Patch-clamp Pipette Research Articles

In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi

Studying the membrane physiology of filamentous fungi is key to understanding their interactions with the environment and crucial for developing new therapeutic strategies for disease-causing pathogens. However, their plasma membrane has been inaccessible for a micron-sized patch-clamp pipette for pA current recordings due to the rigid chitinous cell wall. Here, we report the first femtosecond IR laser nanosurgery of the cell wall of the filamentous fungi, which enabled patch-clamp measurements on protoplasts released from hyphae. A reproducible and highly precise (diffraction-limited, submicron resolution) method for obtaining viable released protoplasts was developed. Protoplast release from the nanosurgery-generated incisions in the cell wall was achieved from different regions of the hyphae. The plasma membrane of the obtained protoplasts formed tight and high-resistance (GΩ) contacts with the recording pipette. The entire nanosurgical procedure followed by the patch-clamp technique could be completed in less than 1 hour. Compared to previous studies using heterologously expressed channels, this technique provides the opportunity to identify new ionic currents and to study the properties of the ion channels in the protoplasts of filamentous fungi in their native environment.

Immediate reuse of patch-clamp pipettes after ultrasonic cleaning

The patch-clamp technique has revolutionized neurophysiology by allowing to study single neuronal excitability, synaptic connectivity, morphology, and the transcriptomic profile. However, the throughput in recordings is limited because of the manual replacement of patch-pipettes after each attempt which are often also unsuccessful. This has been overcome by automated cleaning the tips in detergent solutions, allowing to reuse the pipette for further recordings. Here, we developed a novel method of automated cleaning by sonicating the tips within the bath solution wherein the cells are placed, reducing the risk of contaminating the bath solution or internal solution of the recording pipette by any detergent and avoiding the necessity of a separate chamber for cleaning. We showed that the patch-pipettes can be used consecutively at least ten times and that the cleaning process does not negatively impact neither the brain slices nor other patched neurons. This method, combined with automated patch-clamp, highly improves the throughput for single and especially multiple recordings.

Diacylglycerol Activates the Drosophila Light Sensitive Channel TRPL Expressed in HEK Cells.

Physiological activation by light of the Drosophila TRP and TRP-like (TRPL) channels requires the activation of phospholipase Cβ (PLC). The hydrolysis of phosphatidylinositol 4,5, bisphosphate (PIP2) by PLC is a crucial step in the still-unclear light activation, while the generation of Diacylglycerol (DAG) by PLC seems to be involved. In this study, we re-examined the ability of a DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) to activate the TRPL channels expressed in HEK cells. Unlike previous studies, we added OAG into the cytosol via a patch-clamp pipette and observed robust activation of the expressed TRPL channels. However, TRPL channel activation was much slower than the physiologically activated TRPL by light. Therefore, we used a picosecond-fast optically activated DAG analogue, OptoDArG. Inactive OptoDArG was added into the intracellular solution with the patch-clamp pipette, and it slowly accumulated on the surface membrane of the recorded HEK cell in the dark. A fast application of intense UV light to the recorded cell resulted in a robust and relatively fast TRPL-dependent current that was greatly accelerated by the constitutively active TRPLF557I pore-region mutation. However, this current of the mutant channel was still considerably slower than the native light-induced TRPL current, suggesting that DAG alone is not sufficient for TRPL channel activation under physiological conditions.

There is no F in APC: Using physiological fluoride-free solutions for high throughput automated patch clamp experiments.

Fluoride has been used in the internal recording solution for manual and automated patch clamp experiments for decades because it helps to improve the seal resistance and promotes longer lasting recordings. In manual patch clamp, fluoride has been used to record voltage-gated Na (NaV) channels where seal resistance and access resistance are critical for good voltage control. In automated patch clamp, suction is applied from underneath the patch clamp chip to attract a cell to the hole and obtain a good seal. Since the patch clamp aperture cannot be moved to improve the seal like the patch clamp pipette in manual patch clamp, automated patch clamp manufacturers use internal fluoride to improve the success rate for obtaining GΩ seals. However, internal fluoride can affect voltage-dependence of activation and inactivation, as well as affecting internal second messenger systems and therefore, it is desirable to have the option to perform experiments using physiological, fluoride-free internal solution. We have developed an approach for high throughput fluoride-free recordings on a 384-well based automated patch clamp system with success rates >40% for GΩ seals. We demonstrate this method using hERG expressed in HEK cells, as well as NaV1.5, NaV1.7, and KCa3.1 expressed in CHO cells. We describe the advantages and disadvantages of using fluoride and provide examples of where fluoride can be used, where caution should be exerted and where fluoride-free solutions provide an advantage over fluoride-containing solutions.

