Modulation of plant TPC channels by polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) are powerful modulators of several animal ion channels. It is shown here that PUFAs strongly affect the activity of the Slow Vacuolar (SV) channel encoded by the plant TPC1 gene. The patch-clamp technique was applied to isolated vacuoles from carrot taproots and Arabidopsis thaliana mesophyll cells and arachidonic acid (AA) was chosen as a model molecule for PUFAs. Our study was extended to different PUFAs including the endogenous alpha-linolenic acid (ALA). The addition of micromolar concentrations of AA reversibly inhibited the SV channel decreasing the maximum open probability and shifting the half activation voltage to positive values. Comparing the effects of different PUFAs, it was found that the length of the lipophilic acyl chain, the number of double bonds and the polar head were critical for channel modulation.The experimental data can be reproduced by a simple three-state model, in which PUFAs do not interact directly with the voltage sensors but affect the voltage-independent transition that leads the channel from the open state to the closed configuration. The results indicate that lipids play an important role in co-ordinating ion channel activities similar to what is known from animal cell

The vacuolar channel VvALMT9 mediates malate and tartrate accumulation in berries of Vitis vinifera

Vitis vinifera L. represents an economically important fruit species. Grape and wine flavour is made from a complex set of compounds. The acidity of berries is a major parameter in determining grape berry quality for wine making and fruit consumption. Despite the importance of malic and tartaric acid (TA) storage and transport for grape berry acidity, no vacuolar transporter for malate or tartrate has been identified so far. Some members of the aluminium-activated malate transporter (ALMT) anion channel family from Arabidopsis thaliana have been shown to be involved in mediating malate fluxes across the tonoplast. Therefore, we hypothesised that a homologue of these channels could have a similar role in V. vinifera grape berries. We identified homologues of the Arabidopsis vacuolar anion channel AtALMT9 through a TBLASTX search on the V. vinifera genome database. We cloned the closest homologue of AtALMT9 from grape berry cDNA and designated it VvALMT9. The expression profile revealed that VvALMT9 is constitutively expressed in berry mesocarp tissue and that its transcription level increases during fruit maturation. Moreover, we found that VvALMT9 is targeted to the vacuolar membrane. Using patch-clamp analysis, we could show that, besides malate, VvALMT9 mediates tartrate currents which are higher than in its Arabidopsis homologue. In summary, in the present study we provide evidence that VvALMT9 is a vacuolar malate channel expressed in grape berries. Interestingly, in V. vinifera, a tartrate-producing plant, the permeability of the channel is apparently adjusted to T

Novel Electrical Signaling: First Fast Voltage-Gated Sodium Channel Identified Outside of the Animal Kingdom

International audienc

Heterologous expression reveals that GABA does not directly inhibit the vacuolar anion channel AtALMT9

International audienceGABA, a molecule involved in the regulation of the stomata aperture and drought tolerance, does not modify vacuolar anion fluxes mediated by the anion channel AtALMT9. Dear Editor, GABA (gamma-aminobutyric acid) is a well-known neurotransmitter activating Clchannels in synapses and having an inhibitory effect on neural activity. In plants, GABA was proposed to have different functions, for example, in the regulation of the carbon/nitrogen balance and resistance/tolerance to different biotic and abiotic stresses

Caractérisation fonctionnelle du transporteur vacuolaire ClCa de Arabidopsis thaliana (activité d'échange de NO3-/H+ et régulation par les nucléotides)

Le nitrate représente pour la majeure partie des plantes terrestres une importante source d azote. Les plantes absorbent le nitrate du sol, l assimilent dans des composés azotés et en stockent le surplus dans la vacuole centrale. Les protéines responsables du transport intracellulaire du nitrate sont encore inconnues, mais il a été suggéré que des protéines de la famille des CLC (ChLoride Channel) pouvaient être impliquées dans ces fonctions. Ces travaux de thèse démontrent que la protéine AtClCa (Arabidopsis thaliana ClC) est localisée dans la membrane vacuolaire et transporte des anions au travers du tonoplaste. Ils démontrent également que AtClCa est un antiport NO3-/H+ avec une stoechiométrie de 2 NO3- transportés pour chaque H+ transféré. Sa propriété d antiport, ainsi que sa spécificité pour le nitrate, permettent à AtClCa d accumuler le nitrate dans la vacuole. Son activité de transport est inhibée par l ATP, alors que l ADP et l AMP n ont pas d effet sur le courant porté par AtClCa. Cependant, l AMP et l ATP entrent en compétition pour le site d interaction avec AtClCa. Ce site d interaction avec les nucléotides se trouve probablement dans le domaine C-terminal de la protéine. Le domaine C-terminal de AtClCa a été modélisé en utilisant la structure du C-terminal de la protéine humaine hClC-5. Les données de dynamique moléculaire obtenues via ce modèle s reproduisent les propriétés d interaction entre AtCLCa et les nucléotides déterminées expérimentalement. L ensemble de ces données montre que AtClCa est un élément clef de l homéostasie du nitrate intracellulaire, et que son activité de transport est régulée en fonction de l état métabolique de la cellule.Nitrate is the major nitrogen source for plants. Plants absorb nitrate from the soil, assimilate it in nitrogen compounds and stock the surplus of nitrate in the central vacuole. The proteins responsible for the intracellular transport of nitrate are unknown. It has been suggested that proteins that belong to the CLC family (ChLoride Channel) could be involved in nitrate intracellular homeostasis. In the present thesis we showed that AtClCa (Arabidopsis thaliana ClCa) is localized in the vacuolar membrane, and demonstrated its ability to mediate anions currents across the tonoplast. We could also demonstrate that AtClCa is a NO3-/H+ antiporter with a stoichiometry of 2NO3- transported for each H+ transferred. The antiporter property, together with nitrate specificity, enable AtClCa to mediate the accumulation of nitrate in the vacuole. We also showed that the current mediated by AtClCa is inhibited by ATP. ADP and AMP have no effect on AtClCa current, but AMP competes with ATP for the site of interaction with AtClCa. The interaction of nucleotides with AtClCa takes place presumably at its C-terminal. The C-terminal domain of AtClCa has been modelled by homology using the structure of the C-terminal of hClC-5. The data obtained with this model by molecular dynamics simulations can reproduce the experimental data on the interaction properties of AtClCa and the nucleotides. The set of data presented in this thesis shows that AtClCa is a key element for the homeostasis of intracellular nitrate, and that its transport activity is regulated in function of the metabolic state of the cell.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Cytosolic nucleotides block and regulate the Arabidopsis vacuolar anion channel AtALMT9

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al(3+) to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell

Vacuolar transporters in their physiological context

Vacuoles in vegetative tissues allow the plant surface to expand by accumulating energetically cheap inorganic osmolytes, and thereby optimize the plant for absorption of sunlight and production of energy by photosynthesis. Some specialized cells, such as guard cells and pulvini motor cells, exhibit rapid volume changes. These changes require the rapid release and uptake of ions and water by the vacuole and are a prerequisite for plant survival. Furthermore, seed vacuoles are important storage units for the nutrients required for early plant development. All of these fundamental processes rely on numerous vacuolar transporters. During the past 15 years, the transporters implicated in most aspects of vacuolar function have been identified and characterized. Vacuolar transporters appear to be integrated into a regulatory network that controls plant metabolism. However, little is known about the mode of action of these fundamental processes, and deciphering the underlying mechanisms remains a challenge for the future