An N-terminal transport-deficient module of CATION EXCHANGER1 is sufficient to trigger anoxia stress responses.

Expressing a transport-deficient portion of a calcium transporter protein restores stress sensitivity in plants, revealing a signaling role independent of calcium movement.

Electron currents mediated by tonoplast cytochromes b561.

Ascorbate (ASC) is a key redox buffer in plant cells, whose antioxidant capacity depends on its balance with monodehydroascorbate (MDHA), its one-electron oxidation product. In the cytoplasm of Arabidopsis mesophyll cells, ASC is present at high concentrations and interacts with enzymes that oxidize it to MDHA, such as ascorbate peroxidases, as well as with enzymes that regenerate it, like NAD(P)H-dependent MDHA oxidoreductases (MDHAR) and glutathione-dependent dehydroascorbate reductases (DHAR). In vacuoles, ASC is found at lower concentrations and vacuoles lack these enzymes, but it can still undergo non-enzymatic oxidation by phenoxy radicals generated by class III peroxidases.

Ferric reduction by a CYBDOM protein counteracts increased iron availability in root meristems induced by phosphorus deficiency.

To mobilize sparingly available phosphorus (P) in the rhizosphere, many plant species secrete malate to release P sorbed onto (hydr)oxides of aluminum and iron (Fe). In the presence of Fe, malate can provoke Fe over-accumulation in the root apoplast, triggering a series of events that inhibit root growth. Here, we identified HYPERSENSITIVE TO LOW P1 (HYP1), a CYBDOM protein constituted of a DOMON and a cytochrome b561 domain, as critical to maintain cell elongation and meristem integrity under low P.

Sequential removal of cation/H exchangers reveals their additive role in elemental distribution, calcium depletion and anoxia tolerance.

Multiple Arabidopsis H /Cation exchangers (CAXs) participate in high-capacity transport into the vacuole. Previous studies have analysed single and double mutants that marginally reduced transport; however, assessing phenotypes caused by transport loss has proven enigmatic. Here, we generated quadruple mutants (cax1-4: qKO) that exhibited growth inhibition, an 85% reduction in tonoplast-localised H /Ca transport, and enhanced tolerance to anoxic conditions compared to CAX1 mutants.

A commentary on the inhibition of human TPC2 channel by the natural flavonoid naringenin: Methods, experiments, and ideas.

Human endo-lysosomes possess a class of proteins called TPC channels on their membrane, which are essential for proper cell functioning. This protein family can be functionally studied by expressing them in plant vacuoles. Inhibition of hTPC activity by naringenin, one of the main flavonoids present in the human diet, has the potential to be beneficial in severe human diseases such as solid tumor development, melanoma, and viral infections.

Electrophysiology and fluorescence to investigate cation channels and transporters in isolated plant vacuoles.

The plant vacuole plays a fundamental role in cell homeostasis. The successful application of patch-clamp technique on isolated vacuoles allows the determination of the functional characteristics of tonoplast ion channels and transporters. The parallel use of a sensor-based fluorescence approach capable of detecting changes in calcium and proton concentrations opens up new possibilities for investigation.

Tonoplast cytochrome b561 is a transmembrane ascorbate-dependent monodehydroascorbate reductase: functional characterization of electron currents in plant vacuoles.

Ascorbate (Asc) is a major redox buffer of plant cells, whose antioxidant activity depends on the ratio with its one-electron oxidation product monodehydroascorbate (MDHA). The cytoplasm contains millimolar concentrations of Asc and soluble enzymes that can regenerate Asc from MDHA or fully oxidized dehydroascorbate. Also, vacuoles contain Asc, but no soluble Asc-regenerating enzymes.

Current Methods to Unravel the Functional Properties of Lysosomal Ion Channels and Transporters.

A distinct set of channels and transporters regulates the ion fluxes across the lysosomal membrane. Malfunctioning of these transport proteins and the resulting ionic imbalance is involved in various human diseases, such as lysosomal storage disorders, cancer, as well as metabolic and neurodegenerative diseases. As a consequence, these proteins have stimulated strong interest for their suitability as possible drug targets.

The phosphoinositide PI(3,5)P inhibits the activity of plant NHX proton/potassium antiporters: Advantages of a novel electrophysiological approach.

