RESUMO
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. We demonstrate that HYP1 mediates ascorbate-dependent trans-plasma membrane electron transport and can reduce ferric and cupric substrates in Xenopus laevis oocytes and in planta. HYP1 expression is up-regulated in response to P deficiency in the proximal zone of the root apical meristem. Disruption of HYP1 leads to increased Fe and callose accumulation in the root meristem and causes significant transcriptional changes in roots. We further demonstrate that HYP1 activity overcomes malate-induced Fe accumulation, thereby preventing Fe-dependent root growth arrest in response to low P. Collectively, our results uncover an ascorbate-dependent metalloreductase that is critical to protect root meristems of P-deficient plants from increased Fe availability and provide insights into the physiological function of the yet poorly characterized but ubiquitous CYBDOM proteins.
Assuntos
Meristema , Fósforo , Meristema/metabolismo , Fósforo/metabolismo , Malatos/metabolismo , Ferro/metabolismo , Plantas/metabolismo , Ácido Ascórbico/metabolismo , Raízes de Plantas/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
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. Leveraging inductively coupled plasma mass spectrometry (ICP-MS) and synchrotron X-ray fluorescence (SXRF), we demonstrate CAX transporters work together to regulate leaf elemental content: ICP-MS analysis showed that the elemental concentrations in leaves strongly correlated with the number of CAX mutations; SXRF imaging showed changes in element partitioning not present in single CAX mutants and qKO had a 40% reduction in calcium (Ca) abundance. Reduced endogenous Ca may promote anoxia tolerance; wild-type plants grown in Ca-limited conditions were anoxia tolerant. Sequential reduction of CAXs increased mRNA expression and protein abundance changes associated with reactive oxygen species and stress signalling pathways. Multiple CAXs participate in postanoxia recovery as their concerted removal heightened changes in postanoxia Ca signalling. This work showcases the integrated and diverse function of H+ /Cation transporters and demonstrates the ability to improve anoxia tolerance through diminishing endogenous Ca levels.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Cálcio/metabolismo , Antiporters/genética , Antiporters/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cátions/metabolismo , Plantas/metabolismoRESUMO
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. We attempted to identify the molecular basis of the interaction between hTPC2 and naringenin, using ensemble docking on molecular dynamics (MD) trajectories, but the specific binding site remains elusive, posing a challenge that could potentially be addressed in the future by increased computational power in MD and the combined use of microscopy techniques such as cryo-EM.
Assuntos
Endometriose , Flavanonas , Humanos , Feminino , Flavonoides/farmacologia , Flavonoides/uso terapêutico , Flavanonas/farmacologia , Flavanonas/uso terapêutico , Sítios de LigaçãoRESUMO
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. Here, we show that vacuoles isolated from Arabidopsis mesophyll cells contain a tonoplast electron transport system that works as a reversible, Asc-dependent transmembrane MDHA oxidoreductase. Electron currents were measured by patch-clamp on isolated vacuoles and found to depend on the availability of Asc (electron donor) and ferricyanide or MDHA (electron acceptors) on opposite sides of the tonoplast. Electron currents were catalyzed by cytochrome b561 isoform A (CYB561A), a tonoplast redox protein with cytoplasmic and luminal Asc binding sites. The Km for Asc of the luminal (4.5 mM) and cytoplasmic site (51 mM) reflected the physiological Asc concentrations in these compartments. The maximal current amplitude was similar in both directions. Mutant plants with impaired CYB561A expression showed no detectable trans-tonoplast electron currents and strong accumulation of leaf anthocyanins under excessive illumination, suggesting a redox-modulation exerted by CYB561A on the typical anthocyanin response to high-light stress.
Assuntos
Arabidopsis , Vacúolos , Vacúolos/metabolismo , Antocianinas/metabolismo , Elétrons , Ácido Ascórbico , Oxirredução , Plantas/metabolismo , Arabidopsis/metabolismo , Oxirredutases/metabolismoRESUMO
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)P2, 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.
Assuntos
Fosfatidilinositóis , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Potássio , Trocadores de Sódio-Hidrogênio , Fenômenos Eletrofisiológicos , Fosfatidilinositóis/metabolismo , Potássio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
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. A detailed functional characterization of many lysosomal channels and transporters is lacking, mainly due to technical difficulties in applying the standard patch-clamp technique to these small intracellular compartments. In this review, we focus on current methods used to unravel the functional properties of lysosomal ion channels and transporters, stressing their advantages and disadvantages and evaluating their fields of applicability.
