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Mechanical forces shape living matter from the macro- to the microscale as both eukaryotic and prokaryotic cells are force wielders and sensors. However, whereas such forces have been used to control mechanically dependent behaviors in mammalian cells, we lack the same level of understanding in bacteria. Surface adhesion, the initial stages of biofilm formation and surface biofouling, is a mechanically dependent process, which makes it an ideal target for mechano-control. In this study, we employed nanometer surface vibrations to mechanically stimulate bacteria and investigate their effect on adhesion. We discovered that vibrational stimulation at the nanoscale consistently reduces surface adhesion by altering cell membrane potential. Our findings identify a link between bacteria electrophysiology and surface adhesion and provide evidence that the nanometric mechanical "tickling" of bacteria can inhibit surface adhesion.

In the developing cochlea, just before the onset of hearing on postnatal day 12, the medial olivocochlear efferent axons in synaptic contact with the inner hair cells (IHCs) start withdrawing and new efferent synaptic connections are formed on the outer hair cells (OHCs), thereby progressing towards the adult pattern of medial olivocochlear efferent innervation. The synapses are inhibitory, calcium influx through the α9α10 nicotinic acetylcholine receptors (nAChRs) driving opening of calcium-dependent potassium channels. The nAChRs appear to function similarly in IHCs and OHCs, although with probable kinetic differences. Our aim was to assess their functional similarity in the neonatal mouse cochlea by making whole-cell recordings from both hair cell types between postnatal day 7 and 10 when nAChRs are expressed. ACh was applied to voltage-clamped hair cells by pressure-ejection from a pipette. The cells were dialysed with a Cs + -based solution designed to eliminate calcium-dependent potassium currents. There were differences in amplitude, voltage-sensitivity and reversal potential of the nAChR currents between IHCs and OHCs. There was also some indication that IHC nAChRs have slower activation and desensitization kinetics, although the relatively slow ACh application limited interpretation of this result. These differences, particularly concerning the reversal potential, might indicate the presence of different auxiliary protein subunits of the α9α10 receptor in neonatal IHCs and OHCs., Competing Interests: Declarations of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Voltage imaging is an important complement to traditional methods for probing cellular physiology, such as electrode-based patch clamp techniques. Unlike the related Ca 2+ imaging, voltage imaging provides a direct visualization of bioelectricity changes. We have been exploring the use of sulfonated silicon rhodamine dyes (Berkeley Red Sensor of Transmembrane potential, BeRST) for voltage imaging. In this study, we explore the effect of converting BeRST to diEt BeRST, by replacing the dimethyl aniline of BeRST with a diethyl aniline group. The new dye, diEt BeRST, has a voltage sensitivity of 40% Δ F / F per 100 mV, a 33% increase compared to the original BeRST dye, which has a sensitivity of 30% Δ F / F per 100 mV. In neurons, the cellular brightness of diEt BeRST is about 20% as bright as that of BeRST, which may be due to the lower solubility of diEt BeRST (300 μM) compared to that of BeRST (800 μM). Despite this lower cellular brightness, diEt BeRST is able to record spontaneous and evoked action potentials from multiple neurons simultaneously and in single trials. Far-red excitation and emission profiles enable diEt BeRST to be used alongside existing fluorescent indicators of cellular physiology, like Ca 2+ -sensitive Oregon Green BAPTA. In hippocampal neurons, simultaneous voltage and Ca 2+ imaging reveals neuronal spiking patterns and frequencies that cannot be resolved with traditional Ca 2+ imaging methods. This study represents a first step toward describing the structural features that define voltage sensitivity and brightness in silicon rhodamine-based BeRST indicators.

Efflux pumps are well known to be an important mechanism for removing noxious substances such as antibiotics from bacteria. Given that many antibiotics function by accumulating inside bacteria, efflux pumps contribute to resistance. Efflux pump inactivation is a potential strategy to combat antimicrobial resistance, as bacteria would not be able to pump out antibiotics. We recently discovered that the impact of loss of efflux function is only apparent in actively growing cells. We demonstrated that the global transcriptome of Salmonella Typhimurium is drastically altered during slower growth leading to stationary-phase cells having a remodeled, less permeable envelope that prevents antibiotics entering the cell. Here, we investigated the effects of deleting the major efflux pump of Salmonella Typhimurium, AcrB, on global gene transcription across growth. We revealed that an acrB knockout entered stationary phase later than the wild-type strain SL1344 and displayed increased and prolonged expression of genes responsible for anaerobic energy metabolism. We devised a model linking efflux and membrane potential, whereby deactivation of AcrB prevents influx of protons across the inner membrane and thereby hyperpolarization. Knockout or deactivation of AcrB was demonstrated to increase membrane potential. We propose that the global transcription regulator ArcBA senses changes to the redox state of the quinol pool (linked to the membrane potential of the bacterium) and coordinates the shift from exponential to stationary phase via the key master regulators RpoS, Rsd, and Rmf. Inactivation of efflux pumps therefore influences the fundamental physiology of Salmonella , with likely impacts on multiple phenotypes.IMPORTANCEWe demonstrate for the first time that deactivation of efflux pumps brings about changes to gross bacterial physiology and metabolism. Rather than simply being a response to noxious substances, efflux pumps appear to play a key role in maintenance of membrane potential and thereby energy metabolism. This discovery suggests that efflux pump inhibition or inactivation might have unforeseen positive consequences on antibiotic activity. Given that stationary-phase bacteria are more resistant to antibiotic uptake, late entry into stationary phase would prolong antibiotic accumulation by bacteria. Furthermore, membrane hyperpolarization could result in increased generation of reactive species proposed to be important for the activity of some antibiotics. Finally, changes in gross physiology could also explain the decreased virulence of efflux mutants., Competing Interests: The authors declare no conflict of interest.