In the present work, we discuss the way in which the parallel application of the patch-clamp technique and the 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence detection for recording luminal proton changes allows the functional characterization of nonelectrogenic potassium/proton vacuolar antiporters of the NHX (Na/H exchanger) family. Moreover, we review the functional role of the tonoplast-specific phosphoinositide PI(3,5)P, able to simultaneously inhibit the activity of NHXs and CLC-a transporters, whose coordinated action can play an important role in the water balance of plant cells.

The Discovery of Naringenin as Endolysosomal Two-Pore Channel Inhibitor and Its Emerging Role in SARS-CoV-2 Infection.

The flavonoid naringenin (Nar), present in citrus fruits and tomatoes, has been identified as a blocker of an emerging class of human intracellular channels, namely the two-pore channel (TPC) family, whose role has been established in several diseases. Indeed, Nar was shown to be effective against neoangiogenesis, a process essential for solid tumor progression, by specifically impairing TPC activity. The goal of the present review is to illustrate the rationale that links TPC channels to the mechanism of coronavirus infection, and how their inhibition by Nar could be an efficient pharmacological strategy to fight the current pandemic plague COVID-19.

Beyond the patch-clamp resolution: functional activity of nonelectrogenic vacuolar NHX proton/potassium antiporters and inhibition by phosphoinositides.

We combined the patch-clamp technique with ratiometric fluorescence imaging using the proton-responsive dye BCECF as a luminal probe. Upon application of a steep cytosol-directed potassium ion (K ) gradient in Arabidopsis mesophyll vacuoles, a strong and reversible acidification of the vacuolar lumen was detected, whereas no associated electrical currents were observed, in agreement with electroneutral cation/H exchange. Our data show that this acidification was generated by NHX antiport activity, because: it did not distinguish between K and sodium (Na ) ions; it was sensitive to the NHX inhibitor benzamil; and it was completely absent in vacuoles from nhx1 nhx2 double knockout plants.

The mechanism and energetics of a ligand-controlled hydrophobic gate in a mammalian two pore channel.

In the last decade two-pore intracellular channels (TPCs) attracted the interest of researchers, still some key questions remain open. Their importance for vacuolar (plants) and endo-lysosomal (animals) function highlights them as a very attractive system to study, both theoretically and experimentally. Indicated as key players in the trafficking of the cell, today they are considered a new potential target for avoiding virus infections, including those from coronaviruses.

TMEM16E/ANO5 mutations related to bone dysplasia or muscular dystrophy cause opposite effects on lipid scrambling.

Mutations in the human TMEM16E/ANO5 gene are causative for gnathodiaphyseal dysplasia (GDD), a rare bone malformation and fragility disorder, and for two types of muscular dystrophy (MD). Previous studies have demonstrated that TMEM16E/ANO5 is a Ca -activated phospholipid scramblase and that the mutation c.1538C>T (p.

Understanding the Molecular Basis of Salt Sequestration in Epidermal Bladder Cells of Chenopodium quinoa.

Soil salinity is destroying arable land and is considered to be one of the major threats to global food security in the 21st century. Therefore, the ability of naturally salt-tolerant halophyte plants to sequester large quantities of salt in external structures, such as epidermal bladder cells (EBCs), is of great interest. Using Chenopodium quinoa, a pseudo-cereal halophyte of great economic potential, we have shown previously that, upon removal of salt bladders, quinoa becomes salt sensitive.

Modulation of calcium and potassium permeation in plant TPC channels.

Plant two-pore channels (TPCs) are non-selective cation channels permeable both to monovalent potassium and divalent calcium. We previously developed a technique that allowed the simultaneous determination of the fluxes of these two ions across the channel by a combined use of patch-clamp and fluorescence. In this paper we studied how potassium and calcium fluxes were influenced by modification of cytosolic concentrations of K and Ca.

Gain of function of TMEM16E/ANO5 scrambling activity caused by a mutation associated with gnathodiaphyseal dysplasia.

Mutations in the human TMEM16E (ANO5) gene are associated both with the bone disease gnathodiaphyseal dysplasia (GDD; OMIM: 166260) and muscle dystrophies (OMIM: 611307, 613319). However, the physiological function of TMEM16E has remained unclear. We show here that human TMEM16E, when overexpressed in mammalian cell lines, displayed partial plasma membrane localization and gave rise to phospholipid scrambling (PLS) as well as non-selective ionic currents with slow time-dependent activation at highly depolarized membrane potentials.