Preparation of Pipettes and Pipette-Filling Devices for Patch-Clamping Drosophila Neurons.

An essential requirement of every laboratory procedure is to have all materials ready when they are needed, so that the experimental flow is not disrupted. This is particularly true for patch clamping; therefore, effort must be devoted in advance to produce materials such as patch pipettes. This can be a fiddly business; hence, this protocol provides step-by-step advice on how to pull and polish patch-clamp pipettes. It also includes a brief description on how to prepare homemade filling devices to deliver saline efficiently and inexpensively into the pipettes. The protocol ends with guidelines on how to change the filament of a Sutter horizontal puller, a dreaded yet necessary activity that should be learned by anyone who wishes to become an expert patch clamper.

Machine Learning-Based Pipette Positional Correction for Automatic Patch Clamp In Vitro.

Patch clamp electrophysiology is a common technique used in neuroscience to understand individual neuron behavior, allowing one to record current and voltage changes with superior spatiotemporal resolution compared with most electrophysiology methods. While patch clamp experiments produce high fidelity electrophysiology data, the technique is onerous and labor intensive. Despite the emergence of patch clamp systems that automate key stages in the typical patch clamp procedure, full automation remains elusive. Patch clamp pipettes can miss the target cell during automated experiments because of positioning errors in the robotic manipulators, which can easily exceed the diameter of a neuron. Further, when patching in acute brain slices, the inherent light scattering from non-uniform brain tissue can complicate pipette tip identification. We present a convolutional neural network (CNN), based on ResNet101, to identify and correct pipette positioning errors before each patch clamp attempt, thereby preventing the deleterious effects of and accumulation of positioning errors. This deep-learning-based pipette detection method enabled superior localization of the pipette within 0.62 ± 0.58 μm, resulting in improved cell detection success rate and whole-cell patch clamp success rates by 71% and 59%, respectively, compared with the state-of-the-art cross-correlation method. Furthermore, this technique reduced the average time for pipette correction by 81%. This technique enables real-time correction of pipette position during patch clamp experiments with similar accuracy and quality of recording to manual patch clamp, making notable progress toward full human-out-of-the-loop automation for patch clamp electrophysiology.

PEDOT:PSS organic electrochemical transistors for electrical cell-substrate impedance sensing down to single cells

The adhesion of cells on organic electrochemical transistors (OECT) is investigated down to a single cell resolution using an impedimetric readout method of the transistors. For this purpose a fabrication protocol for micro-sized OECTs based on Poly(3,4-ethylenedioxythiophene):poly(styrene sulfonic acid) (PEDOT:PSS) was developed. OECTs with gate dimensions of 20 μm × 20 μm with cut-off frequencies up to 10 kHz at −3 dB were fabricated. Impedance spectra of the OECTs changed drastically when HEK 293 cells were adhered to the OECT gates. To confirm the single-cell sensitivity, individual cells were removed from the device surface with patch-clamp pipettes while impedance measurements were performed. In addition, the calcium chelator EGTA was used to demonstrate the reproducible activation and deactivation of tight gap junctions in Madin Darby Canine Kidney cells adhered on the OECT gates. We applied an analytical mathematical model combined with an electrically equivalent circuit model to describe the measured impedance spectra and to calculate the cell-related parameters of the adherent cells. The novel technique of impedimetric readout of OECTs for the detection of single cell adhesion offers various future applications.

Method for Rapid Enzymatic Cleaning for Reuse of Patch Clamp Pipettes: Increasing Throughput by Eliminating Manual Pipette Replacement between Patch Clamp Attempts.

