The rectification control and physiological relevance of potassium channel OsAKT2

AKT2 potassium (K⁺) channels are members of the plant Shaker family which mediate dual-directional K⁺ transport with weak voltage-dependency. Here we show that OsAKT2 of rice (Oryza sativa) functions mainly as an inward rectifier with strong voltage-dependency and acutely suppressed outward activity. This is attributed to the presence of a unique K191 residue in the S4 domain. The typical bi-directional leak-like property was restored by a single K191R mutation, indicating that this functional distinction is an intrinsic characteristic of OsAKT2. Furthermore, the opposite R195K mutation of AtAKT2 changed the channel to an inward-rectifier similar to OsAKT2. OsAKT2 was modulated by OsCBL1/OsCIPK23, evoking the outward activity and diminishing the inward current. The physiological relevance in relation to the rectification diversity of OsAKT2 was addressed by functional assembly in the Arabidopsis (Arabidopsis thaliana) akt2 mutant. Overexpression (OE) of OsAKT2 complemented the K⁺ deficiency in the phloem sap and leaves of the mutant plants but did not significantly contribute to the transport of sugars. However, the expression of OsAKT2-K191R overcame both the shortage of phloem K⁺ and sucrose of the akt2 mutant, which was comparable to the effects of the OE of AtAKT2, while the expression of the inward mutation AtAKT2-R195K resembled the effects of OsAKT2. Additionally, OE of OsAKT2 ameliorated the salt tolerance of Arabidopsis.

The presence of a unique K191 residue retains the activity of rice potassium channel OsAKT2 mainly as an inward rectifier (Mode I) that emphasizes its in planta role of phloem K⁺ translocation.     

Studies on Arabidopsis athak5, atakt1 double mutants disclose the range of concentrations at which AtHAK5, AtAKT1 and unknown systems mediate K+ uptake

The high‐affinity K⁺ transporter AtHAK5 and the inward‐rectifier K⁺ channel AtAKT1 have been described to contribute to K⁺ uptake in Arabidopsis thaliana. Studies with T‐DNA insertion lines showed that both systems participate in the high‐affinity range of concentrations and only AtAKT1 in the low‐affinity range. However the contribution of other systems could not be excluded with the information and plant material available. The results presented here with a double knock‐out athak5, atakt1 mutant show that AtHAK5 is the only system mediating K⁺ uptake at concentrations below 0.01 mM. In the range between 0.01 and 0.05 mM K⁺ AtHAK5 and AtAKT1 are the only contributors to K⁺ acquisition. At higher K⁺ concentrations, unknown systems come into operation and participate together with AtAKT1 in low‐affinity K⁺ uptake. These systems can supply sufficient K⁺ to promote plant growth even in the absence of AtAKT1 or in the presence of 10 mM K⁺ where AtAKT1 is not essential.     

Potassium transport in developing fleshy fruits: the grapevine inward K+ channel VvK1.2 is activated by CIPK–CBL complexes and induced in ripening berry flesh cells

The grape berry provides a model for investigating the physiology of non‐climacteric fruits. Increased K⁺ accumulation in the berry has a strong negative impact on fruit acidity (and quality). In maturing berries, we identified a K⁺ channel from the Shaker family, VvK1.2, and two CBL‐interacting protein kinase (CIPK)/calcineurin B‐like calcium sensor (CBL) pairs, VvCIPK04–VvCBL01 and VvCIPK03–VvCBL02, that may control the activity of this channel. VvCBL01 and VvCIPK04 are homologues of Arabidopsis AtCBL1 and AtCIPK23, respectively, which form a complex that controls the activity of the Shaker K⁺ channel AKT1 in Arabidopsis roots. VvK1.2 remained electrically silent when expressed alone in Xenopus oocytes, but gave rise to K⁺ currents when co‐expressed with the pairs VvCIPK03–VvCBL02 or VvCIPK04–VvCBL01, the second pair inducing much larger currents than the first one. Other tested CIPK–CBL pairs expressed in maturing berries were found to be unable to activate VvK1.2. When activated by its CIPK–CBL partners, VvK1.2 acts as a voltage‐gated inwardly rectifying K⁺ channel that is activated at voltages more negative than –100 mV and is stimulated upon external acidification. This channel is specifically expressed in the berry, where it displays a very strong induction at veraison (the inception of ripening) in flesh cells, phloem tissues and perivascular cells surrounding vascular bundles. Its expression in these tissues is further greatly increased upon mild drought stress. VvK1.2 is thus likely to mediate rapid K⁺ transport in the berry and to contribute to the extensive re‐organization of the translocation pathways and transport mechanisms that occurs at veraison.     