The regulation of the cell membrane potential plays a crucial role in governing the transmembrane transport of various ions and cellular life processes. However, in situ and on-demand modulation of cell membrane potential for ion channel regulation is challenging. Herein, we have constructed a supramolecular assembly system based on water-soluble cationic oligo(phenylenevinylene) (OPV) and cucurbit[7]uril (CB[7]). The controllable disassembly of OPV/4CB[7] combined with the subsequent click reaction provides a step-by-step adjustable surface positive potential. These processes can be employed in situ on the plasma membrane to modulate the membrane potential on-demand for precisely controlling the activation of the transient receptor potential vanilloid 1 (TRPV1) ion channel and up-regulating exogenous calcium-responsive gene expression. Compared with typical optogenetics, electrogenetics, and mechanogenetics, our strategy provides a perspective supramolecular genetics toolbox for the regulation of membrane potential and downstream intracellular gene regulation events.

Electrophysiology typically deals with the electrical properties of excitable cells like neurons and muscles. However, all other cells (non-excitable) also possess bioelectric membrane potentials for intracellular and extracellular communications. These membrane potentials are generated by different ions present in fluids available in and outside the cell, playing a vital role in communication and coordination between the cell and its organelles. Bioelectric membrane potential variations disturb cellular ionic homeostasis and are characteristic of many diseases, including cancers. A rapidly increasing interest has emerged in sorting out the electrophysiology of cancer cells. Compared to healthy cells, the distinct electrical properties exhibited by cancer cells offer a unique way of understanding cancer development, migration, and progression. Decoding the altered bioelectric signals influenced by fluctuating electric fields benefits understanding cancer more closely. While cancer research has predominantly focussed on genetic and molecular traits, the delicate area of electrophysiological characteristics has increasingly gained prominence. This review explores the historical exploration of electrophysiology in the context of cancer cells, shedding light on how alterations in bioelectric membrane potentials, mediated by ion channels and gap junctions, contribute to the pathophysiology of cancer., Competing Interests: Declarations. Conflict of Interest: The authors declare no conflicts of interest for the studies reported in the article., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective. The aim of this study is to address the limitations in reconstructing the electrical activity of the heart from the body surface electrocardiogram, which is an ill-posed inverse problem. Current methods often assume values commonly used in the literature in the absence of a priori knowledge, leading to errors in the model. Furthermore, most methods ignore the dynamic activation process inherent in cardiomyocytes during the cardiac cycle. Approach. To overcome these limitations, we propose an extended Kalman filter (EKF)-based neural network approach to dynamically reconstruct cardiac transmembrane potential (TMP). Specifically, a recurrent neural network is used to establish the state estimation equation of the EKF, while a convolutional neural network is used as the measurement equation. The Jacobi matrix of the network undergoes a correction feedback process to obtain the Kalman gain. Main results. After repeated iterations, the final estimated state vector, i.e. the reconstructed image of the TMP, is obtained. The results from both the final simulation and real experiments demonstrate the robustness and accurate quantification of the model. Significance. This study presents a new approach to cardiac TMP reconstruction that offers higher accuracy and robustness compared to traditional methods. The use of neural networks and EKFs allows dynamic modelling that takes into account the activation processes inherent in cardiomyocytes and does not require a priori knowledge of inputs such as forward transition matrices., (© 2024 Institute of Physics and Engineering in Medicine.)

Subtle changes in the membrane potential of pulmonary arterial smooth muscle cells (PASMCs) are pivotal for controlling pulmonary vascular tone, e.g., for initiating Hypoxic Pulmonary Vasoconstriction, a vital mechanism of the pulmonary circulation. In our study, we evaluated the ability of the fluorescence resonance energy transfer (FRET)-based voltage-sensor Mermaid to detect such subtle changes in membrane potential. Mouse PASMCs were isolated and transduced with Mermaid-encoding lentiviral vectors before the acceptor/donor emission ratio was assessed via live cell FRET-imaging. Mermaid's sensitivity was tested by applying specific potassium chloride (KCl) concentrations. These KCl concentrations were previously validated by patch clamp recordings to induce depolarization with predefined amplitudes that physiologically occur in PASMCs. Mermaid's emission ratio dose-dependently increased upon depolarization with KCl. However, Mermaid formed unspecific intracellular aggregates, which limited the usefulness of this voltage sensor. When analyzing the membrane rim only to circumvent these unspecific signals, Mermaid was not suitable to resolve subtle changes in the membrane potential of ≤10 mV. In summary, we found Mermaid to be a suitable alternative for reliably detecting qualitative membrane voltage changes of more than 10 mV in primary mouse PASMCs. However, one should be aware of the limitations associated with this voltage sensor.