The whole-cell patch-clamp method is a gold standard for single-cell analysis of electrical activity, cellular morphology, and gene expression. Prior to our discovery that patch-clamp pipettes could be cleaned and reused, experimental throughput and automation were limited by the need to replace pipettes manually after each experiment. This article presents an optimized protocol for pipette cleaning, which enables it to be performed quickly (< 30 s), resulting in a high yield of whole-cell recording success rate (> 90%) for over 100 reuses of a single pipette. For most patch-clamp experiments (< 30 whole-cell recordings per day), this method enables a single pipette to be used for an entire day of experiments. In addition, we describe easily implementable hardware and software as well as troubleshooting tips to help other labs implement this method in their own experiments. Pipette cleaning enables patch-clamp experiments to be performed with higher throughput, whether manually or in an automated fashion, by eliminating the tedious and skillful task of replacing pipettes. From our experience with numerous electrophysiology laboratories, pipette cleaning can be integrated into existing patch-clamp setups in approximately one day using the hardware and software described in this article. Graphic abstract: Rapid enzymatic cleaning for reuse of patch-clamp pipettes.

Integrative analysis of in vivo recording with single-cell RNA-seq data reveals molecular properties of light-sensitive neurons in mouse V1

Vision formation is classically based on projections from retinal ganglion cells (RGC) to the lateral geniculate nucleus (LGN) and the primary visual cortex (V1). Neurons in the mouse V1 are tuned to light stimuli. Although the cellular information of the retina and the LGN has been widely studied, the transcriptome profiles of single light-stimulated neuron in V1 remain unknown. In our study, in vivo calcium imaging and whole-cell electrophysiological patch-clamp recording were utilized to identify 53 individual cells from layer 2/3 of V1 as light-sensitive (LS) or non-light-sensitive (NS) by single-cell light-evoked calcium evaluation and action potential spiking. The contents of each cell after functional tests were aspirated in vivo through a patch-clamp pipette for mRNA sequencing. Moreover, the three-dimensional (3-D) morphological characterizations of the neurons were reconstructed in a live mouse after the whole-cell recordings. Our sequencing results indicated that V1 neurons with a high expression of genes related to transmission regulation, such as Rtn4r and Rgs7, and genes involved in membrane transport, such as Na+/K+ ATPase and NMDA-type glutamatergic receptors, preferentially responded to light stimulation. Furthermore, an antagonist that blocks Rtn4r signals could inactivate the neuronal responses to light stimulation in live mice. In conclusion, our findings of the vivo-seq analysis indicate the key role of the strength of synaptic transmission possesses neurons in V1 of light sensory.

Photodynamic Modification of Native HCN Channels Expressed in Thalamocortical Neurons

The photodynamic process requires three elements: light, oxygen, and photosensitizer, and involves the formation of singlet oxygen, the molecular oxygen in excited electronic states. Previously, we reported that heterologously expressed hyperpolarization-activated cAMP-gated (HCN) channels in excised membrane patches are sensitive to photodynamic modification (PDM). Here we extend this study to native HCN channels expressed in thalamocortical (TC) neurons in the ventrobasal (VB) complex of the thalamus and dopaminergic neurons (DA) of the ventral tegmental area (VTA). To do this, we introduced the photosensitizer FITC-cAMP into TCs or DAs of rodent brain slices via a whole-cell patch-clamp recording pipette. After illumination with blue light pulses, we observed an increase in the voltage-insensitive, instantaneous Iinst component, accompanied by a long-lasting decrease in the hyperpolarization-dependent Ih component. Both Ih and the increased Iinst after PDM could be blocked by the HCN blockers Cs+ and ZD7288. When FITC and cAMP were dissociated and loaded into neurons as two separate chemicals, light application did not result in any long-lasting changes of the HCN currents. In contrast, light pulses applied to HCN2-/- neurons loaded with FITC-cAMP generated a much greater reduction in the Iinst component compared to that of WT neurons. Next, we investigated the impact of the long-lasting increases in Iinst after PDM on the cellular physiology of VB neurons. Consistent with an upregulation of HCN channel function, PDM elicited a depolarization of the resting membrane potential (RMP). Importantly, Trolox-C, an effective quencher for singlet oxygen, could block the PDM-dependent increase in Iinst and depolarization of the RMP. We propose that PDM of native HCN channels under physiological conditions may provide a photodynamic approach to alleviate HCN channelopathy in certain pathological conditions.