Functional characterization and physiological roles of the single Shaker outward K+ channel in Medicago truncatula

The model legume Medicago truncatula possesses a single outward Shaker K⁺ channel, whereas Arabidopsis thaliana possesses two channels of this type, named AtSKOR and AtGORK, with AtSKOR having been shown to play a major role in K⁺ secretion into the xylem sap in the root vasculature and with AtGORK being shown to mediate the efflux of K⁺ across the guard cell membrane, leading to stomatal closure. Here we show that the expression pattern of the single M. truncatula outward Shaker channel, which has been named MtGORK, includes the root vasculature, guard cells and root hairs. As shown by patch‐clamp experiments on root hair protoplasts, besides the Shaker‐type slowly activating outwardly rectifying K⁺ conductance encoded by MtGORK, a second K⁺‐permeable conductance, displaying fast activation and weak rectification, can be expressed by M. truncatula. A knock‐out (KO) mutation resulting in an absence of MtGORK activity is shown to weakly reduce K⁺ translocation to shoots, and only in plants engaged in rhizobial symbiosis, but to strongly affect the control of stomatal aperture and transpirational water loss. In legumes, the early electrical signaling pathway triggered by Nod‐factor perception is known to comprise a short transient depolarization of the root hair plasma membrane. In the absence of the functional expression of MtGORK, the rate of the membrane repolarization is found to be decreased by a factor of approximately two. This defect was without any consequence on infection thread development and nodule production in plants grown in vitro, but a decrease in nodule production was observed in plants grown in soil.     

Involvement of the S4-S5 linker and the C-linker domain regions to voltage-gating in plant Shaker channels: Comparison with animal HCN and Kv channels

Among the different transport systems present in plant cells, Shaker channels constitute the major pathway for K⁺ in the plasma membrane. Plant Shaker channels are members of the 6 transmembrane-1 pore (6TM-1P) cation channel superfamily as the animal Shaker (Kv) and HCN channels. All these channels are voltage-gated K⁺ channels: Kv channels are outward-rectifiers, opened at depolarized voltages and HCN channels are inward-rectifiers, opened by membrane hyperpolarization. Among plant Shaker channels, we can find outward-rectifiers, inward-rectifiers and also weak-rectifiers, with weak voltage dependence. Despite the absence of crystal structures of plant Shaker channels, functional analyses coupled to homology modeling, mostly based on Kv and HCN crystals, have permitted the identification of several regions contributing to plant Shaker channel gating. In the present mini-review, we make an update on the voltage-gating mechanism of plant Shaker channels which seem to be comparable to that proposed for HCN channels.     

Clustering of the K+ channel GORK of Arabidopsis parallels its gating by extracellular K+

GORK is the only outward‐rectifying Kv‐like K⁺ channel expressed in guard cells. Its activity is tightly regulated to facilitate K⁺ efflux for stomatal closure and is elevated in ABA in parallel with suppression of the activity of the inward‐rectifying K⁺ channel KAT1. Whereas the population of KAT1 is subject to regulated traffic to and from the plasma membrane, nothing is known about GORK, its distribution and traffic in vivo. We have used transformations with fluorescently‐tagged GORK to explore its characteristics in tobacco epidermis and Arabidopsis guard cells. These studies showed that GORK assembles in puncta that reversibly dissociated as a function of the external K⁺ concentration. Puncta dissociation parallelled the gating dependence of GORK, the speed of response consistent with the rapidity of channel gating response to changes in the external ionic conditions. Dissociation was also suppressed by the K⁺ channel blocker Ba²⁺. By contrast, confocal and protein biochemical analysis failed to uncover substantial exo‐ and endocytotic traffic of the channel. Gating of GORK is displaced to more positive voltages with external K⁺, a characteristic that ensures the channel facilitates only K⁺ efflux regardless of the external cation concentration. GORK conductance is also enhanced by external K⁺ above 1 mm . We suggest that GORK clustering in puncta is related to its gating and conductance, and reflects associated conformational changes and (de)stabilisation of the channel protein, possibly as a platform for transmission and coordination of channel gating in response to external K⁺.     