Assuntos
Canais Iônicos , Doenças por Armazenamento dos Lisossomos , Humanos , Membranas Intracelulares/metabolismo , Canais Iônicos/metabolismo , Íons/metabolismo , Doenças por Armazenamento dos Lisossomos/metabolismo , Lisossomos/metabolismo , Técnicas de Patch-ClampRESUMO
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. In excised patch, the presence of fura-2 in the vacuolar solution reveals the direct permeation of calcium in plant TPC channels. In whole-vacuole, the activity of non-electrogenic NHX potassium proton antiporters can be measured by using the proton sensitive dye BCECF loaded in the vacuolar lumen by the patch pipette. Both vacuolar NHXs and CLCa (chloride/nitrate antiporter) are inhibited by the phosphoinositide PI(3,5)P2, suggesting a coordinated role of these proteins in salt accumulation. Increased knowledge in the molecular mechanisms of vacuolar ion channels and transporters has the potential to improve our understanding on how plants cope with a rapidly changing environment.
RESUMO
Subcellular and organellar mechanisms have manifested a prominent importance for a broad variety of processes that maintain cellular life at its most basic level. Mammalian two-pore channels (TPCs) appear to be cornerstones of these processes in endo-lysosomes by controlling delicate ion-concentrations in their interiors. With evolutionary remarkable architecture and one-of-a-kind selectivity filter, TPCs are an extremely attractive topic per se. In the light of the current COVID-19 pandemic, hTPC2 emerges to be more than attractive. As a key regulator of the endocytosis pathway, it is potentially essential for diverse viral infections in humans, as demonstrated. Here, by means of multiscale molecular simulations, we propose a model of sodium transport from the lumen to the cytosol where the central cavity works as a reservoir. Since the inhibition of hTPC2 is proven to stop SARS-CoV2 in vitro, shedding light on the hTPC2 function and mechanism is the first step towards the selection of potential inhibiting candidates.
Assuntos
Ativação do Canal Iônico , Canais Iônicos de Abertura Ativada por Ligante/fisiologia , Sódio/metabolismo , COVID-19 , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Ligantes , SARS-CoV-2/isolamento & purificaçãoRESUMO
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.
Assuntos
Tratamento Farmacológico da COVID-19 , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Flavanonas/farmacologia , Neoplasias/tratamento farmacológico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Arabidopsis/metabolismo , COVID-19/epidemiologia , COVID-19/patologia , COVID-19/virologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Endossomos/virologia , Flavanonas/uso terapêutico , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/virologia , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Pandemias/prevenção & controle , SARS-CoV-2/patogenicidade , Vacúolos/metabolismo , Internalização do Vírus/efeitos dos fármacosRESUMO
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. Our data further show that NHX activity could be reversed, was voltage-independent and specifically impaired by the low-abundance signaling lipid PI(3,5)P2 , which may regulate salt accumulation in plants by acting as a common messenger to coordinately shut down secondary active carriers responsible for cation and anion uptake inside the vacuole. Finally, we developed a theory based on thermodynamics, which supports the data obtained by our novel experimental approach. This work, therefore, represents a proof-of-principle that can be applied to the study of proton-dependent exchangers from plants and animals, which are barely detectable using conventional techniques.
Assuntos
Antiporters , Arabidopsis/fisiologia , Potássio , Vacúolos , Concentração de Íons de Hidrogênio , Íons , Fosfatidilinositóis , Potássio/metabolismo , Prótons , Vacúolos/metabolismoAssuntos
COVID-19 , Flavanonas , Preparações Farmacêuticas , Flavanonas/farmacologia , Humanos , SARS-CoV-2RESUMO
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. A particular boost for theoretical examinations has been made with recent high-resolution X-ray and cryo-EM structures. These findings have opened the way for efficient and precise computational studies at the atomistic level. Here we report a set of multiscale-calculations performed on the mTPC1, a ligand- and voltage-gated sodium selective channel. The molecular dynamics and enhanced molecular dynamics simulations were used for a thorough analysis of the mammalian TPC1 behaviour in the presence and absence of the ligand molecule, with a special accent on the supposed bottleneck, the hydrophobic gate. Moreover, from the reconstructed free energy obtained from enhanced simulations, we have calculated the macroscopic conductance of sodium ions through the mTPC1, which we compared with measured single-channel conductance values. The hydrophobic gate works as a steric barrier and the key parameters are its flexibility and the dimension of the sodium first hydration shell.
