Inverse regulation of SOS1 and HKT1 protein localization and stability by SOS3/CBL4 in Arabidopsis thaliana.

To control net sodium (Na+) uptake, Arabidopsis plants utilize the plasma membrane (PM) Na+/H+ antiporter SOS1 to achieve Na+ efflux at the root and Na+ loading into the xylem, and the channel-like HKT1;1 protein that mediates the reverse flux of Na+ unloading off the xylem. Together, these opposing transport systems govern the partition of Na+ within the plant yet they must be finely co-regulated to prevent a futile cycle of xylem loading and unloading. Here, we show that the Arabidopsis SOS3 protein acts as the molecular switch governing these Na+ fluxes by favoring the recruitment of SOS1 to the PM and its subsequent activation by the SOS2/SOS3 kinase complex under salt stress, while commanding HKT1;1 protein degradation upon acute sodic stress. SOS3 achieves this role by direct and SOS2-independent binding to previously unrecognized functional domains of SOS1 and HKT1;1. These results indicate that roots first retain moderate amounts of salts to facilitate osmoregulation, yet when sodicity exceeds a set point, SOS3-dependent HKT1;1 degradation switches the balance toward Na+ export out of the root. Thus, SOS3 functionally links and co-regulates the two major Na+ transport systems operating in vascular plants controlling plant tolerance to salinity.

The Arabidopsis protein NPF6.2/NRT1.4 is a plasma membrane nitrate transporter and a target of protein kinase CIPK23.

Nitrate and potassium nutrition is tightly coordinated in vascular plants. Physiological and molecular genetics studies have demonstrated that several NPF/NRT1 nitrate transporters have a significant impact on both uptake and the root-shoot partition of these nutrients. However, how these traits are biochemically connected remain controversial since some NPF proteins, e.g. NPF7.3/NRT1.5, have been suggested to mediate K+/H+ exchange instead of nitrate fluxes. Here we show that NPF6.2/NRT1.4, a protein that gates nitrate accumulation at the leaf petiole of Arabidopsis thaliana, also affects the root/shoot distribution of potassium. We demonstrate that NPF6.2/NRT1.4 is a plasma membrane nitrate transporter phosphorylated at threonine-98 by the CIPK23 protein kinase that is a regulatory hub for nitrogen and potassium nutrition. Heterologous expression of NPF6.2/NRT1.4 and NPF7.3/NRT1.5 in yeast mutants with altered potassium uptake and efflux systems showed no evidence of nitrate-dependent potassium transport by these proteins.

HKT sodium and potassium transporters in Arabidopsis thaliana and related halophyte species.

High salinity induces osmotic stress and often leads to sodium ion-specific toxicity, with inhibitory effects on physiological, biochemical and developmental pathways. To cope with increased Na+ in soil water, plants restrict influx, compartmentalize ions into vacuoles, export excess Na+ from the cell, and distribute ions between the aerial and root organs. In this review, we discuss our current understanding of how high-affinity K+ transporters (HKT) contribute to salinity tolerance, focusing on HKT1-like family members primarily involved in long-distance transport, and in the recent research in the model plant Arabidopsis and its halophytic counterparts of the Eutrema genus. Functional characterization of the salt overly sensitive (SOS) pathway and HKT1-type transporters in these species indicate that they utilize similar approaches to deal with salinity, regardless of their tolerance.

A folding reaction at the C-terminal domain drives temperature sensing in TRPM8 channels.

In mammals, temperature-sensitive TRP channels make membrane conductance of cells extremely temperature dependent, allowing the detection of temperature ranging from noxious cold to noxious heat. We progressively deleted the distal carboxyl terminus domain (CTD) of the cold-activated melastatin receptor channel, TRPM8. We found that the enthalpy change associated with channel gating is proportional to the length of the CTD. Deletion of the last 36 amino acids of the CTD transforms TRPM8 into a reduced temperature-sensitivity channel (Q10 ∼4). Exposing the intracellular domain to a denaturing agent increases the energy required to open the channel indicating that cold drives channel gating by stabilizing the folded state of the CTD. Experiments in the presence of an osmoticant agent suggest that channel gating involves a change in solute-inaccessible volume in the CTD of ∼1,900 Å3 This volume matches the void space inside the coiled coil according to the cryogenic electron microscopy structure of TRPM8. The results indicate that a folding-unfolding reaction of a specialized temperature-sensitive structure is coupled to TRPM8 gating.

Coordinated Transport of Nitrate, Potassium, and Sodium.