Distinct Amino Acids in the C-Linker Domain of the Arabidopsis K+ Channel KAT2 Determine Its Subcellular Localization and Activity at the Plasma Membrane

Shaker K⁺ channels form the major K⁺ conductance of the plasma membrane in plants. They are composed of four subunits arranged around a central ion-conducting pore. The intracellular carboxy-terminal region of each subunit contains several regulatory elements, including a C-linker region and a cyclic nucleotide-binding domain (CNBD). The C-linker is the first domain present downstream of the sixth transmembrane segment and connects the CNBD to the transmembrane core. With the aim of identifying the role of the C-linker in the Shaker channel properties, we performed subdomain swapping between the C-linker of two Arabidopsis (Arabidopsis thaliana) Shaker subunits, K⁺ channel in Arabidopsis thaliana2 (KAT2) and Arabidopsis thaliana K⁺ rectifying channel1 (AtKC1). These two subunits contribute to K⁺ transport in planta by forming heteromeric channels with other Shaker subunits. However, they display contrasting behavior when expressed in tobacco mesophyll protoplasts: KAT2 forms homotetrameric channels active at the plasma membrane, whereas AtKC1 is retained in the endoplasmic reticulum when expressed alone. The resulting chimeric/mutated constructs were analyzed for subcellular localization and functionally characterized. We identified two contiguous amino acids, valine-381 and serine-382, located in the C-linker carboxy-terminal end, which prevent KAT2 surface expression when mutated into the equivalent residues from AtKC1. Moreover, we demonstrated that the nine-amino acid stretch ₃₁₂TVRAASEFA₃₂₀ that composes the first C-linker α-helix located just below the pore is a crucial determinant of KAT2 channel activity. A KAT2 C-linker/CNBD three-dimensional model, based on animal HCN (for Hyperpolarization-activated, cyclic nucleotide-gated K⁺) channels as structure templates, has been built and used to discuss the role of the C-linker in plant Shaker inward channel structure and function.In plants, potassium channels from the Shaker family dominate the plasma membrane (PM) conductance to K⁺ in most cell types and play crucial roles in sustained K⁺ transport (Blatt et al., 2012; Hedrich, 2012; Sharma et al., 2013). Plant Shaker channels, like their homologs in animals (Craven and Zagotta, 2006; Wahl-Schott and Biel, 2009), belong to the six transmembrane-one pore (6TM-1P) cation channel superfamily. Functional channels are tetrameric proteins arranged around a central pore (Daram et al., 1997; Urbach et al., 2000; Dreyer et al., 2004). These channels can result from the assembly of Shaker subunits encoded by the same gene (homotetramers) or by different Shaker genes (heterotetramers). Heterotetramerization has been extensively reported within the inwardly rectifying Shaker channel group (five members in Arabidopsis [Arabidopsis thaliana]) and increased channel functional diversity (Jeanguenin et al., 2008; Lebaudy et al., 2008a).Based on in silico sequence analyses, plant Shaker subunits display a short cytosolic N-terminal domain, followed by the 6TM-1P hydrophobic core, and a long C-terminal cytosolic region in which several domains can be identified. The first one, named C-linker (about 80 amino acids in length), is followed by a cyclic nucleotide-binding domain (CNBD), an ankyrin domain (involved in protein-protein interaction; Lee et al., 2007, Grefen and Blatt, 2012), and a domain named K_HA (Ehrhardt et al., 1997) rich in hydrophobic and acidic residues. Sequence analysis of plant Shaker channels indicates that, among these cytosolic domains, the highest levels of similarity are displayed by the C-linker and the CNBD domains. Interestingly, both domains are also highly conserved in some members from the animal K⁺ channel superfamily, like Hyperpolarization-activated, cyclic nucleotide-gated K⁺ channel (HCN), K⁺ voltage-gated channel, subfamily H (KCNH), and Cyclic-nucleotide-gated ion channel (CNGC). In these animal 6TM-1P channels, the roles of C-linker and CNBD domains have been extensively investigated via crystal structure analyses (Zagotta et al., 2003; Brelidze et al., 2012), whereas plant Shaker channels are still poorly characterized at the structural level (Dreyer et al., 2004; Gajdanowicz et al., 2009; Naso et al., 2009; Garcia-Mata et al., 2010).Aiming at investigating the structure-function relationship of plant Shaker channels, we have used the Arabidopsis Shaker subunit K⁺ channel in Arabidopsis thaliana2 (KAT2) as a model. We developed a subdomain-swapping strategy between KAT2 and another Shaker subunit displaying distinctive features, Arabidopsis thaliana K⁺ rectifying channel1 (AtKC1). The KAT2 subunit can form homomeric or heteromeric inwardly rectifying K⁺ channels at the PM and has been shown to be strongly expressed in guard cells, where it provides a major contribution to the membrane conductance to K⁺ (Pilot et al., 2001; Lebaudy et al., 2008b). In contrast, the behavior of AtKC1 is more complex. In planta, this subunit is coexpressed with other inwardly rectifying Shaker subunits, including KAT2, in different plant tissues (Jeanguenin et al., 2011), and in roots, direct evidence has been obtained that AtKC1 is involved in functional heterotetrameric channel formation with AKT1 (Reintanz et al., 2002; Honsbein et al., 2009). However, experiments performed in tobacco (Nicotiana tabacum) mesophyll protoplasts have revealed that when expressed alone, AtKC1 is entrapped in the endoplasmic reticulum (ER). However, in tobacco protoplasts and Xenopus laevis oocytes, coexpression of AtKC1 with KAT2 or other inwardly rectifying Shaker subunits (AKT1, KAT1, or AKT2) gives rise to functional heteromeric channels (Duby et al., 2008; Jeanguenin et al., 2011). In Arabidopsis, it is interesting that evidence of the AtKC1 retention in the ER compartment, in the absence of other Shaker subunits, is lacking, since in the native tissues, AtKC1 is always expressed with its inward partners, with which it is able to form heteromeric channels.Here, we took advantage of the unique behavior of AtKC1 when expressed in heterologous systems to investigate the structure-function relationship of the C-linker of KAT2 by sequence exchange between these two channel subunits and by site-directed mutagenesis. The C-linker domain, which, to our knowledge, had never been studied as such in plant Shaker channels before, could be predicted to play crucial roles in channel properties due to its strategic location between the channel transmembrane core and the cytoplasmic CNBD domain. The resulting KAT2-AtKC1 chimeras were expressed in tobacco mesophyll protoplasts and in X. laevis oocytes for investigating their subcellular localization and measuring their activity at the cell membrane. Here, we show that two amino acids present in the C-linker are important for channel subcellular location and that a stretch of nine amino acids forming a short helix just below the membrane, downstream of the sixth transmembrane segment of the channel hydrophobic core, is involved in channel gating. The obtained experimental results are discussed in relation with a KAT2 C-linker/CNBD three-dimensional (3D) model based on animal HCN channels as structure templates.     

