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Ca2+-dependent protein kinases (CPKs) form a large family of 34 genes in Arabidopsis (Arabidopsis thaliana). Based on their dependence on Ca2+, CPKs can be sorted into three types: strictly Ca2+-dependent CPKs, Ca2+-stimulated CPKs (with a significant basal activity in the absence of Ca2+), and essentially calcium-insensitive CPKs. Here, we report on the third type of CPK, CPK13, which is expressed in guard cells but whose role is still unknown. We confirm the expression of CPK13 in Arabidopsis guard cells, and we show that its overexpression inhibits light-induced stomatal opening. We combine several approaches to identify a guard cell-expressed target. We provide evidence that CPK13 (1) specifically phosphorylates peptide arrays featuring Arabidopsis K+ Channel KAT2 and KAT1 polypeptides, (2) inhibits KAT2 and/or KAT1 when expressed in Xenopus laevis oocytes, and (3) closely interacts in plant cells with KAT2 channels (Förster resonance energy transfer-fluorescence lifetime imaging microscopy). We propose that CPK13 reduces stomatal aperture through its inhibition of the guard cell-expressed KAT2 and KAT1 channels. [ABSTRACT FROM AUTHOR]

Calcium (Ca2+) is a second messenger involved in many plant signaling processes. Biotic and abiotic stimuli induce Ca2+ signals within plant cells, which, when decoded, enable these cells to adapt in response to environmental stresses. Multiple examples of Ca2+ signals from plants containing the fluorescent yellow cameleon sensor (YC) have contributed to the definition of the Ca2+ signature in some cell types such as root hairs, pollen tubes and guard cells. YC is, however, of limited use in highly autofluorescent plant tissues, in particular mesophyll cells. Alternatively, the bioluminescent reporter aequorin enables Ca2+ imaging in the whole plant, including mesophyll cells, but this requires specific devices capable of detecting the low amounts of emitted light. Another type of Ca2+ sensor, referred to as GFP-aequorin (G5A), has been engineered as a chimeric protein, which combines the two photoactive proteins from the jellyfish Aequorea victoria, the green fluorescent protein (GFP) and the bioluminescent protein aequorin. The Ca2+-dependent light-emitting property of G5A is based on a bioluminescence resonance energy transfer (BRET) between aequorin and GFP. G5A has been used for over 10 years for enhanced in vivo detection of Ca2+ signals in animal tissues. Here, we apply G5A in Arabidopsis and show that G5A greatly improves the imaging of Ca2+ dynamics in intact plants. We describe a simple method to image Ca2+ signals in autofluorescent leaves of plants with a cooled charge-coupled device (cooled CCD) camera. We present data demonstrating how plants expressing the G5A probe can be powerful tools for imaging of Ca2+ signals. It is shown that Ca2+ signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins. [ABSTRACT FROM AUTHOR]

The essential mineral nutrient potassium (K+) is the most impor- tant inorganic cation for plants and is recognized as a limiting factor for crop yield and quality. Nonetheless, it is only partially understood how K+ contributes to plant productivity. K+ is used as a major active solute to maintain turgor and to drive irreversible and reversible changes in cell volume. K+ also plays an important role in numerous metabolic processes, for example, by serving as an essential cofactor of enzymes. Here, we provide evidence for an additional, previously unrecognized role of K+ in plant growth. By combining diverse experimental approaches with computational cell simulation, we show that K+ circulating in the phloem serves as a decentralized energy storage that can be used to overcome local energy limitations. Posttranslational modification of the phloem-expressed Arabidopsis K+ channel AKT2 taps this "potassium battery," which then efficiently assists the plasma membrane H+-ATPase in energizing the transmembrane phloem (re) loading processes. [ABSTRACT FROM AUTHOR]

Grapevine ( Vitis vinifera), the genome sequence of which has recently been reported, is considered as a model species to study fleshy fruit development and acid fruit physiology. Grape berry acidity is quantitatively and qualitatively affected upon increased K+ accumulation, resulting in deleterious effects on fruit (and wine) quality. Aiming at identifying molecular determinants of K+ transport in grapevine, we have identified a K+ channel, named VvK1.1, from the Shaker family. In silico analyses indicated that VvK1.1 is the grapevine counterpart of the Arabidopsis AKT1 channel, known to dominate the plasma membrane inward conductance to K+ in root periphery cells, and to play a major role in K+ uptake from the soil solution. VvK1.1 shares common functional properties with AKT1, such as inward rectification (resulting from voltage sensitivity) or regulation by calcineurin B-like (CBL)-interacting protein kinase (CIPK) and Ca2+-sensing CBL partners (shown upon heterologous expression in Xenopus oocytes). It also displays distinctive features such as activation at much more negative membrane voltages or expression strongly sensitive to drought stress and ABA (upregulation in aerial parts, downregulation in roots). In roots, VvK1.1 is mainly expressed in cortical cells, like AKT1. In aerial parts, VvK1.1 transcripts were detected in most organs, with expression levels being the highest in the berries. VvK1.1 expression in the berry is localized in the phloem vasculature and pip teguments, and displays strong upregulation upon drought stress, by about 10-fold. VvK1.1 could thus play a major role in K+ loading into berry tissues, especially upon drought stress. [ABSTRACT FROM AUTHOR]