Robust RNA-Seq of aRNA-amplified single cell material collected by patch clamp

Most single cell RNA sequencing protocols start with single cells dispersed from intact tissue. High-throughput processing of the separated cells is enabled using microfluidics platforms. However, dissociation of tissue results in loss of information about cell location and morphology and potentially alters the transcriptome. An alternative approach for collecting RNA from single cells is to re-purpose the electrophysiological technique of patch clamp recording. A hollow patch pipette is attached to individual cells, enabling the recording of electrical activity, after which the cytoplasm may be extracted for single cell RNA-Seq (“Patch-Seq”). Since the tissue is not disaggregated, the location of cells is readily determined, and the morphology of the cells is maintained, making possible the correlation of single cell transcriptomes with cell location, morphology and electrophysiology. Recent Patch-Seq studies utilizes PCR amplification to increase amount of nucleic acid material to the level required for current sequencing technologies. PCR is prone to create biased libraries – especially with the extremely high degrees of exponential amplification required for single cell amounts of RNA. We compared a PCR-based approach with linear amplifications and demonstrate that aRNA amplification (in vitro transcription, IVT) is more sensitive and robust for single cell RNA collected by a patch clamp pipette.

Functional recruitment of dynamin requires multimeric interactions for efficient endocytosis

During clathrin mediated endocytosis (CME), the concerted action of dynamin and its interacting partners drives membrane scission. Essential interactions occur between the proline/arginine-rich domain of dynamin (dynPRD) and the Src-homology domain 3 (SH3) of various proteins including amphiphysins. Here we show that multiple SH3 domains must bind simultaneously to dynPRD through three adjacent motifs for dynamin’s efficient recruitment and function. First, we show that mutant dynamins modified in a single motif, including the central amphiphysin SH3 (amphSH3) binding motif, partially rescue CME in dynamin triple knock-out cells. However, mutating two motifs largely prevents that ability. Furthermore, we designed divalent dynPRD-derived peptides. These ligands bind multimers of amphSH3 with >100-fold higher affinity than monovalent ones in vitro. Accordingly, dialyzing living cells with these divalent peptides through a patch-clamp pipette blocks CME much more effectively than with monovalent ones. We conclude that dynamin drives vesicle scission via multivalent interactions in cells.

α-Synuclein (αSyn) Preformed Fibrils Induce Endogenous αSyn Aggregation, Compromise Synaptic Activity and Enhance Synapse Loss in Cultured Excitatory Hippocampal Neurons.

Synucleinopathies are characterized by the accumulation of insoluble α-synuclein (αSyn). To test whether αSyn aggregates modulate synaptic activity, we used a recently developed model in primary neurons for inducing αSyn pathology. We demonstrated that preformed fibrils (PFFs) generated with recombinant human αSyn compromised synaptic activity in a time- and dose-dependent manner and that the magnitude of these deficits correlated with the formation of αSyn pathology in cultured excitatory hippocampal neurons from both sexes of mice. Remarkably, acute passive infusion of αSyn PFFs from whole-cell patch-clamp pipette decreased mEPSC frequency within 10 min followed by induction of αSyn pathology within 1 d. Moreover, by direct addition of αSyn PFFs into culture medium, the formation of misfolded αSyn inclusions dramatically compromised the colocalization of synaptic markers and altered dynamic changes of dendritic spines, but the viability of neurons was not affected up to 7 d post-treatment with αSyn PFFs. Our data indicate that intraneuronal αSyn fibrils impaired the initiation of synaptogenesis and their physiological functions, thereby suggesting that targeting synaptic dysfunction in synucleinopathies may provide a promising therapeutic direction.SIGNIFICANCE STATEMENT Under pathological conditions, the presynaptic protein α-synuclein (αSyn) aggregates to form intraneuronal inclusions. To understand how and to what extent αSyn aggregates modulate synaptic activity before neuron loss, we demonstrate that αSyn preformed fibrils (PFFs) reduced synaptic activity in a dose- and time-dependent manner. The magnitude of these deficits correlated with the deposition of αSyn pathology, which dramatically compromised the colocalization of synaptic markers and altered the dendritic spine dynamics. The present work further highlights the impact of αSyn PFFs on synaptogenesis and physiological function, which may be applicable to other types of synucleinopathies.

Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow

Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow

Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow.