The K+ channel KZM2 is involved in stomatal movement by modulating inward K+ currents in maize guard cells

Stomata are the major gates in plant leaf that allow water and gas exchange, which is essential for plant transpiration and photosynthesis. Stomatal movement is mainly controlled by the ion channels and transporters in guard cells. In Arabidopsis, the inward Shaker K⁺ channels, such as KAT1 and KAT2, are responsible for stomatal opening. However, the characterization of inward K⁺ channels in maize guard cells is limited. In the present study, we identified two KAT1‐like Shaker K⁺ channels, KZM2 and KZM3, which were highly expressed in maize guard cells. Subcellular analysis indicated that KZM2 and KZM3 can localize at the plasma membrane. Electrophysiological characterization in HEK293 cells revealed that both KZM2 and KZM3 were inward K⁺ (K_in) channels, but showing distinct channel kinetics. When expressed in Xenopus oocytes, only KZM3, but not KZM2, can mediate inward K⁺ currents. However, KZM2 can interact with KZM3 forming heteromeric K_in channel. In oocytes, KZM2 inhibited KZM3 channel conductance and negatively shifted the voltage dependence of KZM3. The activation of KZM2–KZM3 heteromeric channel became slower than the KZM3 channel. Patch‐clamping results showed that the inward K⁺ currents of maize guard cells were significantly increased in the KZM2 RNAi lines. In addition, the RNAi lines exhibited faster stomatal opening after light exposure. In conclusion, the presented results demonstrate that KZM2 functions as a negative regulator to modulate the K_in channels in maize guard cells. KZM2 and KZM3 may form heteromeric K_in channel and control stomatal opening in maize.     

