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

Festa, Margherita; Minicozzi, Velia; Boccaccio, Anna; Lagostena, Laura; Gradogna, Antonella; Qi, Tianwen; Costa, Alex; Larisch, Nina; Hamamoto, Shin; Milenkovic, Stefan; Scholz‐Starke, Joachim; Ceccarelli, Matteo; Vitale, Alessandro; Dietrich, Petra; Uozumi, Nobuyuki; Gambale, Franco; Carpaneto, Armando

doi:10.3390/cells11060921

Cited by 8 publications

(6 citation statements)

References 203 publications

(277 reference statements)

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“…Mesophyll protoplasts were isolated from well‐expanded leaves from 7‐wk‐old plants and transiently transformed using the polyethylene glycol method as described elsewhere (Yoo et al ., 2007; Festa et al ., 2022; Gradogna & Carpaneto, 2022). Confocal microscopy analysis of vacuoles expressing the CYB561A‐EGFP protein was performed using a Leica SP2 (http://www.leica-microsystems.com) laser‐scanning confocal imaging system.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2023

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Summary 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.

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Section: Methodsmentioning

confidence: 99%

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

et al. 2023

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“…Several ion channels are found in organelles and their dysfunction can lead to disease, including metabolic and neurodegenerative diseases and cancers ( Festa et al, 2022 ; Gururaja Rao et al, 2020 ; for reviews on organellar ion channels and cancer see: Leanza et al, 2013 , Leanza et al, 2019 , Peruzzo et al, 2016 ; Szabo et al, 2021 ). Table 1 summarizes the key organelles, ion channels and their roles in physiology and cancer.…”

Section: Primary Research Example 2—organellar Kv13 Ion Channels In C...mentioning

confidence: 99%

“…Our capabilities to record organelle ion channel (OIC) currents has made significant technological progress in recent years: unlike typical cell plasma membrane channel recordings, organelle ion channel recordings on membranes inside the cell require highly developed and adapted manual patch clamp (MPC) techniques ( Chen et al, 2017 ; Shapovalov et al, 2017 ; Bednarczyk et al, 2021 ; Festa et al, 2022 ). Another area that has sprung from this foundational MPC research is the technological advancement in OIC recordings made via automated patch clamp (APC) platforms.…”

Section: Primary Research Example 2—organellar Kv13 Ion Channels In C...mentioning

confidence: 99%

News and views on ion channels in cancer: is cancer a channelopathy?

Bell,

Leanza,

Gentile

et al. 2023

Front. Pharmacol.

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Ion channels are key signaling proteins found throughout the body; they are critical in many, wide-ranging physiological processes, from gene expression, sensory perception and processing to the cardiac action potential. When ion channel activity goes awry, for example, via mutation, damage or disrupted homeostasis, the outcome can result in causation, development and/or maintenance of disease. Ion channel dependent diseases have been dubbed channelopathies. Recent studies on the role of ion channels in cancer biology suggest that cancer is one such channelopathy. Many ion channels have now been implicated in the cellular processes that are affected in a multitude of cancers. In the last two decades, the field of ion channel and cancer research has been growing exponentially: a combination of developments in molecular biology, genetics, electrophysiology and automation have driven an explosion in our capabilities to interrogate ion channel pathways; how, why and where they go wrong and therapeutic interventions to correct their pathophysiology in cancer. A review of this vast and rapidly developing field would require a titanic tome to merely dimple the surface of research that has ballooned recently. In lieu of that huge undertaking—for the benefit of both authors and readers - this review discusses select examples of primary, applied and clinical research, aiming to shine a light on some of the more innovative and novel findings that this exciting field is excavating.

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“…It is also worth noting that the plant vacuole can be used to study the properties of toxins produced by plant pathogens (Carpaneto et al 2002) or as a heterologous system for the study of lysosomal animal channels and transporters including human TPC channels (Festa et al 2022), which share the same inhibitors (Benkerrou et al 2019;Filippini et al 2020) but not the same agonists (Boccaccio et al 2014;Kirsch et al 2018) with the plant homolog.…”

Section: The Patch-clamp Technique Applied On Isolated Vacuolesmentioning

confidence: 99%

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

Gradogna

Carpaneto

2022

Stress Biology

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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.

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

Cited by 8 publications

References 203 publications

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

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

News and views on ion channels in cancer: is cancer a channelopathy?

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

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