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J B Thibaud

Publications and source records attributed to J B Thibaud.

9 recordsLinked to original sources
PubMed1998

Evidence for a multi-ion pore behavior in the plant potassium channel KAT1.

KAT1 is a cloned voltage-gated K+ channel from the plant Arabidopsis thaliana L., which displays an inward rectification reminiscent of 'anomalous' rectification of the if pacemaker current recorded in animal cells. Macroscopic conductance of KAT1 expressed in Xenopus oocytes was 5-fold less in pure Rb+ solution than in pure K+ solution, and negligible in pure Na+ solution. Experiments in different K+/Na+ or K+/Rb+ mixtures revealed deviations from the principle of independence and notably two anomalous effects of the K+/Rb+ mole fraction (i.e., the ratio [K+]/([K+]+[Rb+])). First, the KAT1 deactivation time constant was both voltage- and mole fraction-dependent (a so-called 'foot in the door' effect was thus observed in KAT1 channel). Second, when plotted against the K+/Rb+ mole fraction, KAT1 conductance values passed through a minimum. This minimum is more important for two pore mutants of KAT1 (T259S and T260S) that displayed an increase in PRb/PK. These results are consistent with the idea that KAT1 conduction requires several ions to be present simultaneously within the pore. Therefore, this atypical 'green' member of the Shaker superfamily of K+ channels further shows itself to be an interesting model as well for permeation as for gating mechanism studies.

Amino Acid Sequence↗
PubMed1998

Identification and disruption of a plant shaker-like outward channel involved in K+ release into the xylem sap.

SKOR, a K+ channel identified in Arabidopsis, displays the typical hydrophobic core of the Shaker channel superfamily, a cyclic nucleotide-binding domain, and an ankyrin domain. Expression in Xenopus oocytes identified SKOR as the first member of the Shaker family in plants to be endowed with outwardly rectifying properties. SKOR expression is localized in root stelar tissues. A knockout mutant shows both lower shoot K+ content and lower xylem sap K+ concentration, indicating that SKOR is involved in K+ release into the xylem sap toward the shoots. SKOR expression is strongly inhibited by the stress phytohormone abscisic acid, supporting the hypothesis that control of K+ translocation toward the shoots is part of the plant response to water stress.

Amino Acid Sequence↗
PubMed1998

Magnesium ions promote assembly of channel-like structures from beticolin 0, a non-peptide fungal toxin purified from Cercospora beticola.

Beticolins are toxins produced by the fungus Cercospora beticola. Using beticolin 0 (B0), we have produced a strong and Mg(2+)-dependent increase in the membrane conductance of Arabidopsis protoplasts and Xenopus oocytes. In protein-free artificial bilayers, discrete deflexions of current were observed (12 pS unitary conductance in symmetrical 100 mM KCl) in the presence of B0 (approximately 10 microM) and in the presence of nominal Mg2+. Addition of 50 microM Mg2+ induced a macroscopic current which could be reversed to single channel current by chelating Mg2+ with EDTA. Both unitary and macroscopic currents were ohmic. The increase in conductance of biological membranes triggered by B0 is therefore likely to originate from the ability of this toxin to organize itself into transmembrane pores in the presence of Mg2+. The pore is poorly selective, displaying permeability ratios PCl/PK, PNa/PK and PCa/PK close to 0.3, 0.65 and 0.4, respectively. Such channel-like activity could be involved in the deleterious biological activity of the toxin, by causing the collapse of ionic and electrical gradients through biological membranes together with Ca2+ influx and scrambling of cellular signals.

Animals↗
PubMed1996

The baculovirus/insect cell system as an alternative to Xenopus oocytes. First characterization of the AKT1 K+ channel from Arabidopsis thaliana.

Two plant (Arabidopsis thaliana) K+ transport systems, KAT1 and AKT1, have been expressed in insect cells (Sf9 cell line) using recombinant baculoviruses. Microscopic observation after immunogold staining revealed that the expressed AKT1 and KAT1 polypeptides were mainly associated with internal membranes, but that a minute fraction was targeted to the cell membrane. KAT1 was known, from earlier electrophysiological characterization in Xenopus oocytes, to be an inwardly rectifying voltage-gated channel highly selective for K+, while similar experiments had failed to characterize AKT1. Insect cells expressing KAT1 displayed an exogenous inwardly rectifying K+ conductance reminiscent of that described previously in Xenopus oocytes expressing KAT1. Under similar conditions, cells expressing AKT1 showed a disturbed cell membrane electrical stability that precluded electrophysiological analysis. Use of a baculovirus transfer vector designed so as to decrease the expression level allowed the first electrophysiological characterization of AKT1. The baculovirus system can thus be used as an alternative method when expression in Xenopus oocytes is unsuccessful for electrophysiological characterization of the ion channel of interest. The plant AKT1 protein has been shown in this way to be an inwardly rectifying voltage-gated channel highly selective for K+ ions and sensitive to cGMP.