Prokaryotic type III CRISPR-Cas systems provide immunity against viruses and plasmids using CRISPR-associated Rossman fold (CARF) protein effectors 1-5 . Recognition of transcripts of these invaders with sequences that are complementary to CRISPR RNA guides leads to the production of cyclic oligoadenylate second messengers, which bind CARF domains and trigger the activity of an effector domain 6,7 . Whereas most effectors degrade host and invader nucleic acids, some are predicted to contain transmembrane helices without an enzymatic function. Whether and how these CARF-transmembrane helix fusion proteins facilitate the type III CRISPR-Cas immune response remains unknown. Here we investigate the role of cyclic oligoadenylate-activated membrane protein 1 (Cam1) during type III CRISPR immunity. Structural and biochemical analyses reveal that the CARF domains of a Cam1 dimer bind cyclic tetra-adenylate second messengers. In vivo, Cam1 localizes to the membrane, is predicted to form a tetrameric transmembrane pore, and provides defence against viral infection through the induction of membrane depolarization and growth arrest. These results reveal that CRISPR immunity does not always operate through the degradation of nucleic acids, but is instead mediated via a wider range of cellular responses., (© 2024. The Author(s).)

It is common to say that the origin of the membrane potential is attributed to transmembrane ion transport, but it is theoretically possible to explain its generation by the mechanism of ion adsorption. It has been previously suggested that the ion adsorption mechanism even leads to potential formulae identical to the famous Nernst equation or the Goldman-Hodgkin-Katz equation. Our further analysis, presented in this paper, indicates that the potential formula based on the ion adsorption mechanism leads to an equation that is a function of the surface charge density of the material and the surface potential of the material. Furthermore, we have confirmed that the equation holds in all the different experimental systems that we have studied. This equation appears to be a key equation that governs the characteristics of the membrane potential in all systems., (© 2023. Prof. Dr. Jan van der Hoeven stichting voor theoretische biologie.)

Subthreshold depolarization enhances neurotransmitter release evoked by action potentials and plays a key role in modulating synaptic transmission by combining analog and digital signals. This process is known to be Ca 2+ dependent. However, the underlying mechanism of how small changes in basal Ca 2+ caused by subthreshold depolarization can regulate transmitter release triggered by a large increase in local Ca 2+ is not well understood. This study aimed to investigate the source and signaling mechanisms of Ca 2+ that couple subthreshold depolarization with the enhancement of glutamate release in hippocampal cultures and CA3 pyramidal neurons. Subthreshold depolarization increased presynaptic Ca 2+ levels, the frequency of spontaneous release, and the amplitude of evoked release, all of which were abolished by blocking L-type Ca 2+ channels. A high concentration of intracellular Ca 2+ buffer or blockade of calmodulin abolished depolarization-induced increases in transmitter release. Estimation of the readily releasable pool size using hypertonic sucrose showed depolarization-induced increases in readily releasable pool size, and this increase was abolished by the blockade of calmodulin. Our results provide mechanistic insights into the modulation of transmitter release by subthreshold potential change and highlight the role of L-type Ca 2+ channels in coupling subthreshold depolarization to the activation of Ca 2+ -dependent signaling molecules that regulate transmitter release.

Membrane theory makes it possible to compute the membrane potential of living cells accurately. The principle is that the plasma membrane is selectively permeable to ions and that its permeability to mobile ions determines the characteristics of the membrane potential. However, an artificial experimental cell system with an impermeable membrane can exhibit a nonzero membrane potential, and its characteristics are consistent with the prediction of the Goldman-Hodgkin-Katz eq., which is a noteworthy concept of membrane theory, despite the membrane's impermeability to mobile ions. We noticed this troublesome facet of the membrane theory. We measured the potentials through permeable and impermeable membranes where we used the broad varieties of membranes. Then we concluded that the membrane potential must be primarily, although not wholly, governed by the ion adsorption-desorption process rather than by the passage of ions across the cell membrane. A theory based on the Association-Induction Hypothesis seems to be a more plausible mechanism for the generation of the membrane potential and to explain this unexpected physiological fact. The Association-Induction Hypothesis states that selective ion permeability of the membrane is not a condition for the generation of the membrane potential in living cells, which contradicts the prediction of the membrane theory. Therefore, the Association-Induction Hypothesis is the actual cause of membrane potential. We continued the theoretical analysis by taking into account the Association-Induction Hypothesis and saw that its universality as a cause of potential generation mechanism. We then concluded that the interfacial charge distribution is one of the fundamental causes of the membrane potential., (© 2022. Akadémiai Kiadó Zrt.)