Molecular biology of K transport across the plant cell membrane: What do we learn from comparison between plant species?

Cloning and characterizations of plant K⁺ transport systems aside from Arabidopsis have been increasing over the past decade, favored by the availability of more and more plant genome sequences. Information now available enables the comparison of some of these systems between species. In this review, we focus on three families of plant K⁺ transport systems that are active at the plasma membrane: the Shaker K⁺ channel family, comprised of voltage-gated channels that dominate the plasma membrane conductance to K⁺ in most environmental conditions, and two families of transporters, the HAK/KUP/KT K⁺ transporter family, which includes some high-affinity transporters, and the HKT K⁺ and/or Na⁺ transporter family, in which K⁺-permeable members seem to be present in monocots only. The three families are briefly described, giving insights into the structure of their members and on functional properties and their roles in Arabidopsis or rice. The structure of the three families is then compared between plant species through phylogenic analyses. Within clusters of ortologues/paralogues, similarities and differences in terms of expression pattern, functional properties and, when known, regulatory interacting partners, are highlighted. The question of the physiological significance of highlighted differences is also addressed.     

Relative contribution of AtHAK5 and AtAKT1 to K+ uptake in the high‐affinity range of concentrations

The relative contribution of the high‐affinity K⁺ transporter AtHAK5 and the inward rectifier K⁺ channel AtAKT1 to K⁺ uptake in the high‐affinity range of concentrations was studied in Arabidopsis thaliana ecotype Columbia (Col‐0). The results obtained with wild‐type lines, with T‐DNA insertion in both genes and specific uptake inhibitors, show that AtHAK5 and AtAKT1 mediate the ‐sensitive and the Ba²⁺‐sensitive components of uptake, respectively, and that they are the two major contributors to uptake in the high‐affinity range of Rb⁺ concentrations. Using Rb⁺ as a K⁺ analogue, it was shown that AtHAK5 mediates absorption at lower Rb⁺ concentrations than AtAKT1 and depletes external Rb⁺ to values around 1 μM. Factors such as the presence of K⁺ or during plant growth determine the relative contribution of each system. The presence of in the growth solution inhibits the induction of AtHAK5 by K⁺ starvation. In K⁺‐starved plants grown without , both systems are operative, but when is present in the growth solution, AtAKT1 is probably the only system mediating Rb⁺ absorption, and the capacity of the roots to deplete Rb⁺ is reduced.     

K+ Channels and the Intracellular Calcium Signal in Human Melanoma Cell Proliferation

K⁺ channels, membrane voltage, and intracellular free Ca²⁺ are involved in regulating proliferation in a human melanoma cell line (SK MEL 28). Using patch-clamp techniques, we found an inwardly rectifying K⁺ channel and a calcium-activated K⁺ channel. The inwardly rectifying K⁺ channel was calcium independent, insensitive to charybdotoxin, and carried the major part of the whole-cell current. The K⁺ channel blockers quinidine, tetraethylammonium chloride and Ba²⁺ and elevated extracellular K⁺ caused a dose-dependent membrane depolarization. This depolarization was correlated to an inhibition of cell proliferation. Charybdotoxin affected neither membrane voltage nor proliferation. Basic fibroblast growth factor and fetal calf serum induced a transient peak in intracellular Ca²⁺ followed by a long-lasting Ca²⁺ influx. Depolarization by voltage clamp decreased and hyperpolarization increased intracellular Ca²⁺, illustrating a transmembrane flux of Ca²⁺ following its electrochemical gradient. We conclude that K⁺ channel blockers inhibit cell-cycle progression by membrane depolarization. This in turn reduces the driving force for the influx of Ca²⁺, a messenger in the mitogenic signal cascade of human melanoma cells. Received: 9 May 1995/Revised: 30 January 1996     