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. Although the molecular mechanisms for the biological responses to fluid flow/shear force are not fully understood, fluid flow-mediated regulation of ion channel gating may contribute critically. Therefore, fluid flow/shear force sensitivity of ion channels has been studied using the patch-clamp technique. However, depending on the experimental protocol, the outcomes and interpretation of data can be erroneous. Here, we present experimental and theoretical evidence for fluid flow-related errors and provide methods for estimating, preventing, and correcting these errors. Changes in junction potential between the Ag/AgCl reference electrode and bathing fluid were measured with an open pipette filled with 3 M KCl. Fluid flow could then shift the liquid/metal junction potential to approximately 7 mV. Conversely, by measuring the voltage shift induced by fluid flow, we estimated the ion concentration in the unstirred boundary layer. In the static condition, the real ion concentrations adjacent to the Ag/AgCl reference electrode or ion channel inlet at the cell-membrane surface can reach as low as approximately 30% of that in the flow condition. Placing an agarose 3 M KCl bridge between the bathing fluid and reference electrode may have prevented this problem of junction potential shifting. However, the unstirred layer effect adjacent to the cell membrane surface could not be fixed in this way. Here, we provide a method for measuring real ion concentrations in the unstirred boundary layer with an open patch-clamp pipette, emphasizing the importance of using an agarose salt-bridge while studying fluid flow-induced regulation of ion currents. Therefore, this novel approach, which takes into consideration the real concentrations of ions in the unstirred boundary layer, may provide useful insight on the experimental design and data interpretation related to fluid shear stress regulation of ion channels.

Methods for Investigating TRP Channel Gating.

A complete characterization of temperature -and voltage-activated TRP channel gating requires a precise determination of the absolute probability of opening in a wide range of voltages, temperatures, and agonist concentrations. We have achieved this in the case of the TRPM8 channel expressed in Xenopus laevis oocytes. Measurements covered an extensive range of probabilities and unprecedented applied voltages up to 500mV. In this chapter, we describe animal care protocols of patch-clamp pipette preparation, temperature control methods, and analysis of ionic currents to obtain reliable absolute open channel probabilities.

Quick visualization of neurons in brain tissues using an optical clearing technique.

Neurons are classified into several morphological types according to the locations of their somata and the branching patterns of their axons and dendrites. Recent studies suggest that these morphological features are related to their physiological properties, including firing characteristics, responses to neuromodulators, and wiring patterns. Therefore, rapid morphological identification of electrophysiologically recorded neurons promises to advance our understanding of neuronal circuits. One of the most common anatomical cell identification methods is neuronal reconstruction with biocytin delivered through whole-cell patch-clamp pipettes. However, conventional reconstruction methods usually take longer than 24h and limit the throughput of electrophysiological experiments. Here, we developed a quick, simple cell reconstruction method by optimizing the tissue clearing protocol ScaleSQ. We found that adding 200mM NaCl almost entirely prevented tissue swelling without compromising optical clearing ability. This solution, termed IsoScaleSQ, allowed us to increase the transparency of the gray matter of 500-µm-thick slices within 30min, meaning that the total time required to reconstruct whole-cell recorded neurons was reduced to 1h. This novel method will improve the efficacy and effectiveness of electrophysiological experiments linked to cell morphology.

Real-time local oxygen measurements for high resolution cellular imaging

Single-cell metabolic investigations are hampered by the absence of flexible tools to measure local partial pressure of O2 (pO2) at high spatial-temporal resolution. To this end, we developed an optical sensor capable of measuring local pericellular pO2 for subcellular resolution measurements with confocal imaging while simultaneously carrying out electrophysiological and/or chemo-mechanical single cell experiments. Here we present the OxySplot optrode, a ratiometric fluorescent O2-micro-sensor created by adsorbing O2-sensitive and O2-insensitive fluorophores onto micro-particles of silica. To protect the OxySplot optrode from the components and reactants of liquid environment without compromising access to O2, the micro-particles are coated with an optically clear silicone polymer (PDMS, polydimethylsiloxane). The PDMS coated OxySplot micro-particles are used alone or in a thin (~50 μm) PDMS layer of arbitrary shape referred to as the OxyMat. Additional top coatings on the OxyMat (e.g., fibronectin, laminin, polylysine, special photoactivatable surfaces etc.) facilitate adherence of cells. The OxySplots report the cellular pO2 and micro-gradients of pO2 without disrupting the flow of extracellular solutions or interfering with patch-clamp pipettes, mechanical attachments, and micro-superfusion. Since OxySplots and a cell can be imaged and spatially resolved, calibrated changes of pO2 and intracellular events can be imaged simultaneously. In addition, the response-time (t0.5 = 0.7 s, 0–160 mmHg) of OxySplots is ~100 times faster than amperometric Clark-type polarization microelectrodes. Two usage example of OxySplots with cardiomyocytes show (1) OxySplots measuring pericellular pO2 while tetramethylrhodamine methyl-ester (TMRM) was used to measure mitochondrial membrane potential (ΔΨm); and (2) OxySplots measuring pO2 during ischemia and reperfusion while rhod-2 was used to measure cytosolic [Ca2+]i levels simultaneously. The OxySplot/OxyMat optrode system provides an affordable and highly adaptable optical sensor system for monitoring pO2 with a diverse array of imaging systems, including high-speed, high-resolution confocal microscopes while physiological features are measured simultaneously.