Voltage-sensing phosphatase (VSP) consists of a voltage sensor domain (VSD) and a cytoplasmic catalytic region (CCR), which is similar to phosphatase and tensin homolog (PTEN). How the VSD regulates the innate enzyme component of VSP remains unclear. Here, we took a combined approach that entailed the use of electrophysiology, fluorometry, and structural modeling to study the electrochemical coupling in Ciona intestinalis VSP. We found that two hydrophobic residues at the lowest part of S4 play an essential role in the later transition of VSD-CCR coupling. Voltage clamp fluorometry and disulfide bond locking indicated that S4 and its neighboring linker move as one helix (S4-linker helix) and approach the hydrophobic spine in the CCR, a structure located near the cell membrane and also conserved in PTEN. We propose that the hydrophobic spine operates as a hub for translating an electrical signal into a chemical one in VSP.

Knowledge of the electrical potentials within different compartments of a biological cell induced by applied alternating fields is needed for assessing the effects of electromagnetic radiation on cells, understanding electroporation and other electric field-induced effects, and deriving expressions for the complex permittivity of suspensions of cells. In the work presented in this paper, closed-form analytical expressions have been derived for the electrical potentials within different layers of an inhomogeneous particle consisting of four different dielectric layers and suspended in a homogeneous medium. Those expressions have been used to derive, for the case of a realistic model of a cell containing a large concentric organelle, expressions for the transmembrane potentials (at cell and organelle level) and electric fields within the cell compartments induced by applied fields. The results of the present theoretical model indicate points of departure between the present and previous theoretical models. The present theory also confirms the validity of the equivalence approach introduced by Irimajiri and co-workers for computing the complex permittivity for suspensions of multi-shelled particles. In addition, it shows that the electric field is amplified at the level of the cell and organelle membranes, but not within other cell compartments., Competing Interests: Declaration of Competing Interest The author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is caused by a developmentally regulated silencing of the FMR1 gene, but its effect on human neuronal network development and function is not fully understood. Here, we isolated isogenic human embryonic stem cell (hESC) subclones-one with a full FX mutation and one that is free of the mutation (control) but shares the same genetic background-differentiated them into induced neurons (iNs) by forced expression of NEUROG-1 , and compared the functional properties of the derived neuronal networks. High-throughput image analysis demonstrates that FX-iNs have significantly smaller cell bodies and reduced arborizations than the control. Both FX- and control-neurons can discharge repetitive action potentials, and FX neuronal networks are also able to generate spontaneous excitatory synaptic currents with slight differences from the control, demonstrating that iNs generate more mature neuronal networks than the previously used protocols. MEA analysis demonstrated that FX networks are hyperexcitable with significantly higher spontaneous burst-firing activity compared to the control. Most importantly, cross-correlation analysis enabled quantification of network connectivity to demonstrate that the FX neuronal networks are significantly less synchronous than the control, which can explain the origin of the development of intellectual dysfunction associated with FXS.

Genetically encoded voltage indicators (GEVIs) allow optical recordings of membrane potential changes in defined cell populations. Transgenic reporter animals that facilitate precise and repeatable targeting with high expression levels would further the use of GEVIs in the in vivo mammalian brain. However, the literature on developing and applying transgenic mouse lines as vehicles for GEVI expression is limited. Here we report the first in vivo experiments using a transgenic reporter mouse for the GEVI ArcLight, which utilizes a Cre/tTA dependent expression system (TIGRE 1.0). We developed two mouse lines with ArcLight expression restricted to either olfactory receptor neurons, or a subpopulation of interneurons located in the granule and glomerular layers in the olfactory bulb. The ArcLight expression in these lines was sufficient for in vivo imaging of odorant responses in single trials using epifluorescence and 2-photon imaging. The voltage responses were odor-specific and concentration-dependent, which supported earlier studies about perceptual transformations carried out by the bulb that used calcium sensors of neural activity. This study demonstrates that the ArcLight transgenic line is a flexible genetic tool that can be used to record the neuronal electrical activity of different cell types with a signal-to-noise ratio that is comparable to previous reports using viral transduction., (© 2022. The Author(s).)