Involvement of extracellular oxidative burst in salicylic acid-induced stomatal closure in Arabidopsis

Salicylic acid (SA), a ubiquitous phenolic phytohormone, is involved in many plant physiological processes including stomatal movement. We analysed SA‐induced stomatal closure, production of reactive oxygen species (ROS) and nitric oxide (NO), cytosolic calcium ion ([Ca²⁺]_cyt) oscillations and inward‐rectifying potassium (K⁺_in) channel activity in Arabidopsis. SA‐induced stomatal closure was inhibited by pre‐treatment with catalase (CAT) and superoxide dismutase (SOD), suggesting the involvement of extracellular ROS. A peroxidase inhibitor, SHAM (salicylhydroxamic acid) completely abolished SA‐induced stomatal closure whereas neither an inhibitor of NADPH oxidase (DPI) nor atrbohD atrbohF mutation impairs SA‐induced stomatal closures. 3,3′‐Diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) stainings demonstrated that SA induced H₂O₂ and O₂^– production. Guard cell ROS accumulation was significantly increased by SA, but that ROS was suppressed by exogenous CAT, SOD and SHAM. NO scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO) suppressed the SA‐induced stomatal closure but did not suppress guard cell ROS accumulation whereas SHAM suppressed SA‐induced NO production. SA failed to induce [Ca²⁺]_cyt oscillations in guard cells whereas K⁺_in channel activity was suppressed by SA. These results indicate that SA induces stomatal closure accompanied with extracellular ROS production mediated by SHAM‐sensitive peroxidase, intracellular ROS accumulation and K⁺_in channel inactivation.     

An inwardly rectifying potassium channel in the basolateral membrane of sheep parotid secretory cells

Summary Using whole-cell patch-clamp techniques, we demonstrate that sheep parotid secretory cells have both inwardly and outwardly rectifying currents. The outwardly rectifying current, which is blocked by 10 mmol/liter tetraethylammonium (TEA) applied extracellularly, is probably carried by the 250 pS Ca²⁺-and voltage-activated K⁺ (BK) channel which has been described in previous studies. In contrast, the inwardly rectifying current, which is also carried by K⁺ ions, is not sensitive to TEA. It is similar to the inwardly rectifying currents observed in many excitable tissues in that (i) its conductance is dependent on the square root of the extracellular K⁺, (ii) the voltage range over which it is activated is influenced by the extracellular K⁺ concentration and (iii) it is blocked by the addition of Cs⁺ ions (670 µmol/liter) to the bathing solution. Our previously published cell-attached patch studies have shown that the channel type most commonly observed in the basolateral membrane of unstimulated sheep parotid secretory cells is a K⁺ channel with a conductance of 30 pS and, in this study, we find that its conductance also depends on the square root of the extracellular K⁺ concentration. It thus seems likely that it carries the inwardly rectifying K⁺ current seen in the whole-cell studies.     

HvAKT2 and HvHAK1 confer drought tolerance in barley through enhanced leaf mesophyll H+ homoeostasis

