Honeybee locomotion is impaired by Am-CaV3 low voltage-activated Ca2+ channel antagonist.

Voltage-gated Ca2+ channels are key transducers of cellular excitability and participate in several crucial physiological responses. In vertebrates, 10 Ca2+ channel genes, grouped in 3 families (CaV1, CaV2 and CaV3), have been described and characterized. Insects possess only one member of each family. These genes have been isolated in a limited number of species and very few have been characterized although, in addition to their crucial role, they may represent a collateral target for neurotoxic insecticides. We have isolated the 3 genes coding for the 3 Ca2+ channels expressed in Apis mellifera. This work provides the first detailed characterization of the honeybee T-type CaV3 Ca2+ channel and demonstrates the low toxicity of inhibiting this channel. Comparing Ca2+ currents recorded in bee neurons and myocytes with Ca2+ currents recorded in Xenopus oocytes expressing the honeybee CaV3 gene suggests native expression in bee muscle cells only. High-voltage activated Ca2+ channels could be recorded in the somata of different cultured bee neurons. These functional data were confirmed by in situ hybridization, immunolocalization and in vivo analysis of the effects of a CaV3 inhibitor. The biophysical and pharmacological characterization and the tissue distribution of CaV3 suggest a role in honeybee muscle function.

A procedure for localisation and electrophysiological characterisation of ion channels heterologously expressed in a plant context.

BACKGROUND: In silico analyses based on sequence similarities with animal channels have identified a large number of plant genes likely to encode ion channels. The attempts made to characterise such putative plant channels at the functional level have most often relied on electrophysiological analyses in classical expression systems, such as Xenopus oocytes or mammalian cells. In a number of cases, these expression systems have failed so far to provide functional data and one can speculate that using a plant expression system instead of an animal one might provide a more efficient way towards functional characterisation of plant channels, and a more realistic context to investigate regulation of plant channels. RESULTS: With the aim of developing a plant expression system readily amenable to electrophysiological analyses, we optimised experimental conditions for preparation and transformation of tobacco mesophyll protoplasts and engineered expression plasmids, that were designed to allow subcellular localisation and functional characterisation of ion channels eventually in presence of their putative (possibly over-expressed) regulatory partners. Two inward K+ channels from the Shaker family were functionally expressed in this system: not only the compliant KAT1 but also the recalcitrant AKT1 channel, which remains electrically silent when expressed in Xenopus oocytes or in mammalian cells. CONCLUSION: The level of endogenous currents in control protoplasts seems compatible with the use of the described experimental procedures for the characterisation of plant ion channels, by studying for instance their subcellular localisation, functional properties, structure-function relationships, interacting partners and regulation, very likely in a more realistic context than the classically used animal systems.

A plant Shaker-like K+ channel switches between two distinct gating modes resulting in either inward-rectifying or "leak" current.

Among the Shaker-like plant potassium channels, AKT2 is remarkable because it mediates both instantaneous "leak-like" and time-dependent hyperpolarisation-activated currents. This unique gating behaviour has been analysed in Xenopus oocytes and in COS and Chinese hamster ovary cells. Whole-cell and single-channel data show that (i) AKT2 channels display two distinct gating modes, (ii) the gating of a given AKT2 channel can change from one mode to the other and (iii) this conversion is under the control of post-translational factor(s). This behaviour is strongly reminiscent of that of the KCNK2 channel, recently reported to be controlled by its phosphorylation state.

Scattering by a slab containing randomly located cylinders: comparison between radiative transfer and electromagnetic simulation.

This study is devoted to the examination of scattering of waves by a slab containing randomly located cylinders. For the first time to our knowledge, the complete transmission problem has been solved numerically. We have compared the radiative transfer theory with a numerical solution of the wave equation. We discuss the coherent effects, such as forward-scattering dip and backscattering enhancement. It is seen that the radiative transfer equation can be used with great accuracy even for optically thin systems whose geometric thickness is comparable with the wavelength. We have also shown the presence of dependent scattering.

Guard cell inward K+ channel activity in arabidopsis involves expression of the twin channel subunits KAT1 and KAT2.

