Wendy González

Potassium (K+) gradients serve as a mobile energy source in plant vascular tissues

The essential mineral nutrient potassium (K+) is the most important 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.

Neutralization of a single arginine residue gates open a two-pore domain, alkali-activated K+ channel.

Potassium channels share a common selectivity filter that determines the conduction characteristics of the pore. Diversity in K+ channels is given by how they are gated open. TASK-2, TALK-1, and TALK-2 are two-pore region (2P) KCNK K+ channels gated open by extracellular alkalinization. We have explored the mechanism for this alkalinization-dependent gating using molecular simulation and site-directed mutagenesis followed by functional assay. We show that the side chain of a single arginine residue (R224) near the pore senses pH in TASK-2 with an unusual pKa of 8.0, a shift likely due to its hydrophobic environment. R224 would block the channel through an electrostatic effect on the pore, a situation relieved by its deprotonation by alkalinization. A lysine residue in TALK-2 fulfills the same role but with a largely unchanged pKa, which correlates with an environment that stabilizes its positive charge. In addition to suggesting unified alkaline pH-gating mechanisms within the TALK subfamily of channels, our results illustrate in a physiological context the principle that hydrophobic environment can drastically modulate the pKa of charged amino acids within a protein.

A minimal cysteine motif required to activate the SKOR K+ channel of Arabidopsis by the reactive oxygen species H2O2

Reactive oxygen species (ROS) are essential for development and stress signaling in plants. They contribute to plant defense against pathogens, regulate stomatal transpiration, and influence nutrient uptake and partitioning. Although both Ca2+ and K+ channels of plants are known to be affected, virtually nothing is known of the targets for ROS at a molecular level. Here we report that a single cysteine (Cys) residue within the Kv-like SKOR K+ channel of Arabidopsis thaliana is essential for channel sensitivity to the ROS H2O2. We show that H2O2 rapidly enhanced current amplitude and activation kinetics of heterologously expressed SKOR, and the effects were reversed by the reducing agent dithiothreitol (DTT). Both H2O2 and DTT were active at the outer face of the membrane and current enhancement was strongly dependent on membrane depolarization, consistent with a H2O2-sensitive site on the SKOR protein that is exposed to the outside when the channel is in the open conformation. Cys substitutions identified a single residue, Cys168 located within the S3 α-helix of the voltage sensor complex, to be essential for sensitivity to H2O2. The same Cys residue was a primary determinant for current block by covalent Cys S-methioylation with aqueous methanethiosulfonates. These, and additional data identify Cys168 as a critical target for H2O2, and implicate ROS-mediated control of the K+ channel in regulating mineral nutrient partitioning within the plant.

TASK-2: a K2P K+ channel with complex regulation and diverse physiological functions

TASK-2 (K2P5.1) is a two-pore domain K+ channel belonging to the TALK subgroup of the K2P family of proteins. TASK-2 has been shown to be activated by extra- and intracellular alkalinization. Extra- and intracellular pH-sensors reside at arginine 224 and lysine 245 and might affect separate selectivity filter and inner gates respectively. TASK-2 is modulated by changes in cell volume and a regulation by direct G-protein interaction has also been proposed. Activation by extracellular alkalinization has been associated with a role of TASK-2 in kidney proximal tubule bicarbonate reabsorption, whilst intracellular pH-sensitivity might be the mechanism for its participation in central chemosensitive neurons. In addition to these functions TASK-2 has been proposed to play a part in apoptotic volume decrease in kidney cells and in volume regulation of glial cells and T-lymphocytes. TASK-2 is present in chondrocytes of hyaline cartilage, where it is proposed to play a central role in stabilizing the membrane potential. Additional sites of expression are dorsal root ganglion neurons, endocrine and exocrine pancreas and intestinal smooth muscle cells. TASK-2 has been associated with the regulation of proliferation of breast cancer cells and could become target for breast cancer therapeutics. Further work in native tissues and cells together with genetic modification will no doubt reveal the details of TASK-2 functions that we are only starting to suspect.

Intrinsic Electrostatic Potential in the BK Channel Pore: Role in Determining Single Channel Conductance and Block

The internal vestibule of large-conductance Ca2+ voltage-activated K+ (BK) channels contains a ring of eight negative charges not present in K+ channels of lower conductance (Glu386 and Glu389 in hSlo) that modulates channel conductance through an electrostatic mechanism (Brelidze, T.I., X. Niu, and K.L. Magleby. 2003. Proc. Natl. Acad. Sci. USA. 100:9017–9022). In BK channels there are also two acidic amino acid residues in an extracellular loop (Asp326 and Glu329 in hSlo). To determine the electrostatic influence of these charges on channel conductance, we expressed wild-type BK channels and mutants E386N/E389N, D326N, E329Q, and D326N/E329Q channels on Xenopus laevis oocytes, and measured the expressed currents under patch clamp. Contribution of E329 to the conductance is negligible and single channel conductance of D326N/E329Q channels measured at 0 mV in symmetrical 110 mM K+ was 18% lower than the control. Current–voltage curves displayed weak outward rectification for D326N and the double mutant. The conductance differences between the mutants and wild-type BK were caused by an electrostatic effect since they were enhanced at low K+ (30 mM) and vanished at high K+ (1 M K+). We determine the electrostatic potential change, Δφ, caused by the charge neutralization using TEA+ block for the extracellular charges and Ba2+ for intracellular charges. We measured 13 ± 2 mV for Δφ at the TEA+ site when turning off the extracellular charges, and 17 ± 2 mV for the Δφ at the Ba2+ site when the intracellular charges were turned off. To understand the electrostatic effect of charge neutralizations, we determined Δφ using a BK channel molecular model embedded in a lipid bilayer and solving the Poisson-Boltzmann equation. The model explains the experimental results adequately and, in particular, gives an economical explanation to the differential effect on the conductance of the neutralization of charges D326 and E329.