Ligand-gated ion channels (LGIC, also referred to as ionotropic receptors) are important transmembrane proteins that open to allow ions to flow across the membrane and locally modify the membrane potential in response to the binding of a ligand. For more than a decade, a tremendous effort has been carried out in the determination of many LGIC structures in high resolution, leading to an unprecedented molecular description of channel gating. However, it is sometimes difficult to classify experimentally derived structures to their corresponding functional states, and alternative methods may help resolve or refine this issue. In this review, we focus on the application of photo-isomerizable tweezers (PIT) as a powerful strategy to interrogate molecular mechanisms of LGIC while assessing their functionality by electrophysiology., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Alzheimer's disease (AD) is the most common cause of dementia affecting millions of people. Neuronal death in AD is initiated by oligomeric amyloid-β (Aβ) peptides. Recently, we proposed the amyloid degradation toxicity hypothesis, which explains multiple major observations associated with AD including autophagy failure and a decreased metabolism. According to the hypothesis, the key event in the cellular toxicity of amyloid is the formation of non-selective membrane channels in lysosomal membranes by amyloid fragments that are produced by the digestion of Aβ previously absorbed by endocytosis. Electrophysiological data suggest that amyloid-formed channels have different sizes, which can be explained by the fact that channel creating barrel-shaped amyloid aggregates can consist of different number of monomers. To estimate the ability of channels to leak molecules of various molecular weights, we modeled the channels as saline-filled cylinders in non-conductive membranes that pass spheres with a density of average globular proteins. As a basis, we used the conductance distribution taken from the previously published experimental dataset, in which single channels with electrical conductance of up to one nanosiemens were registered. Our calculations show that channels with such a giant conductance can allow for passing macromolecules such as large as lysosomal cathepsins implicated in the activation of apoptosis. The formation of giant channels is disproportionally promoted in an acidic environment. Also, amyloid fragments leaking from permeabilized lysosomes can reach the internal leaflet of the plasma membrane and permeabilize it. We conclude that while dissipation of the proton gradient by any (even smallest) amyloid channels readily explains lysosomal failure, the relatively rare events of lysosomal permeabilization to large macromolecules can be an additional mechanism of cellular death induced by exposure to Aβ., (Copyright © 2021 Elsevier B.V. All rights reserved.)

S-Adenosyl-L-methionine (SAM) was used to probe the functional effects exerted via the cardiac RyR isoform (RyR2) adenine nucleotide binding site. Single channel experiments revealed that SAM applied to the cytoplasmic face of RyR2 had complex voltage dependent effects on channel gating and conductance. At positive transmembrane holding potentials, SAM caused a striking reduction in channel openings and a reduced channel conductance. In contrast, at negative potentials, SAM promoted a clearly resolved subconductance state. At membrane potentials between -75 and -25 mV, the open probability of the subconductance state was independent of voltage. ATP, but not the non-adenosine-based ryanodine receptor (RyR) activator 4-chloro-m-cresol, interfered with the effects of SAM at both negative and positive potentials. This suggests that ATP and SAM interact with a common binding site. Molecular docking showed SAM bound to the adenine nucleotide binding site and formed a hydrogen bond to Glu4886 in the C-terminal end of the S6 alpha helix. In this configuration, SAM may alter the conformation of the RyR2 ion conduction pathway. This work provides novel insightGraphical Abstract into potential functional outcomes of ligand binding to the RyR adenine nucleotide binding site., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Excitation-contraction coupling (ECC) is the process by which electrical excitation of muscle is converted into force generation. Depolarization of skeletal muscle resting potential contributes to failure of ECC in diseases such as periodic paralysis, intensive care unit acquired weakness and possibly fatigue of muscle during vigorous exercise. When extracellular K + is raised to depolarize the resting potential, failure of ECC occurs suddenly, over a narrow range of resting potentials. Simultaneous imaging of Ca 2+ transients and recording of action potentials (APs) demonstrated failure to generate Ca 2+ transients when APs peaked at potentials more negative than -30mV. An AP property that closely correlated with failure of the Ca 2+ transient was the integral of AP voltage with respect to time. Simultaneous recording of Ca 2+ transients and APs with electrodes separated by 1.6mm revealed AP conduction fails when APs peak below -21mV. We hypothesize propagation of APs and generation of Ca 2+ transients are governed by distinct AP properties: AP conduction is governed by AP peak, whereas Ca 2+ release from the sarcoplasmic reticulum is governed by AP integral. The reason distinct AP properties may govern distinct steps of ECC is the kinetics of the ion channels involved. Na channels, which govern propagation, have rapid kinetics and are insensitive to AP width (and thus AP integral) whereas Ca 2+ release is governed by gating charge movement of Cav1.1 channels, which have slower kinetics such that Ca 2+ release is sensitive to AP integral. The quantitative relationships established between resting potential, AP properties, AP conduction and Ca 2+ transients provide the foundation for future studies of failure of ECC induced by depolarization of the resting potential., Competing Interests: XW, MN, CD, JM, SB, RB, BF, AV, MR No competing interests declared, (© 2022, Wang et al.)