Adenosine Triphosphate↗
PubMed1996

Functional expression of the plant K+ channel KAT1 in insect cells.

Following the biophysical analysis of plant K+ channels in their natural environment, three members from the green branch of the evolutionary tree of life KAT1, AKT1, and KST1 have recently been identified on the molecular level. Among them, we focussed on the expression and characterization of the Arabidopsis thaliana K+ channel KAT1 in the insect cell line Sf9. The infection of Sf9 cells with KAT1-recombinant baculovirus resulted in functional expression of KAT1 channels, which was monitored by inward-rectifying, K+-selective (impermeable to Na+ and even NH4+) ionic conductance in whole-cell patch-clamp recordings. A voltage threshold as low as -60 to -80mV for voltage activation compared to other plant inward rectifiers in vivo, and to in vitro expression of KAT1 in Xenopus oocytes or yeast, may be indicative for channel modulation by the expression system. A rise in cytoplasmic Ca2+ concentration (up to 1 mM), a regulator of the inward rectifier in Vicia faba guard cells, did not modify the voltage dependence of KAT1 in Sf9 cells. The access to channel function on one side and channel protein on the other make Sf9 cells a suitable heterologous system for studies on the biophysical properties, post-traditional modification and assembly of a green inward rectifier.

Animals↗
PubMed1995

Expression of a cloned plant K+ channel in Xenopus oocytes: analysis of macroscopic currents.

The open reading frame from the Arabidopsis thaliana KAT1 cDNA was cloned in a transcription plasmid between the 3' and 5' untranslated regions of a beta-globin cDNA from Xenopus oocyte. The polyadenylated transcripts resulting from in vitro transcription gave rise to high levels of expression of KAT1 channel when injected in Xenopus oocytes. Upon hyperpolarization, a slow activating current could be recorded, inwardly- or outwardly-directed, depending on K+ external concentration. Predictions of the voltage-gated channel theory were shown to fit the data well. The equivalent gating charge and the half-activation potential ranged around 2 and -145 mV, respectively. KAT1 gating characteristics did not depend on K+ external concentration nor on external pH in the 5.0-7.5 range. KAT1 conductance was, however, increased (40%) when external pH was decreased from 6.5 to 5.0. The apparent affinity constant of KAT1 for K+ lay in the range 15-30 mM, at external pH 7.4. As for many K+ channels of animal cells, external caesium caused a voltage-dependent blockage of inward (but not outward) KAT1 current, whereas tetraethylammonium caused a voltage-independent block of both inward and outward KAT1 currents. In conclusion, high levels of expression made it possible to carry out the first quantitative analysis of KAT1 macroscopic currents. KAT1 channel was shown to display features similar to those of as yet uncloned inward-rectifying voltage-gated channels described in both plant cells (namely guard cells) and animal cells.

Animals↗
PubMed1994

Level of expression in Xenopus oocytes affects some characteristics of a plant inward-rectifying voltage-gated K+ channel.

The plant K+ channel KAT1 shows some similarity to animal voltage-gated channels of the Shaker superfamily. Contrary to these animal counterparts, this plant channel is inwardly rectifying, being gated upon hyperpolarization. Different levels of expression of KAT1 in Xenopus oocytes could be obtained by increasing the amount of injected cRNA. The resulting KAT1 gating and sensitivity to external caesium were significantly changed. Similar findings have been published regarding animal voltage-gated channels. The present data show that plant channels may also undergo modification of their activity upon modification of their level of expression.

Animals↗
PubMed1993

A test for screening monoclonal antibodies to membrane proteins based on their ability to inhibit protein reconstitution into vesicles.

The hypothesis that the binding of an antibody to a membrane protein is likely to prevent the reconstitution of the protein into liposomes was checked, by using the plant plasma membrane H(+)-ATPase (EC 3.6.1.35) as a model system, and two reconstitution procedures: spontaneous insertion (SI) of purified H(+)-ATPase into preformed liposomes, and a detergent-mediated reconstitution (DMR) procedure allowing the reconstitution of the whole membrane protein content. Nine monoclonal antibodies (MABs) raised against H(+)-ATPase were tested. None affected the functioning of the enzyme reconstituted in liposomes, suggesting that the probability to obtain an inhibitory MAB is low. Five MABs inhibited its SI, and seven inhibited its reconstitution in the DMR procedure. These results indicate that it is possible to screen antibodies directed against membrane protein, by making use of their ability to inhibit the reconstitution of these proteins.

Antibodies, Monoclonal↗