Potassium (K+) and nitrogen (N) are essential nutrients, and their absorption and distribution within the plant must be coordinated for optimal growth and development. Potassium is involved in charge balance of inorganic and organic anions and macromolecules, control of membrane electrical potential, pH homeostasis and the regulation of cell osmotic pressure, whereas nitrogen is an essential component of amino acids, proteins, and nucleic acids. Nitrate (NO3 -) is often the primary nitrogen source, but it also serves as a signaling molecule to the plant. Nitrate regulates root architecture, stimulates shoot growth, delays flowering, regulates abscisic acid-independent stomata opening, and relieves seed dormancy. Plants can sense K+/NO3 - levels in soils and adjust accordingly the uptake and root-to-shoot transport to balance the distribution of these ions between organs. On the other hand, in small amounts sodium (Na+) is categorized as a "beneficial element" for plants, mainly as a "cheap" osmolyte. However, at high concentrations in the soil, Na+ can inhibit various physiological processes impairing plant growth. Hence, plants have developed specific mechanisms to transport, sense, and respond to a variety of Na+ conditions. Sodium is taken up by many K+ transporters, and a large proportion of Na+ ions accumulated in shoots appear to be loaded into the xylem by systems that show nitrate dependence. Thus, an adequate supply of mineral nutrients is paramount to reduce the noxious effects of salts and to sustain crop productivity under salt stress. In this review, we will focus on recent research unraveling the mechanisms that coordinate the K+-NO3 -; Na+-NO3 -, and K+-Na+ transports, and the regulators controlling their uptake and allocation.

Recognition and Activation of the Plant AKT1 Potassium Channel by the Kinase CIPK23.

Plant growth largely depends on the maintenance of adequate intracellular levels of potassium (K+). The families of 10 Calcineurin B-Like (CBL) calcium sensors and 26 CBL-Interacting Protein Kinases (CIPKs) of Arabidopsis (Arabidopsis thaliana) decode the calcium signals elicited by environmental inputs to regulate different ion channels and transporters involved in the control of K+ fluxes by phosphorylation-dependent and -independent events. However, the detailed molecular mechanisms governing target specificity require investigation. Here, we show that the physical interaction between CIPK23 and the noncanonical ankyrin domain in the cytosolic side of the inward-rectifier K+ channel AKT1 regulates kinase docking and channel activation. Point mutations on this domain specifically alter binding to CIPK23, enhancing or impairing the ability of CIPK23 to regulate channel activity. Our data demonstrate the relevance of this protein-protein interaction that contributes to the formation of a complex between CIPK23/CBL1 and AKT1 in the membrane for the proper regulation of K+ transport.

Regulation of K+ Nutrition in Plants.

Modern agriculture relies on mineral fertilization. Unlike other major macronutrients, potassium (K+) is not incorporated into organic matter but remains as soluble ion in the cell sap contributing up to 10% of the dry organic matter. Consequently, K+ constitutes a chief osmoticum to drive cellular expansion and organ movements, such as stomata aperture. Moreover, K+ transport is critical for the control of cytoplasmic and luminal pH in endosomes, regulation of membrane potential, and enzyme activity. Not surprisingly, plants have evolved a large ensemble of K+ transporters with defined functions in nutrient uptake by roots, storage in vacuoles, and ion translocation between tissues and organs. This review describes critical transport proteins governing K+ nutrition, their regulation, and coordinated activity, and summarizes our current understanding of signaling pathways activated by K+ starvation.

The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2.

The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H+-ATPase-energized K+ uptake. Moreover, through reversible post-translational modifications it can also function as an open, K+-selective channel, which taps a 'potassium battery', providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals.

Cloning and functional characterization of HKT1 and AKT1 genes of Fragaria spp.-Relationship to plant response to salt stress.

Commercial strawberry, Fragaria x ananassa Duch., is a species sensitive to salinity. Under saline conditions, Na+ uptake by the plant is increased, while K+ uptake is significantly reduced. Maintaining an adequate K+/Na+ cytosolic ratio determines the ability of the plant to survive in saline environments. The goal of the present work was to clone and functionally characterize the genes AKT1 and HKT1 involved in K+ and Na+ transport in strawberry and to determine the relationship of these genes with the responses of three Fragaria spp. genotypes having different ecological adaptations to salt stress. FaHKT1 and FcHKT1 proteins from F. x ananassa and F. chiloensis have 98.1% of identity, while FaAKT1 and FcAKT1 identity is 99.7%. FaHKT1 and FaAKT1 from F. x ananassa, were functionally characterized in Xenopus oocytes. FaHKT1, belongs to the group I of HKT transporters and is selective for Na+. Expression of FaAKT1 in oocytes showed that the protein is a typical inward-rectifying and highly K+-selective channel. The relative expression of Fragaria HKT1 and AKT1 genes was studied in roots of F. x ananassa cv. Camarosa and of F. chiloensis (accessions Bau and Cucao) grown under salt stress. The expression of AKT1 was transiently increased in 'Camarosa', decreased in 'Cucao' and was not affected in 'Bau' upon salt stress. HKT1 expression was significantly increased in roots of 'Cucao' and was not affected in the other two genotypes. The increased relative expression of HKT1 and decreased expression of AKT1 in 'Cucao' roots correlates with the higher tolerance to salinity of this genotype in comparison with 'Camarosa' and 'Bau'.

A Single Amino-Acid Substitution in the Sodium Transporter HKT1 Associated with Plant Salt Tolerance.