Two mathematically distinct physiological concepts, the Goldman-Hodgkin-Katz eq. (GHK eq.) and the Hodgkin-Huxley model (HH model) were successfully associated with each other in a prior work. The previous work was performed on the following premises (i) The membrane potential is generated by ion adsorption, as opposed to the classical ion transport mechanisms, (ii) The living cell is a thermodynamically real system rather than an ideal system, and (iii) The conductance employed in the HH model is replaced by the ion activity coefficient, which is weighted with the role of conductance. Consequently, the GHK eq. was mathematically associated with the HH model through the intermediary of Boltzmann ion distribution and mass action law. To verify if our theoretical formularization could afford a physiologically, physically and chemically viable model, we performed computational analysis using the formulae (quantitative correlations between various variables) we derived in the previous work. The computational results obtained through associating the GHK eq. with the HH model validated our model and its predictions. This outcome suggests that the current prevailing physiological concepts could be expanded further, to incorporate the newly proposed mechanisms. That is, GHK eq. and HH model could be interpreted via another set of founding principles that incorporate the ubiquitous phenomena of ion-adsorption., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Ruminants such as cattle and sheep depend on the breakdown of carbohydrates from plant-based feedstuff, which is accomplished by the microbial community in the rumen. Roughly 40% of the members of the rumen microbiota belong to the family Prevotellaceae , which ferments sugars to organic acids such as acetate, propionate, and succinate. These substrates are important nutrients for the ruminant. In a metaproteome analysis of the rumen of cattle, proteins that are homologous to the Na + -translocating NADH:quinone oxidoreductase (NQR) and the quinone:fumarate reductase (QFR) were identified in different Prevotella species. Here, we show that fumarate reduction to succinate in anaerobically growing Prevotella bryantii is coupled to chemiosmotic energy conservation by a supercomplex composed of NQR and QFR. This s odium-translocating N ADH: f umarate oxido r eductase (SNFR) supercomplex was enriched by blue native PAGE (BN-PAGE) and characterized by in-gel enzyme activity staining and mass spectrometry. High NADH oxidation (850 nmol min -1  mg -1 ), quinone reduction (490 nmol min -1  mg -1 ), and fumarate reduction (1,200 nmol min -1  mg -1 ) activities, together with high expression levels, demonstrate that SNFR represents a charge-separating unit in P. bryantii . Absorption spectroscopy of SNFR exposed to different substrates revealed intramolecular electron transfer from the flavin adenine dinucleotide (FAD) cofactor in NQR to heme b cofactors in QFR. SNFR catalyzed the stoichiometric conversion of NADH and fumarate to NAD + and succinate. We propose that the regeneration of NAD + in P. bryantii is intimately linked to the buildup of an electrochemical gradient which powers ATP synthesis by electron transport phosphorylation. IMPORTANCE Feeding strategies for ruminants are designed to optimize nutrient efficiency for animals and to prevent energy losses like enhanced methane production. Key to this are the fermentative reactions of the rumen microbiota, dominated by Prevotella spp. We show that succinate formation by P. bryantii is coupled to NADH oxidation and sodium gradient formation by a newly described supercomplex consisting of Na + -translocating NADH:quinone oxidoreductase (NQR) and fumarate reductase (QFR), representing the s odium-translocating N ADH: f umarate oxido r eductase (SNFR) supercomplex. SNFR is the major charge-separating module, generating an electrochemical sodium gradient in P. bryantii . Our findings offer clues to the observation that use of fumarate as feed additive does not significantly increase succinate production, or decrease methanogenesis, by the microbial community in the rumen.

The cellular resting membrane potential (V m ) not only determines electrical responsiveness of excitable cells but also plays pivotal roles in non-excitable cells, mediating membrane transport, cell-cycle progression, and tumorigenesis. Studying these processes requires estimation of V m , ideally over long periods of time. Here, we introduce two ratiometric genetically encoded V m indicators, rArc and rASAP, and imaging and analysis procedures for measuring differences in average resting V m between cell groups. We investigated the influence of ectopic expression of K + channels and their disease-causing mutations involved in Andersen-Tawil (Kir2.1) and Temple-Baraitser (K V 10.1) syndrome on median resting V m of HEK293T cells. Real-time long-term monitoring of V m changes allowed to estimate a 40-50 min latency from induction of transcription to functional Kir2.1 channels in HEK293T cells. The presented methodology is readily implemented with standard fluorescence microscopes and offers deeper insights into the role of the resting V m in health and disease., (© 2021. The Author(s).)