Assuntos
Canais de Cálcio/química , Simulação de Acoplamento Molecular , Animais , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ativação do Canal Iônico , Ligantes , CamundongosRESUMO
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. In this work, we analyzed the molecular mechanism underlying the unique salt dumping capabilities of bladder cells in quinoa. The transporters differentially expressed in the EBC transcriptome and functional electrophysiological testing of key EBC transporters in Xenopus oocytes revealed that loading of Na+ and Cl- into EBCs is mediated by a set of tailored plasma and vacuole membrane-based sodium-selective channel and chloride-permeable transporter.
Assuntos
Chenopodium quinoa/metabolismo , Plantas Tolerantes a Sal/metabolismo , Vacúolos/metabolismo , Células Epidérmicas/metabolismo , Células Epidérmicas/fisiologia , Proteínas de Membrana Transportadoras , Proteínas de Plantas/metabolismo , Salinidade , Tolerância ao Sal/fisiologia , Sódio/metabolismo , Canais de Sódio/metabolismo , Solo/química , Estresse Fisiológico , TranscriptomaRESUMO
Mammalian two-pore channels (TPCs) are activated by the low-abundance membrane lipid phosphatidyl-(3,5)-bisphosphate (PI(3,5)P2) present in the endo-lysosomal system. Malfunction of human TPC1 or TPC2 (hTPC) results in severe organellar storage diseases and membrane trafficking defects. Here, we compared the lipid-binding characteristics of hTPC2 and of the PI(3,5)P2-insensitive TPC1 from the model plant Arabidopsis thaliana. Combination of simulations with functional analysis of channel mutants revealed the presence of an hTPC2-specific lipid-binding pocket mutually formed by two channel regions exposed to the cytosolic side of the membrane. We showed that PI(3,5)P2 is simultaneously stabilized by positively charged amino acids (K203, K204, and K207) in the linker between transmembrane helices S4 and S5 and by S322 in the cytosolic extension of S6. We suggest that PI(3,5)P2 cross links two parts of the channel, enabling their coordinated movement during channel gating.
Assuntos
Canais de Cálcio/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Canais de Cálcio/química , Canais de Cálcio/genética , Humanos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Fosfatos de Fosfatidilinositol/química , Estrutura Terciária de Proteína , Protoplastos/metabolismo , Alinhamento de SequênciaRESUMO
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 Ca2+. A decrease in cytosolic calcium from 2 to 0.5â¯mM led to a shift of the activation curve of about +60â¯mV; although at positive potentials currents were very similar, calcium ion permeation was significantly reduced and the ratio between the total and calcium-mediated current increased about two-fold. Upon removal of cytosolic potassium, in the presence of 2â¯mM cytosolic calcium, the voltage-dependent activation curve was not modified but a dramatic reduction of the currents at positive voltages was apparent. However, calcium permeation did not change significantly in this condition. This work demonstrated that the electrophysiological measurements alone were not capable to predict the extent of the flow of different ions through cation channels. The parallel use of calcium detection by fluorescent dyes proved to be a valuable tool for the correct quantification of the permeation mechanisms in non-selective ion channels.
Assuntos
Cálcio/metabolismo , Canais Iônicos/metabolismo , Plantas/metabolismo , Potássio/metabolismoRESUMO
Our research introduces the natural flavonoid naringenin as a novel inhibitor of an emerging class of intracellular channels, Two-Pore Channel 2 (TPC2), as shown by electrophysiological evidence in a heterologous system, i.e. Arabidopsis vacuoles lacking endogenous TPCs. In view of the control exerted by TPC2 on intracellular calcium signaling, we demonstrated that naringenin dampens intracellular calcium responses of human endothelial cells stimulated with VEGF, histamine or NAADP-AM, but not with ATP or Angiopoietin-1 (negative controls). The ability of naringenin to impair TPC2-dependent biological activities was further explored in an established in vivo model, in which VEGF-containing matrigel plugs implanted in mice failed to be vascularized in the presence of naringenin. Overall, the present data suggest that naringenin inhibition of TPC2 activity and the observed inhibition of angiogenic response to VEGF are linked by impaired intracellular calcium signaling. TPC2 inhibition is emerging as a key therapeutic step in a range of important pathological conditions including the progression and metastatic potential of melanoma, Parkinson's disease, and Ebola virus infection. The identification of naringenin as an inhibitor of TPC2-mediated signaling provides a novel and potentially relevant tool for the advancement of this field of research.