Plant K⁺ uptake typically consists low—affinity mechanisms mediated by Shaker K⁺ channels (AKT/KAT/KC) and high‐affinity mechanisms regulated by HAK/KUP/KT transporters, which are extensively studied. However, the evolutionary and genetic roles of both K⁺ uptake mechanisms for drought tolerance are not fully explored in crops adapted to dryland agriculture. Here, we employed evolutionary bioinformatics, biotechnological and electrophysiological approaches to determine the role of two important K⁺ transporters HvAKT2 and HvHAK1 in drought tolerance in barley. HvAKT2 and HvHAK1 were cloned and functionally characterized using barley stripe mosaic virus‐induced gene silencing (BSMV‐VIGS) in drought‐tolerant wild barley XZ5 and agrobacterium‐mediated gene transfer in the barley cultivar Golden Promise. The hallmarks of the K⁺ selective filters of AKT2 and HAK1 are both found in homologues from strepotophyte algae, and they are evolutionarily conserved in strepotophyte algae and land plants. HvAKT2 and HvHAK1 are both localized to the plasma membrane and have high selectivity to K⁺ and Rb⁺ over other tested cations. Overexpression of HvAKT2 and HvHAK1 enhanced K⁺ uptake and H⁺ homoeostasis leading to drought tolerance in these transgenic lines. Moreover, HvAKT2‐ and HvHAK1‐overexpressing lines showed distinct response of K⁺, H⁺ and Ca²⁺ fluxes across plasma membrane and production of nitric oxide and hydrogen peroxide in leaves as compared to the wild type and silenced lines. High‐ and low‐affinity K⁺ uptake mechanisms and their coordination with H⁺ homoeostasis play essential roles in drought adaptation of wild barley. These findings can potentially facilitate future breeding programs for resilient cereal crops in a changing global climate.     

Calcineurin B‐like protein CBL10 directly interacts with AKT1 and modulates K+ homeostasis in Arabidopsis

Potassium transporters and channels play crucial roles in K⁺ uptake and translocation in plant cells. These roles are essential for plant growth and development. AKT1 is an important K⁺ channel in Arabidopsis roots that is involved in K⁺ uptake. It is known that AKT1 is activated by a protein kinase CIPK23 interacting with two calcineurin B‐like proteins CBL1/CBL9. The present study showed that another calcineurin B‐like protein (CBL10) may also regulate AKT1 activity. The CBL10‐over‐expressing lines showed a phenotype as sensitive as that of the akt1 mutant under low‐K⁺ conditions. In addition, the K⁺ content of both CBL10‐over‐expressing lines and akt1 mutant plants were significantly reduced compared with wild‐type plants. Moreover, CBL10 directly interacted with AKT1, as verified in yeast two‐hybrid, BiFC and co‐immunoprecipitation experiments. The results of electrophysiological analysis in both Xenopus oocytes and Arabidopsis root cell protoplasts demonstrated that CBL10 impairs AKT1‐mediated inward K⁺ currents. Furthermore, the results from the yeast two‐hybrid competition assay indicated that CBL10 may compete with CIPK23 for binding to AKT1 and negatively modulate AKT1 activity. The present study revealed a CBL‐interacting protein kinase‐independent regulatory mechanism of calcineurin B‐like proteins in which CBL10 directly regulates AKT1 activity and affects ion homeostasis in plant cells.     

Potassium Translocation into the Root Xylem

Abstract: Potassium is the most abundant cation in cells of higher plants and plays vital roles in plant growth and develop ment. Since the soil is the only source of potassium, plant roots are well adapted to exploit the soil for potassium and supply it to the leaves. Transport across the root can be divided into three stages: uptake into the root symplast, transport across the symplast and release into the xylem. Uptake kinetics of potassium have been studied extensively in the past and sug gested the presence of high and low affinity systems. Molecular and electrophysiological techniques have now confirmed the existence of discrete transporters encoded by a number of genes. Surprisingly, detailed characterisation of the transpor ters using reverse genetics and heterologous expression shows that a number of the transporters (AKT and AtKUP family) func tion both in the low (μM) and high (mM) K⁺ range. Electrophy siological studies indicate that K⁺ uptake by roots is coupled to H⁺, to drive uptake from micromolar K⁺. However, thus far only Na⁺ coupled K⁺ transport has been demonstrated (HKT1). Ion channels play a major role in the exchange of potassium be tween the symplast and the xylem. An outward rectifying chan nel (KORC) mediates potassium release. Cloning of the gene en coding this channel (SKOR) shows that it belongs to the Shaker super-family. Both electrophysiological and genetic studies demonstrate that K⁺ release through this channel is controlled by the stress hormone abscisic acid. Interestingly, xylem par enchyma cells of young barley roots also contain a number of in ward rectifying K⁺ channels that are controlled by G-proteins. The involvement of G-proteins emphasises once more that po tassium transport at the symplast/xylem boundary is under hor monal control. The role of the electrical potential difference across the symplastxylem boundary in controlling potassium release is discussed.     