ClC-5 mutations associated with Dent’s disease: a major role of the dimer interface

Dent’s disease is an X-linked recessive disorder affecting the proximal tubules. Mutations in the 2Cl−/H+ exchanger ClC-5 gene CLCN5 are frequently associated with Dent’s disease. Functional characterization of mutations of CLCN5 have helped to elucidate the physiopathology of Dent’s disease and provided evidence that several different mechanisms underlie the ClC-5 dysfunction in Dent’s disease. Modeling studies indicate that many CLCN5 mutations are located at the interface between the monomers of ClC-5, demonstrating that this protein region plays an important role in Dent’s disease. On the basis of functional data, CLCN5 mutations can be divided into three different classes. Class 1 mutations impair processing and folding, and as a result, the ClC-5 mutants are retained within the endoplasmic reticulum and targeted for degradation by quality control mechanisms. Class 2 mutations induce a delay in protein processing and reduce the stability of ClC-5. As a consequence, the cell surface expression and currents of the ClC-5 mutants are lower. Class 3 mutations do not alter the trafficking of ClC-5 to the cell surface and early endosomes but induce altered electrical activity. Here, we discuss the functional consequences of the three classes of CLCN5 mutations on ClC-5 structure and function.

Distinct roles of the last transmembrane domain in controlling Arabidopsis K+ channel activity

The family of voltage-gated potassium channels in plants presumably evolved from a common ancestor and includes both inward-rectifying (K(in)) channels that allow plant cells to accumulate K(+) and outward-rectifying (K(out)) channels that mediate K(+) efflux. Despite their close structural similarities, the activity of K(in) channels is largely independent of K(+) and depends only on the transmembrane voltage, whereas that of K(out) channels responds to the membrane voltage and the prevailing extracellular K(+) concentration. Gating of potassium channels is achieved by structural rearrangements within the last transmembrane domain (S6). Here we investigated the functional equivalence of the S6 helices of the K(in) channel KAT1 and the K(out) channel SKOR by domain-swapping and site-directed mutagenesis. Channel mutants and chimeras were analyzed after expression in Xenopus oocytes. We identified two discrete regions that influence gating differently in both channels, demonstrating a lack of functional complementarity between KAT1 and SKOR. Our findings are supported by molecular models of KAT1 and SKOR in the open and closed states. The role of the S6 segment in gating evolved differently during specialization of the two channel subclasses, posing an obstacle for the transfer of the K(+)-sensor from K(out) to K(in) channels.

Mutation Update of the CLCN5 Gene Responsible for Dent Disease 1

Dent disease is a rare X‐linked tubulopathy characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressive renal failure, and variable manifestations of other proximal tubule dysfunctions. It often progresses over a few decades to chronic renal insufficiency, and therefore molecular characterization is important to allow appropriate genetic counseling. Two genetic subtypes have been described to date: Dent disease 1 is caused by mutations of the CLCN5 gene, coding for the chloride/proton exchanger ClC‐5; and Dent disease 2 by mutations of the OCRL gene, coding for the inositol polyphosphate 5‐phosphatase OCRL‐1. Herein, we review previously reported mutations (n = 192) and their associated phenotype in 377 male patients with Dent disease 1 and describe phenotype and novel (n = 42) and recurrent mutations (n = 24) in a large cohort of 117 Dent disease 1 patients belonging to 90 families. The novel missense and in‐frame mutations described were mapped onto a three‐dimensional homology model of the ClC‐5 protein. This analysis suggests that these mutations affect the dimerization process, helix stability, or transport. The phenotype of our cohort patients supports and extends the phenotype that has been reported in smaller studies.

Gating of two-pore domain K+ channels by extracellular pH.

Potassium channels have a conserved selectivity filter that is important in determining which ions are conducted and at what rate. Although K+ channels of different conductance characteristics are known, they differ more widely in the way their opening and closing, the gating, is governed. TASK and TALK subfamily proteins are two-pore region KCNK K+ channels gated open by extracellular pH. We discuss the mechanism for this gating in terms of electrostatic effects on the pore changing the occupancy and open probability of the channels in a way reminiscent of C-type inactivation gating at the selectivity filter. Essential to this proposed mechanism is the replacement of two highly conserved aspartate residues at the pore mouth by asparagine or histidine residues in the TALK and TASK channels.

An extracellular ion pathway plays a central role in the cooperative gating of a K(2P) K+ channel by extracellular pH.

Background: TASK-3 is gated cooperatively by extracellular pH. Results: Mutual electrostatic interaction between K+ ions and two pH-sensing histidines occurs in a recently discovered extracellular ion pathway. Conclusion: Channel opening requires neutralization of both sensing histidines, with neutralization of the second sensor becoming favored by an electrostatic effect K+ ions. Significance: The work suggests a central role for the extracellular ion pathway in the gating of K2P K+ channels. Proton-gated TASK-3 K+ channel belongs to the K2P family of proteins that underlie the K+ leak setting the membrane potential in all cells. TASK-3 is under cooperative gating control by extracellular [H+]. Use of recently solved K2P structures allows us to explore the molecular mechanism of TASK-3 cooperative pH gating. Tunnel-like side portals define an extracellular ion pathway to the selectivity filter. We use a combination of molecular modeling and functional assays to show that pH-sensing histidine residues and K+ ions mutually interact electrostatically in the confines of the extracellular ion pathway. K+ ions modulate the pKa of sensing histidine side chains whose charge states in turn determine the open/closed transition of the channel pore. Cooperativity, and therefore steep dependence of TASK-3 K+ channel activity on extracellular pH, is dependent on an effect of the permeant ion on the channel pHo sensors.