Assuntos
Canais de Cálcio/metabolismo , Flavanonas/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Canais de Cálcio/genética , Sinalização do Cálcio/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , NADP/análogos & derivados , NADP/farmacologiaRESUMO
Living cells exploit the electrical properties of matter for a multitude of fundamental physiological processes, such as accumulation of nutrients, cellular homeostasis, signal transmission. While ion channels and transporters (able to couple ions to various substrates) have been extensively studied, direct measurements of electron currents mediated by specific proteins are just at the beginning. Here, we present the various electrophysiological approaches that have allowed recordings of electron currents and highlight the future potential of such experiments.
Assuntos
Fenômenos Eletrofisiológicos , Xenopus/fisiologia , Animais , Grupo dos Citocromos b/genética , Grupo dos Citocromos b/metabolismo , Elétrons , Ferricianetos/química , Íons/química , Íons/metabolismo , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Oócitos/fisiologia , Técnicas de Patch-Clamp , Xenopus/crescimento & desenvolvimentoRESUMO
Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) is a low-abundance signaling lipid associated with endo-lysosomal and vacuolar membranes in eukaryotic cells. Recent studies on Arabidopsis indicated a critical role of PI(3,5)P2 in vacuolar acidification and morphology during ABA-induced stomatal closure, but the molecular targets in plant cells remained unknown. By using patch-clamp recordings on Arabidopsis vacuoles, we show here that PI(3,5)P2 does not affect the activity of vacuolar H+-pyrophosphatase or vacuolar H+-ATPase. Instead, PI(3,5)P2 at low nanomolar concentrations inhibited an inwardly rectifying conductance, which appeared upon vacuolar acidification elicited by prolonged H+ pumping activity. We provide evidence that this novel conductance is mediated by chloride channel a (CLC-a), a member of the anion/H+ exchanger family formerly implicated in stomatal movements in Arabidopsis H+-dependent currents were absent in clc-a knock-out vacuoles, and canonical CLC-a-dependent nitrate/H+ antiport was inhibited by low concentrations of PI(3,5)P2 Finally, using the pH indicator probe BCECF, we show that CLC-a inhibition contributes to vacuolar acidification. These data provide a mechanistic explanation for the essential role of PI(3,5)P2 and advance our knowledge about the regulation of vacuolar ion transport.
Assuntos
Arabidopsis/metabolismo , Canais de Cloreto/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transdução de Sinais , Ânions , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Concentração de Íons de Hidrogênio , Transporte de Íons , Lisossomos/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vacúolos/metabolismoRESUMO
Two-pore channels (TPC) are intracellular endo-lysosomal proteins with only recently emerging roles in organellar signalling and involvement in severe human diseases. Here, we investigated the functional properties of human TPC1 expressed in TPC-free vacuoles from Arabidopsis thaliana cells. Large (20 pA/pF) TPC1 currents were elicited by cytosolic addition of the phosphoinositide phosphatidylinositol-(3,5)-bisphosphate (PI(3,5)P2) with an apparent binding constant of ~15 nM. The channel is voltage-dependent, activating at positive potentials with single exponential kinetics and currents are Na+ selective, with measurable but low permeability to Ca2+. Cytosolic Ca2+ modulated hTPC1 in dual way: low µM cytosolic Ca2+ increased activity by shifting the open probability towards negative voltages and by accelerating the time course of activation. This mechanism was well-described by an allosteric model. Higher levels of cytosolic Ca2+ induced a voltage-dependent decrease of the currents compatible with Ca2+ binding in the permeation pore. Conversely, an increase in luminal Ca2+ decreased hTPC1 activity. Our data point to a process in which Ca2+ permeation in hTPC1 has a positive feedback on channel activity while Na+ acts as a negative regulator. We speculate that the peculiar Ca2+ and Na+ dependence are key for the physiological roles of the channel in organellar homeostasis and signalling.