Stomatal opening, which controls gas exchanges between plants and the atmosphere, results from an increase in turgor of the two guard cells that surround the pore of the stoma. KAT1 was the only inward K(+) channel shown to be expressed in Arabidopsis guard cells, where it was proposed to mediate a K(+) influx that enables stomatal opening. We report that another Arabidopsis K(+) channel, KAT2, is expressed in guard cells. More than KAT1, KAT2 displays functional features resembling those of native inward K(+) channels in guard cells. Coexpression in Xenopus oocytes and two-hybrid experiments indicated that KAT1 and KAT2 can form heteromultimeric channels. The data indicate that KAT2 plays a crucial role in the stomatal opening machinery.

A shaker-like K(+) channel with weak rectification is expressed in both source and sink phloem tissues of Arabidopsis.

RNA gel blot and reverse transcription-polymerase chain reaction experiments were used to identify a single K(+) channel gene in Arabidopsis as expressed throughout the plant. Use of the beta-glucuronidase reporter gene revealed expression of this gene, AKT2/AKT3, in both source and sink phloem tissues. The AKT2/AKT3 gene corresponds to two previously identified cDNAs, AKT2 (reconstructed at its 5' end) and AKT3, the open reading frame of the latter being shorter at its 5' end than that of the former. Rapid amplification of cDNA ends with polymerase chain reaction and site-directed mutagenesis was performed to identify the initiation codon for AKT2 translation. All of the data are consistent with the hypothesis that the encoded polypeptide corresponds to the longest open reading frame previously identified (AKT2). Electrophysiological characterization (macroscopic and single-channel currents) of AKT2 in both Xenopus oocytes and COS cells revealed a unique gating mode and sensitivity to pH (weak inward rectification, inhibition, and increased rectification upon internal or external acidification), suggesting that AKT2 has enough functional plasticity to perform different functions in phloem tissue of source and sink organs. The plant stress hormone abscisic acid was shown to increase the amount of AKT2 transcript, suggesting a role for the AKT2 in the plant response to drought.

pH control of the plant outwardly-rectifying potassium channel SKOR.

SKOR, an Arabidopsis depolarisation-activated K+-selective channel, was expressed in Xenopus oocytes, and external and internal pH effects were analysed. Internal pH was manipulated by injections of alkaline or acidic solutions or by acid load from acetate-containing medium. An internal pH decrease from 7.4 to 7.2 induced a strong (ca. 80%) voltage-independent decrease of the macroscopic SKOR current, the macroscopic gating parameters and the single channel conductance remained unchanged. An external acidification from 7.4 to 6.4 had similar effects. It is proposed that pH changes regulate the number of channels available for activation. Sensitivity of SKOR activity to pH in the physiological range suggests that internal and external pH play a role in the regulation of K+ secretion into the xylem sap.

Beticolins, nonpeptidic, polycyclic molecules produced by the phytopathogenic fungus Cercospora beticola, as a new family of ion channel-forming toxins.

Beticolins are toxins produced by Cercospora beticola, a phytopathogenic fungus responsible for the leaf spot disease of sugar beet. They form a family of 20 nonpeptidic compounds (named B0 to B19) that share the same polycyclic skeleton but differ by isomeric configuration (ortho- or para-) and by a variable residue R (bridging two carbons in one of the six cycles). It has been previously shown that B0 assembles itself into a multimeric structure and forms ion channels into planar lipid bilayers (C. Goudet, A.-A. Very, M.-L. Milat, M. Ildefonse, J.-B. Thibaud, H. Sentenac, and J.-P. Blein, Plant J. 14:359-364, 1998). In the present work, we investigate pore formation by three ortho-beticolins, B0, B2, and B4, and their related (i.e., same R) para-isomers, B13, B1, and B3, respectively, using planar lipid bilayers. All beticolins were able to form ion channels with multiple conductance states, although the type of cyclization (ortho- or para-) and residue (R) result in variations of channel conductance and ionic permeability, respectively. Channel formation by beticolins is likely to be involved in the biological activity of these toxins.

Cluster organization and pore structure of ion channels formed by beticolin 3, a nonpeptidic fungal toxin.