CatSper channels are regulated by protein kinase A

Mammalian sperm must undergo capacitation as a preparation for entering into hyperactivated motility, undergoing the acrosome reaction, and acquiring fertilizing ability. One of the initial capacitation events occurs when sperm encounter an elevated HCO3- concentration. This anion activates the atypical adenylyl cyclase Adcy10, increases intracellular cAMP, and stimulates protein kinase A (PKA). Moreover, an increase in intracellular Ca2+ concentration ([Ca2+] i ) is essential for sperm capacitation. Although a cross-talk between cAMP-dependent pathways and Ca2+ clearly plays an essential role in sperm capacitation, the connection between these signaling events is incompletely understood. Here, using three different approaches, we found that CatSper, the main sperm Ca2+ channel characterized to date, is up-regulated by a cAMP-dependent activation of PKA in mouse sperm. First, HCO3- and the PKA-activating permeable compound 8-Br-cAMP induced an increase in [Ca2+] i , which was blocked by the PKA peptide inhibitor PKI, and H89, another PKA inhibitor, also abrogated the 8-Br-cAMP response. Second, HCO3- increased the membrane depolarization induced upon divalent cation removal by promoting influx of monovalent cations through CatSper channels, which was inhibited by PKI, H89, and the CatSper blocker HC-056456. Third, electrophysiological patch clamp, whole-cell recordings revealed that CatSper activity is up-regulated by HCO3- and by direct cAMP injection through the patch-clamp pipette. The activation by HCO3- and cAMP was also blocked by PKI, H89, Rp-cAMPS, and HC-056456, and electrophysiological recordings in sperm from CatSper-KO mice confirmed CatSper's role in these activation modes. Our results strongly suggest that PKA-dependent phosphorylation regulates [Ca2+] i homeostasis by activating CatSper channel complexes.

Novel roles of ascorbate in plants: induction of cytosolic Ca2+ signals and efflux from cells via anion channels.

Ascorbate is not often considered as a signalling molecule in plants. This study demonstrates that, in Arabidopsis roots, exogenous l-ascorbic acid triggers a transient increase of the cytosolic free calcium activity ([Ca2+]cyt.) that is central to plant signalling. Exogenous copper and iron stimulate the ascorbate-induced [Ca2+]cyt. elevation, while cation channel blockers, free radical scavengers, low extracellular [Ca2+], transition metal chelators, and removal of the cell wall inhibit this reaction. These data show that apoplastic redox-active transition metals are involved in the ascorbate-induced [Ca2+]cyt. elevation. Exogenous ascorbate also induces a moderate increase in programmed cell death symptoms in intact roots, but it does not activate Ca2+ influx currents in patch-clamped root protoplasts. Intriguingly, the replacement of gluconate with ascorbate in the patch-clamp pipette reveals a large ascorbate efflux current, which shows sensitivity to the anion channel blocker, anthracene-9-carboxylic acid (A9C), indicative of the ascorbate release via anion channels. EPR spectroscopy measurements demonstrate that salinity (NaCl) triggers the accumulation of root apoplastic ascorbyl radicals in an A9C-dependent manner, confirming that l-ascorbate leaks through anion channels under depolarization. This mechanism may underlie ascorbate release, signalling phenomena, apoplastic redox reactions, iron acquisition, and control the ionic and electrical equilibrium (together with K+ efflux via GORK channels).