Voltage-gated potassium channels of plants are multimeric proteins built of four α-subunits. In the model plant Arabidopsis thaliana, nine genes coding for K+ channel α-subunits have been identified. When co-expressed in heterologous expression systems, most of them display the ability to form heteromeric K+ channels. Till now it was not clear whether plants use this potential of heteromerization to increase the functional diversity of potassium channels. Here, we designed an experimental approach employing different transgenic plant lines that allowed us to prove the existence of heteromeric K+ channels in plants. The chosen strategy might also be useful for investigating the activity and function of other multimeric channel proteins like, for instance, cyclic-nucleotide gated channels, tandem-pore K+ channels and glutamate receptor channels. [ABSTRACT FROM AUTHOR]

Amongst the nine voltage-gated K+ channel (Kv) subunits expressed in Arabidopsis, AtKC1 does not seem to form functional Kv channels on its own, and is therefore said to be silent. It has been proposed to be a regulatory subunit, and to significantly influence the functional properties of heteromeric channels in which it participates, along with other Kv channel subunits. The mechanisms underlying these properties of AtKC1 remain unknown. Here, the transient (co-)expression of AtKC1, AKT1 and/or KAT1 genes was obtained in tobacco mesophyll protoplasts, which lack endogenous inward Kv channel activity. Our experimental conditions allowed both localization of expressed polypeptides (GFP-tagging) and recording of heterologously expressed Kv channel activity (untagged polypeptides). It is shown that AtKC1 remains in the endoplasmic reticulum unless it is co-expressed with AKT1. In these conditions heteromeric AtKC1-AKT1 channels are obtained, and display functional properties different from those of homomeric AKT1 channels in the same context. In particular, the activation threshold voltage of the former channels is more negative than that of the latter ones. Also, it is proposed that AtKC1-AKT1 heterodimers are preferred to AKT1-AKT1 homodimers during the process of tetramer assembly. Similar results are obtained upon co-expression of AtKC1 with KAT1. The whole set of data provides evidence that AtKC1 is a conditionally-targeted Kv subunit, which probably downregulates the physiological activity of other Kv channel subunits in Arabidopsis. [ABSTRACT FROM AUTHOR]

Plant outward-rectifying K+ channels mediate K+ efflux from guard cells during stomatal closure and from root cells into the xylem for root–shoot allocation of potassium (K). Intriguingly, the gating of these channels depends on the extracellular K+ concentration, although the ions carrying the current are derived from inside the cell . This K+ dependence confers a sensitivity to the extracellular K+ concentration ([K+]) that ensures that the channels mediate K+ efflux only, regardless of the [K+] prevailing outside. We investigated the mechanism of K+-dependent gating of the K+ channel SKOR of Arabidopsis by site-directed mutagenesis. Mutations affecting the intrinsic K+ dependence of gating were found to cluster in the pore and within the sixth transmembrane helix (S6), identifying an ‘S6 gating domain’ deep within the membrane. Mapping the SKOR sequence to the crystal structure of the voltage-dependent K+ channel KvAP from Aeropyrum pernix suggested interaction between the S6 gating domain and the base of the pore helix, a prediction supported by mutations at this site. These results offer a unique insight into the molecular basis for a physiologically important K+-sensory process in plants. [ABSTRACT FROM AUTHOR]

An inward Shaker K+ channel identified in Zea mays (maize), ZmK2.1, displays strong regulation by external K+ when expressed in Xenopus laevis (African clawed frog) oocytes or COS cells. ZmK2.1 is specifically activated by K+ with an apparent Km close to 15 mM independent of the membrane hyperpolarization level. In the absence of K+, ZmK2.1 appears to enter a nonconducting state. Thus, whatever the membrane potential, this maize channel cannot mediate K+ influx in the submillimolar concentration range, unlike its relatives in Arabidopsis thaliana. Its expression is restricted to the shoots, the strongest signal (RT-PCR) being associated with vascular/bundle sheath strands. Based on sequence and gene structure, the closest relatives of ZmK2.1 in Arabidopsis are K+ Arabidopsis Transporter 1 (KAT1) (expressed in guard cells) and KAT2 (expressed in guard cells and leaf phloem). Patch-clamp analyses of guard cell protoplasts reveal a higher functional diversity of K+ channels in maize than in Arabidopsis. Channels endowed with regulation by external K+ similar to that of ZmK2.1 (channel activity regulated by external K+ with a Km close to 15 mM, regulation independent of external Ca2+) constitute a major component of the maize guard cell inward K+ channel population. The presence of such channels in maize might reflect physiological traits of C4 and/or monocotyledonous plants. [ABSTRACT FROM AUTHOR]