To gain insight into the mechanisms of ionophoric activity of usnic acid (UA), we examined the UA-induced generation of potentials on a planar bilayer lipid membrane (BLM) in the presence of concentration gradients of hydrogen and magnesium or calcium ions under open-circuit conditions. Remarkably, the BLM potential generated by UA at the proton concentration gradient of 1 pH unit was approximately twice the Nernst equilibrium level. With a concentration gradient of magnesium or calcium ions, the BLM potential generated by UA had the opposite sign. The observed anomalies in the membrane potentials were consistent with a theory developed by Markin and Sokolov (Bioelectrochem. Bioenerg. 1990) for the case of ionophore-mediated coupled fluxes of several ions across a membrane., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Recently calcium homeostasis modulators (CALHMs) are identified as ATP release channels play crucial role in functioning of neurons including gustatory signaling and neuronal excitability. Pathologies of Alzheimer's disease and depression have been associated with the dysfunction of CALHMs. Recently, CALHMs has been emerged as an important therapeutic research particularly in neurobiological studies. CALHM1 is most extensively studied among CALHMs and is an ATP and ion channel that is activated by membrane depolarization or removal of extracellular Ca 2+ . Despite the emerged role of CALHM5 shown by an recently assembled data; however, the neuronal function remains obscure until the first Cryo-EM structure of CALHM5 was recently solved by various research group which acts as a template to study the hidden functional properties of the CALHM5 protein based on structure function mechanism. It provides insight in some of the different pathophysiological roles. CALHM5 structure showed an abnormally large pore channel structure assembled as an undecamer with four transmembrane helices (TM1-TM4), an N-terminal helix (NTH), an extracellular loop region and an intracellular C-terminal domain (CTD) that consists of three α-helices CH1-3. The TM1 and NTH were always poorly defined among all CALHMs; however, these regions were well defined in CALHM5 channel structure. In this context, this review will provide insight in structure, function and mechanism to understand its significant role in pathological diseases particularly in Alzheimer's disease. Moreover, it focuses on CALHM5 structure and recent associated properties based on Cryo-EM research., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Transient receptor potential cation channel subfamily M member 4 (TRPM4) is a Ca 2+ -activated nonselective cation channel that mediates membrane depolarization. Although, a current with the hallmarks of a TRPM4-mediated current has been previously reported in pancreatic acinar cells (PACs), the role of TRPM4 in the regulation of acinar cell function has not yet been explored. In the present study, we identify this TRPM4 current and describe its role in context of Ca 2+ signaling of PACs using pharmacological tools and TRPM4-deficient mice. We found a significant Ca 2+ -activated cation current in PACs that was sensitive to the TRPM4 inhibitors 9-phenanthrol and 4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic acid (CBA). We demonstrated that the CBA-sensitive current was responsible for a Ca 2+ -dependent depolarization of PACs from a resting membrane potential of -44.4 ± 2.9 to -27.7 ± 3 mV. Furthermore, we showed that Ca 2+ influx was higher in the TRPM4 KO- and CBA-treated PACs than in control cells. As hormone-induced repetitive Ca 2+ transients partially rely on Ca 2+ influx in PACs, the role of TRPM4 was also assessed on Ca 2+ oscillations elicited by physiologically relevant concentrations of the cholecystokinin analog cerulein. These data show that the amplitude of Ca 2+ signals was significantly higher in TRPM4 KO than in control PACs. Our results suggest that PACs are depolarized by TRPM4 currents to an extent that results in a significant reduction of the inward driving force for Ca 2+ . In conclusion, TRPM4 links intracellular Ca 2+ signaling to membrane potential as a negative feedback regulator of Ca 2+ entry in PACs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The two-pore domain K + (K 2P ) channel, which is involved in setting the resting membrane potential in neurons, is an essential target for receptor agonists. Activation of the γ-aminobutyric acid (GABA) receptors (GABA A R and GABA B R) reduces cellular excitability through Cl - influx and K + efflux in neurons. Relatively little is known about the link between GABA A R and the K + channel. The present study was performed to identify the effect of GABAR agonists on K 2P channel expression and activity in the neuroblastic B35 cells that maintain glutamic acid decarboxylase (GAD) activity and express GABA. TASK and TREK/TRAAK mRNA were expressed in B35 cells with a high level of TREK-2 and TRAAK. In addition, TREK/TRAAK proteins were detected in the GABAergic neurons obtained from GABA transgenic mice. Furthermore, TREK-2 mRNA and protein expression levels were markedly upregulated in B35 cells by GABA A R and GABA B R agonists. In particular, muscimol, a GABA A R agonist, significantly increased TREK-2 expression and activity, but the effect was reduced in the presence of the GABA A R antagonist bicuculine or TREK-2 inhibitor norfluoxetine. In the whole-cell and single-channel patch configurations, muscimol increased TREK-2 activity, but the muscimol effect disappeared in the N-terminal deletion mutant. These results indicate that muscimol directly induces TREK-2 activation through the N-terminus and suggest that muscimol can reduce cellular excitability by activating the TREK-2 channel and by inducing Cl - influx in GABAergic neurons.