Subunit-dependent oxidative stress sensitivity of LRRC8 volume-regulated anion channels.

Key Points: Swelling-activated anion currents are modulated by oxidative conditions, but it is unknown if oxidation acts directly on the LRRC8 channel-forming proteins or on regulatory factors. We found that LRRC8A-LRRC8E heteromeric channels are dramatically activated by oxidation of intracellular cysteines, whereas LRRC8A-LRRC8C and LRRC8A-LRRC8D heteromers are inhibited by oxidation. Volume-regulated anion currents in Jurkat T lymphocytes were inhibited by oxidation, in agreement with a low expression of the LRRC8E subunit in these cells.

Cisplatin activates volume sensitive LRRC8 channel mediated currents in Xenopus oocytes.

LRRC8 proteins have been shown to underlie the ubiquitous volume regulated anion channel (VRAC). VRAC channels are composed of the LRRC8A subunit and at least one among the LRRC8B-E subunits. In addition to their role in volume regulation, LRRC8 proteins have been implicated in the uptake of chemotherapeutic agents.

Investigation of LRRC8-Mediated Volume-Regulated Anion Currents in Xenopus Oocytes.

Volume-regulated anion channels (VRACs) play an important role in controlling cell volume by opening upon cell swelling. Recent work has shown that heteromers of LRRC8A with other LRRC8 members (B, C, D, and E) form the VRAC. Here, we used Xenopus oocytes as a simple system to study LRRC8 proteins.

Kidney CLC-K chloride channels inhibitors: structure-based studies and efficacy in hypertension and associated CLC-K polymorphisms.

Objective: Alterations in the handling of renal salt reabsorption may contribute to interindividual differences in blood pressure regulation and susceptibility to hypertension. CLC-K chloride channels and their accessory subunit barttin play a pivotal role in kidney by controlling chloride and water absorption. Compounds selective for CLC-Ks, such as the benzofuran derivative MT-189, may have a significant therapeutic potential.

Regulatory-auxiliary subunits of CLC chloride channel-transport proteins.

The CLC family of chloride channels and transporters is composed by nine members, but only three of them, ClC-Ka/b, ClC-7 and ClC-2, have been found so far associated with auxiliary subunits. These CLC regulatory subunits are small proteins that present few common characteristics among them, both structurally and functionally, and their effects on the corresponding CLC protein are different. Barttin, a protein with two transmembrane domains, is essential for the membrane localization of ClC-K proteins and their activity in the kidney and inner ear.

I-J loop involvement in the pharmacological profile of CLC-K channels expressed in Xenopus oocytes.

CLC-K chloride channels and their subunit, barttin, are crucial for renal NaCl reabsorption and for inner ear endolymph production. Mutations in CLC-Kb and barttin cause Bartter syndrome. Here, we identified two adjacent residues, F256 and N257, that when mutated hugely alter in Xenopus oocytes CLC-Ka's biphasic response to niflumic acid, a drug belonging to the fenamate class, with F256A being potentiated 37-fold and N257A being potently blocked with a KD~1μM.

Targeting kidney CLC-K channels: pharmacological profile in a human cell line versus Xenopus oocytes.

CLC-K chloride channels play a crucial role in kidney physiology and genetic mutations, affecting their function are responsible for severe renal salt loss in humans. Thus, compounds that selectively bind to CLC-Ka and/or CLC-Kb channels and modulate their activity may have a significant therapeutic potential. Here, we compare the biophysical and pharmacological behaviors of human CLC-K channels expressed either in HEK293 cells or in Xenopus oocytes and we show that CLC-K channel properties are greatly influenced by the biochemical environment surrounding the channels.

Alkaline pH block of CLC-K kidney chloride channels mediated by a pore lysine residue.

CLC-K chloride channels are expressed in the kidney and the inner ear, where they are involved in NaCl reabsorption and endolymph production, respectively. These channels require the beta subunit barttin for proper function. Mutations in ClC-Kb and barttin, lead to Bartter's syndrome.