SummaryBeticolins are toxins produced by the fungus Cercospora beticola. Using beticolin 0 (B0), we have produced a strong and Mg2+‐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 μM) and in the presence of nominal Mg2+. Addition of 50 μM 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. [ABSTRACT FROM AUTHOR]

The effect of HCOon ion absorption by young corn roots was studied in conditions allowing the independent control of both the pH of uptake solution and the CO 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 NOaccumulation were slowed down, while Rb uptake and K accumulation rates were increased by HCO. 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 CO/HCO/HCOaccelerated 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. [ABSTRACT FROM AUTHOR]

DSC1, a Drosophila channel with sequence similarity to the voltage-gated sodium channel (NaV), was identified over 20 years ago. This channel was suspected to function as a non-specific cation channel with the ability to facilitate the permeation of calcium ions (Ca2+). A honeybee channel homologous to DSC1 was recently cloned and shown to exhibit strict selectivity for Ca2+, while excluding sodium ions (Na+), thus defining a new family of Ca2+ channels, known as CaVv4In this study, we characterize CaVv4, showing that it exhibits an unprecedented type of inactivation, which depends on both an IFM motif and on the permeating divalent cation, like NaV and CaVv1 channels, respectively. CaVv4 displays a specific pharmacology with an unusual response to the alkaloid veratrine. It also possesses an inactivation mechanism that uses the same structural domains as NaV but permeates Ca2+ ions instead. This distinctive feature may provide valuable insights into how voltage- and calcium- dependent modulation of voltage-gated Ca2+ and Na+ channels occur under conditions involving local changes in intracellular calcium concentrations. Our study underscores the unique profile of CaVv4 and defines this channel as a novel class of voltage- gated Ca2+ channels. [ABSTRACT FROM AUTHOR]

Guard cells adjust their volume by changing their ion content due to intense fluxes that, for K+, are believed to flow through inward or outward Shaker channels. Because Shaker channels can be homo- or heterotetramers and Arabidopsis guard cells express at least five genes encoding inward Shaker subunits, including the two major ones, KAT1 and KAT2, the molecular identity of inward Shaker channels operating therein is not yet completely elucidated. Here, we first addressed the properties of KAT1-KAT2 heteromers by expressing KAT1-KAT2 tandems in Xenopus oocytes. Then, computer analyses of the data suggested that coexpression of free KAT1 and KAT2 subunits resulted mainly in heteromeric channels made of two subunits of each type due to some preferential association of KAT1- KAT2 heterodimers at the first step of channel assembly. This was further supported by the analysis of KAT2 effect on KAT1 targeting in tobacco cells. Finally, patch-clamp recordings of native inward channels in wild-type and mutant genotypes strongly suggested that this preferential heteromerization occurs inpianta and that Arabidopsis guard cell inward Shaker channels are mainly heteromers of KAT1 and KAT2 subunits. [ABSTRACT FROM AUTHOR]

Ectomycorrhizal symbiosis between fungi and woody plants strongly improves plant mineral nutrition and constitutes a major biological process in natural ecosystems. Molecular identification and functional characterization of fungal transport systems involved in nutrient uptake are crucial steps toward understanding the improvement of plant nutrition and the symbiotic relationship itself. In the present report a transporter belonging to the Trk family is identified in the model ectomycorrhizal fungus Hebeloma cylindrosporum and named HcTrk1. The Trk family is still poorly characterized, although it plays crucial roles in K+ transport in yeasts and filamentous fungi. In Saccharomyces cerevisiae K+ uptake is mainly dependent on the activity of Trk transporters thought to mediate H+:K+ symport. The ectomycorrhizal HcTrk1 transporter was functional when expressed in Xenopus oocytes, enabling the first electrophysiological characterization of a transporter from the Trk family. HcTrk1 mediates instantaneously activating inwardly rectifying currents, is permeable to both K+ and Na+, and displays channel-like functional properties. The whole set of data and particularly a phenomenon reminiscent of the anomalous mole fraction effect suggest that the transport does not occur according to the classical alternating access model. Permeation appears to occur through a single-file pore, where interactions between Na+ and K+ might result in Na+:K+ co-transport activity. HcTrk1 is expressed in external hyphae that explore the soil when the fungus grows in symbiotic condition. Thus, it could play a major role in both the K+ and Na+ nutrition of the fungus (and of the plant) in nutrient-poor soils. [ABSTRACT FROM AUTHOR]