Beticolin 3 (B3) belongs to a family of nonpeptidic phytotoxins produced by the fungus Cercospora beticola, which present a broad spectrum of cytotoxic effects. We report here that, at cytotoxic concentration (10 microM), B3 formed voltage-independent, weakly selective ion channels with multiple conductance levels in planar lipid bilayers. In symmetrical standard solutions, conductance values of the first levels were, respectively, 16 +/- 1 pS, 32 +/- 2 pS, and 57 +/- 2 pS (n = 4) and so on, any conductance level being roughly twice the lower one. Whether a cluster organization of elementary channels or different channel structures underlies this particular property was addressed by investigating the ionic selectivity and the pore size corresponding to the first three conductance levels. Both selectivity and pore size were found to be almost independent of the conductance level. This indicated that multiple conductance behavior resulted from a cluster organization of "B3 elementary channels." According to the estimated pore size and analyses of x-ray diffraction of B3 microcrystals, a structural model for "B3 elementary channels" is proposed. The ability to form channels is likely to be involved in the biological activity of beticolins.

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.

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.

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.

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.

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.

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.

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.

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.

Role of apoplast acidification by the H(+) pump : Effect on the sensitivity to pH and CO2 of iron reduction by roots of Brassica napus L.

We have studied the mechanism of the response to iron deficiency in rape (Brassica napus L.), taking into account our previous results: net H(+) extrusion maintains a pH shift between the root apoplast and the solution, and the magnitude of the pH shift decreases as the buffering power in the solution increases. The ferric stress increased the ability of roots to reduce Fe[III]EDTA. Buffering the bulk solution (without change in pH) inhibited Fe[III]EDTA reduction. At constant bulk pH, the inhibition (ratio of the Fe[III]EDTA-reduction rates measured in the presence and in the absence of buffer) increased with the rate of H(+) extrusion (modulated by the length of a pretreatment in 0.2 mM CaSO4). These results support the hypothesis that the apoplastic pH shift caused by H(+) excretion stimulated Fe[III] reduction. The shape of the curves describing the pH-dependency of Fe[III]EDTA reduction in the presence and in the absence of a buffer fitted this hypothesis. When compared to the titration curves of Fe[III]citrate and of Fe[III]EDTA, the curves describing the dependency of the reduction rate of these chelates on pH indicated that the stimulation of Fe[III] reduction by the apoplastic pH shift due to H(+) excretion could result from changes in electrostatic interactions between the chelates and the fixed chargers of the cell wall and-or plasmalemma. Blocking H(+) excretion by vanadate resulted in complete inhibiton of Fe[III] reduction, even in an acidic medium in which there was neither a pH shift nor an inhibitory effect of a buffer. This indicates that the apoplastic pH shift resulting from H(+) pumping is not the only mechanism which is involved in the coupling of Fe[III] reduction to H(+) transport. Our results shed light on the way by which the strong buffering effect of HCO 3 (-) in some soils may be involved in iron deficiency encountered by some of the plants which grow in them.

Effect of HCO - (3) concentration in the absorption solution on the energetic coupling of H(+)-cotransports in roots of Zea mays L.

The effect of HCO 3 (-) on ion absorption by young corn roots was studied in conditions allowing the independent control of both the pH of uptake solution and the CO2 partial pressure in air bubbled through the solution. The surface pH shift in the vicinity of the outer surface of the plasmalemma induced by active H(+) excretion was estimated using the initial uptake rate of acetic acid as a pH probe (Sentenac and Grignon (1987) Plant Physiol. 84, 1367). Acetic acid and orthophosphate uptake rates and NO 3 (-) accumulation were slowed down, while (86)Rb(+) uptake and K(+) accumulation rates were increased by HCO 3 (-) . These effects were similar to those induced by 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid/2-amino-2-(hydroxymethyl)-1,3-propanediol (Hepes-Tris). They were more pronounced when the H(+) excretion was strong, were rapidly reversible and were not additive to those of Hepes-Tris. The hypothesis is advanced that the buffering system CO2/H2CO3/HCO 3 (-) accelerated the diffusion of equivalent H(+) inside the cell wall towards the medium. This attenuated the surface pH shift in the vicinity the plasma membrane and affected the coupling between the proton pump and cotransport systems.