A crucial prerequisite for plant growth and survival is the maintenance of potassium uptake, especially when high sodium surrounds the root zone. The Arabidopsis HIGH-AFFINITY K(+) TRANSPORTER1 (HKT1), and its homologs in other salt-sensitive dicots, contributes to salinity tolerance by removing Na(+) from the transpiration stream. However, TsHKT1;2, one of three HKT1 copies in Thellungiella salsuginea, a halophytic Arabidopsis relative, acts as a K(+) transporter in the presence of Na(+) in yeast (Saccharomyces cerevisiae). Amino-acid sequence comparisons indicated differences between TsHKT1;2 and most other published HKT1 sequences with respect to an Asp residue (D207) in the second pore-loop domain. Two additional T salsuginea and most other HKT1 sequences contain Asn (n) in this position. Wild-type TsHKT1;2 and altered AtHKT1 (AtHKT1(N-D)) complemented K(+)-uptake deficiency of yeast cells. Mutant hkt1-1 plants complemented with both AtHKT1(N) (-) (D) and TsHKT1;2 showed higher tolerance to salt stress than lines complemented by the wild-type AtHKT1 Electrophysiological analysis in Xenopus laevis oocytes confirmed the functional properties of these transporters and the differential selectivity for Na(+) and K(+) based on the n/d variance in the pore region. This change also dictated inward-rectification for Na(+) transport. Thus, the introduction of Asp, replacing Asn, in HKT1-type transporters established altered cation selectivity and uptake dynamics. We describe one way, based on a single change in a crucial protein that enabled some crucifer species to acquire improved salt tolerance, which over evolutionary time may have resulted in further changes that ultimately facilitated colonization of saline habitats.

Gating of thermally activated channels.

A class of ion channels that belongs to the transient receptor potential (TRP) superfamily and is present in specialized neurons that project to the skin has evolved as temperature detectors. These channels are classified into subfamilies, namely canonical (TRPC), melastatin (TRPM), ankyrin (TRPA), and vanilloid (TRPV). Some of these channels are activated by heat (TRPM2/4/5, TRPV1-4), while others by cold (TRPA1, TRPC5, and TRPM8). The general structure of these channels is closely related to that of the voltage-dependent K(+) channels, with their subunits containing six transmembrane segments that form tetramers. Thermal TRP channels are polymodal receptors. That is, they can be activated by temperature, voltage, pH, lipids, and agonists. The high temperature sensitivity in these thermal TRP channels is due to a large enthalpy change (∼100 kcal/mol), which is about five times the enthalpy change in voltage-dependent gating. The characterization of the macroscopic currents and single-channel analysis demonstrated that gating by temperature is complex and best described by branched or allosteric models containing several closed and open states. The identification of molecular determinants of temperature sensitivity in TRPV1, TRPA1, and TRPV3 strongly suggest that thermal sensitivity arises from a specific protein domain.

Temperature and voltage coupling to channel opening in transient receptor potential melastatin 8 (TRPM8).

Expressed in somatosensory neurons of the dorsal root and trigeminal ganglion, the transient receptor potential melastatin 8 (TRPM8) channel is a Ca(2+)-permeable cation channel activated by cold, voltage, phosphatidylinositol 4,5-bisphosphate, and menthol. Although TRPM8 channel gating has been characterized at the single channel and macroscopic current levels, there is currently no consensus regarding the extent to which temperature and voltage sensors couple to the conduction gate. In this study, we extended the range of voltages where TRPM8-induced ionic currents were measured and made careful measurements of the maximum open probability the channel can attain at different temperatures by means of fluctuation analysis. The first direct measurements of TRPM8 channel temperature-driven conformational rearrangements provided here suggest that temperature alone is able to open the channel and that the opening reaction is voltage-independent. Voltage is a partial activator of TRPM8 channels, because absolute open probability values measured with fully activated voltage sensors are less than 1, and they decrease as temperature rises. By unveiling the fast temperature-dependent deactivation process, we show that TRPM8 channel deactivation is well described by a double exponential time course. The fast and slow deactivation processes are temperature-dependent with enthalpy changes of 27.2 and 30.8 kcal mol(-1). The overall Q10 for the closing reaction is about 33. A three-tiered allosteric model containing four voltage sensors and four temperature sensors can account for the complex deactivation kinetics and coupling between voltage and temperature sensor activation and channel opening.

Dissection of the components for PIP2 activation and thermosensation in TRP channels.

Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a central role in the activation of several transient receptor potential (TRP) channels. The role of PIP2 on temperature gating of thermoTRP channels has not been explored in detail, and the process of temperature activation is largely unexplained. In this work, we have exchanged different segments of the C-terminal region between cold-sensitive (TRPM8) and heat-sensitive (TRPV1) channels, trying to understand the role of the segment in PIP2 and temperature activation. A chimera in which the proximal part of the C-terminal of TRPV1 replaces an equivalent section of TRPM8 C-terminal is activated by PIP2 and confers the phenotype of heat activation. PIP2, but not temperature sensitivity, disappears when positively charged residues contained in the exchanged region are neutralized. Shortening the exchanged segment to a length of 11 aa produces voltage-dependent and temperature-insensitive channels. Our findings suggest the existence of different activation domains for temperature, PIP2, and voltage. We provide an interpretation for channel-PIP2 interaction using a full-atom molecular model of TRPV1 and PIP2 docking analysis.