
Xenopus Laevis Oocytes

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.

scholarly journals Induction of apoptosis using sphingolipids activates a chloride current in Xenopus laevis oocytes

2000 ◽  
Vol 279 (1) ◽  
pp. C158-C165 ◽  
Author(s):  
R. Souktani ◽  
A. Berdeaux ◽  
B. Ghaleh ◽  
J. F. Giudicelli ◽  
L. Guize ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Dna Cleavage ◽  
Ionic Currents ◽  
Chloride Current ◽  
Xenopus Laevis Oocytes ◽  
Cell Shrinkage ◽  
Transport Pathways ◽  
Kinase C ◽  
Induction Of Apoptosis ◽  
A Current

The purpose of this study was to investigate whether the cell shrinkage that occurs during apoptosis could be explained by a change of the activity in ion transport pathways. We tested whether sphingolipids, which are potent pro-apoptotic compounds, can activate ionic currents in Xenopus laevis oocytes. Apoptosis was characterized in our model by a decrease in cell volume, a loss of cell viability, and DNA cleavage. Oocytes were studied using voltage-clamp after injection with N, N-dimethyl-d-erythrosphingosine (DMS) or d-sphingosine (DS). DMS and DS activated a fast-activating, slowly inactivating, outwardly rectifying current, similar to I Cl-swell, a swelling-induced chloride current. Lowering the extracellular chloride dramatically reduced the current, and the channel was more selective for thiocyanate and iodide (thiocyanate > iodide) than for chloride. The current was blocked by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and lanthanum but not by niflumic acid. Oocytes injected with a pseudosubstrate inhibitor of protein kinase C (PKC), PKC-(19–31), exhibited the same current. DMS-activated current was abolished by preexposure with phorbol myristate acetate. Our results suggest that induction of apoptosis in X. laevis oocytes, using sphingolipids or PKC inhibitors, activates a current similar to swelling-induced chloride current previously described in oocytes.

Functional expression of the oxytocin receptor in Xenopus laevis oocytes primed with mRNA from bovine endometrium

1988 ◽  
Vol 1 (1) ◽  
pp. 77-81 ◽  
Author(s):  
S. D. Morley ◽  
W. Meyerhof ◽  
J. Schwarz ◽  
D. Richter
Keyword(s):  
Xenopus Laevis ◽  
Receptor Antagonist ◽  
Voltage Clamp ◽  
Functional Expression ◽  
Oxytocin Receptor ◽  
Xenopus Laevis Oocytes ◽  
Induced Membrane ◽  
Membrane Changes ◽  
Dose Dependent ◽  
Voltage Clamp Method

ABSTRACT Synthesis of the uterine receptor for the hypothalamic hormone oxytocin has been induced in oocytes from Xenopus laevis previously primed with bovine endometrium mRNA. The injected oocytes responded to oxytocin by showing dose-dependent oscillations in membrane currents as recorded by the voltage-clamp method. The response was specific in that it was not elicited by several other peptides tested. The oxytocin-induced membrane changes were suppressed when oocytes were pretreated with an oxytocin receptor antagonist.

A calcium-dependent transient outward current in Xenopus laevis oocytes

1982 ◽  
Vol 215 (1201) ◽  
pp. 491-497 ◽  
Keyword(s):  
Membrane Potential ◽  
Xenopus Laevis ◽  
Voltage Clamp ◽  
Outward Current ◽  
Membrane Currents ◽  
Transient Current ◽  
Transient Outward Current ◽  
Xenopus Laevis Oocytes ◽  
Calcium Dependent

Membrane currents were investigated in Xenopus laevis oocytes under voltage clamp. Depolarizing pulses, given from a holding potential of about –100 mV, elicited a transient outward current when the membrane potential was made more positive than about –20 mV. As the potential was made increasingly positive the transient outward current first increased and then decreased. The amplitude of the transient current increased when the external Ca 2+ concentration was raised; and the current was abolished by Mn 2+ . It appears that when the membrane is depolarized Ca 2+ ions enter the oocyte and trigger an outward current, possibly by opening Cl – channels.

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