OsKAT2 is the prevailing functional inward rectifier potassium channels in rice guard cell

AtKAT1 plays roles as a major channel to uptake K⁺ in guard cell when stomata open in dicot model plant Arabidopsis. In a recent publication, we isolated 3 KAT-like potassium channels in rice. We expressed them in CHO cell to identify electrophysiological characteristics of the channels. OsKAT2 showed much bigger inwardly rectifying potassium channel activities among them. The histochemical X-glu staining of transgenic rice leaf blades expressing β-glucuronidase fused with OsKAT2 promoter showed that the OsKAT2 is dominantly expressed in rice guard cell. These findings indicate that OsKAT2 may be a functional ortholog of AtKAT1 in rice. Thus this gene will be the prime target for engineering the guard cell movement to improve drought tolerance in monocot plants, including most major crops.     

The trafficking protein SYP121 of Arabidopsis connects programmed stomatal closure and K⁺ channel activity with vegetative growth

The vesicle‐trafficking protein SYP121 (SYR1/PEN1) was originally identified in association with ion channel control at the plasma membrane of stomatal guard cells, although stomata of the Arabidopsis syp121 loss‐of‐function mutant close normally in ABA and high Ca²⁺. We have now uncovered a set of stomatal phenotypes in the syp121 mutant that reduce CO₂ assimilation, slow vegetative growth and increase water use efficiency in the whole plant, conditional upon high light intensities and low relative humidity. Stomatal opening and the rise in stomatal transpiration of the mutant was delayed in the light and following Ca²⁺‐evoked closure, consistent with a constitutive form of so‐called programmed stomatal closure. Delayed reopening was observed in the syp121, but not in the syp122 mutant lacking the homologous gene product; the delay was rescued by complementation with wild‐type SYP121 and was phenocopied in wild‐type plants in the presence of the vesicle‐trafficking inhibitor Brefeldin A. K⁺ channel current that normally mediates K⁺ uptake for stomatal opening was suppressed in the syp121 mutant and, following closure, its recovery was slowed compared to guard cells of wild‐type plants. Evoked stomatal closure was accompanied by internalisation of GFP‐tagged KAT1 K⁺ channels in both wild‐type and syp121 mutant guard cells, but their subsequently recycling was slowed in the mutant. Our findings indicate that SYP121 facilitates stomatal reopening and they suggest that K⁺ channel traffic and recycling to the plasma membrane underpins the stress memory phenomenon of programmed closure in stomata. Additionally, they underline the significance of vesicle traffic for whole‐plant water use and biomass production, tying SYP121 function to guard cell membrane transport and stomatal control.     

ATP-sensitive potassium channels and vascular function

Fluorescence-based approaches provide powerful techniques to directly report structural dynamics underlying gating processes in Shaker K_V channels. Here, following on from work carried out in Shaker channels, we have used voltage clamp fluorimetry for the first time to study voltage sensor motions in mammalian K_V1.5 channels, by attaching TMRM fluorescent probes to substituted cysteine residues in the S3-S4 linker of K_V1.5 (A397C). Compared with the Shaker channel, there are significant differences in the fluorescence signals that occur on activation of the channel. In addition to a well-understood fluorescence quenching signal associated with S4 movement, we have recorded a unique partial recovery of fluorescence after the quenching that is attributable to gating events at the outer pore mouth,¹ that is not seen in Shaker despite significant homology between it and Kv1.5 channels in the S5-P loop-S6 region. Extracellular potassium is known to modulate C-type inactivation in Shaker and K_V channels at sites in the outer pore mouth, and so here we have measured the concentration-dependence of potassium effects on the fluorescence recovery signals from A397C. Elevation of extracellular K⁺ inhibits the rapid fluorescence recovery, with complete abolition at 99 mM K⁺, and an IC₅₀ of 29 mM K⁺_o. These experiments suggest that the rapid fluorescence recovery reflects early gating movements associated with inactivation, modulated by extracellular K⁺, and further support the idea that outer pore motions occur rapidly after K_V1.5 channel opening and can be observed by fluorophores attached to the S3-S4 linker.