Assembly of plant Shaker subunits as heterotetramers, increasing channel functional diversity, has been reported. Here we focus on a new interaction, between AKT2 and KAT2 subunits. The assembly as AKT2/KAT2 heterotetramers is demonstrated by (i) a strong signal in two-hybrid tests with intracytoplasmic C-terminal regions, (ii) the effect of KAT2 on AKT2 subunit targeting in tobacco cells, (iii) the complete inhibition of AKT2 currents by co-expression with a dominant-negative KAT2 subunit in Xenopus oocytes, and reciprocally, and (iv) the appearance, upon co-expression of wild-type AKT2 and KAT2 subunits, of new channel functional properties that cannot be explained by the co-existence of two kinds of homotetrameric channels. In particular, the instantaneous current, characteristic of AKT2, displayed new functional features when compared with those of AKT2 homotetramers: activation by external acidification (instead of inhibition) and weak inhibition by calcium. Single channel current measurements in oocytes co-expressing AKT2 and KAT2 revealed a strong preference for incorporation of subunits into heteromultimers and a diversity of individual channels. In planta, these new channels, which may undergo specific regulations, are likely to be formed in guard cells and in the phloem, where they could participate in the control of membrane potential and potassium fluxes. [ABSTRACT FROM AUTHOR]

Abstract: Members of the Shaker-like plant K+ channel family share a common structure, but are highly diverse in their function: they behave as either hyperpolarization-activated inward-rectifying (Kin) channels, or leak-like (Kweak) channels, or depolarization-activated outward-rectifying (Kout) channels. Here we created 256 chimeras between the Kin channel KAT1 and the Kout channel SKOR. The chimeras were screened in a potassium-uptake deficient yeast strain to identify those, which mediate potassium inward currents, i.e., which are functionally equivalent to KAT1. This strategy allowed us to identify three chimeras which differ from KAT1 in three parts of the polypeptide: the cytosolic N-terminus, the cytosolic C-terminus, and the putative voltage-sensor S4. Additionally, mutations in the Kout channel SKOR were generated in order to localize molecular entities underlying its depolarization activation. The triple mutant SKOR-D312N-M313L-I314G, carrying amino-acid changes in the S6 segment, was identified as a channel which did not display any rectification in the tested voltage-range. [Copyright &y& Elsevier]

The regulation of the redox status involves the activation of intracellular pathways as Nrf2 which provides hormetic adaptations against oxidative stress in response to environmental stimuli. In the brain, Nrf2 activation upregulates the formation of glutathione (GSH) which is the primary antioxidant system mainly produced by astrocytes. Astrocytes have also been shown to be themselves the target of oxidative stress. However, how changes in the redox status itself could impact the intracellular Ca 2+ homeostasis in astrocytes is not known, although this could be of great help to understand the neuronal damage caused by oxidative stress. Indeed, intracellular Ca 2+ changes in astrocytes are crucial for their regulatory actions on neuronal networks. We have manipulated GSH concentration in astroglioma cells with selective inhibitors and activators of the enzymes involved in the GSH cycle and analyzed how this could modify Ca 2+ homeostasis. IP 3 -mediated store-operated calcium entry (SOCE), obtained after store depletion elicited by G q -linked purinergic P 2 Y receptors activation, are either sensitized or desensitized, following GSH depletion or increase, respectively. The desensitization may involve decreased expression of the proteins STIM2, Orai1, and Orai3 which support SOCE mechanism. The sensitization process revealed by exposing cells to oxidative stress likely involves the increase in the activity of Calcium Release-Activated Channels (CRAC) and/or in their membrane expression. In addition, we observe that GSH depletion drastically impacts P 2 Y receptor-mediated changes in membrane currents, as evidenced by large increases in Ca 2+ -dependent K + currents. We conclude that changes in the redox status of astrocytes could dramatically modify Ca 2+ responses to Gq-linked GPCR activation in both directions, by impacting store-dependent Ca 2+ -channels, and thus modify cellular excitability under purinergic stimulation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mokrane, Snabi, Cens, Guiramand, Charnet, Bertaud, Menard, Rousset, de Jesus Ferreira, Thibaud, Cohen-Solal, Vignes and Roussel.)