Many neurotransmitters are organic ions that carry a net charge, and their release from secretory vesicles is therefore an electrodiffusion process. The selectivity of early exocytotic fusion pores is investigated by combining electrodiffusion theory, measurements of amperometric foot signals from chromaffin cells with anion substitution, and molecular dynamics simulation. The results reveal that very narrow fusion pores are cation selective, but more dilated fusion pores become anion permeable. The transition occurs around a fusion pore conductance of ∼300 pS. The cation selectivity of a narrow fusion pore accelerates the release of positively charged transmitters such as dopamine, noradrenaline, adrenaline, serotonin, and acetylcholine, while glutamate release may require a more dilated fusion pore., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

H + -ATP-ases, which support proton efflux through the plasma membrane, are key molecular transporters for electrogenesis in cells of higher plants. Initial activities of the transporters can influence the thresholds of generation of electrical responses induced by stressors and modify other parameters of these responses. Previously, it was theoretically shown that the stochastic heterogeneity of individual cell thresholds for electrical responses in a system of electrically connected neuronal cells can decrease the total threshold of the system ("diversity-induced resonance", DIR). In the current work, we tested a hypothesis about decreasing the thresholds of generation of cooling-induced electrical responses in a system of electrically connected plant cells with increasing stochastic spatial heterogeny in the initial activities of H + -ATP-ases in these cells. A two-dimensional model of the system of electrically connected excitable cells (simple imitation of plant leaf), which was based on a model previously developed in our works, was used for the present investigation. Simulation showed that increasing dispersion in the distribution of initial activities of H + -ATP-ases between cells decreased the thresholds of generation of cooling-induced electrical responses. In addition, the increasing weakly influenced the amplitudes of electrical responses. Additional analysis showed two different mechanisms of the revealed effect. The increasing spatial heterogeneity in activities of H + -ATP-ases induced a weak positive shift of the membrane potential at rest. The shift decreased the threshold of electrical response generation. However, the decreased threshold induced by increasing the H + -ATP-ase activity heterogeneity was also observed after the elimination of the positive shift. The result showed that the "DIR-like" mechanism also participated in the revealed effect. Finally, we showed that the standard deviation of the membrane potentials before the induction of action potentials could be used for the estimation of thresholds of cooling-induced plant electrical responses. Thus, spatial heterogeneity in the initial activities of H + -ATP-ases can be a new regulatory mechanism influencing the generation of electrical responses in plants under actions of stressors.

As the main theory of carcinogenesis, the Somatic Mutation Theory, increasingly presents difficulties to explain some experimental observations, different theories are being proposed. A major alternative approach is the Tissue Organization Field Theory, which explains cancer origin as a tissue regulation disease instead of having a mainly cellular origin. This work fits in the latter hypothesis, proposing the bioelectric field, in particular the cell membrane polarization state, and ionic exchange through ion channels and gap junctions, as an important mechanism of cell communication and tissue organization and regulation. Taking into account recent experimental results and proposed bioelectric models, a computational model of cancer initiation was developed, including the propagation of a cell depolarization wave in the tissue under consideration. Cell depolarization leads to a change in its state, with the activation and deactivation of several regulation pathways, increasing cell proliferation and motility, changing its epigenetic state to a more stem cell-like behavior without the requirement of genomic mutation. The intercellular communication via gap junctions leads, in certain circumstances, to a bioelectric state propagation to neighbor cells, in a chain-like reaction, till an electric discontinuity is reached. However, this is a reversible process, and it was shown experimentally that, by implementing a therapy targeted on cell ion exchange channels, it is possible to reverse the state and repolarize cells. This mechanism can be an important alternative way in cancer prevention, diagnosis and therapy, and new experiments are proposed to test the presented hypothesis.

Hepatic lipid accumulation in obesity correlates with the severity of hyperinsulinemia and systemic insulin resistance. Obesity-induced hepatocellular lipid accumulation results in hepatocyte depolarization. We have established that hepatocyte depolarization depresses hepatic afferent vagal nerve firing, increases GABA release from liver slices, and causes hyperinsulinemia. Preventing hepatic GABA release or eliminating the ability of the liver to communicate to the hepatic vagal nerve ameliorates the hyperinsulinemia and insulin resistance associated with diet-induced obesity. In people with obesity, hepatic expression of GABA transporters is associated with glucose infusion and disposal rates during a hyperinsulinemic euglycemic clamp. Single-nucleotide polymorphisms in hepatic GABA re-uptake transporters are associated with an increased incidence of type 2 diabetes mellitus. Herein, we identify GABA as a neuro-hepatokine that is dysregulated in obesity and whose release can be manipulated to mute or exacerbate the glucoregulatory dysfunction common to obesity., Competing Interests: Declaration of interests The results presented in this paper have resulted in patent cooperation treaty Application No. 62/511,753 and 62/647,468: METHODS AND COMPOSITIONS FOR REGULATING GLUCOSE HOMEOSTASIS, which has been licensed by Livendocrine, LLC founded by Benjamin Renquist., (Published by Elsevier Inc.)

Cytokines are important neuroinflammatory modulators in neurodegenerative brain disorders including traumatic brain injury (TBI) and stroke. However, their temporal effects on the physiological properties of microglia and neurons during the recovery period have been unclear. Here, using an ATP-induced cortical injury model, we characterized selective effects of ATP injection compared to needle-control. In the damaged region, the fluorescent intensity of CX 3 CR1-GFP (+) cells, as well as the cell density, was increased and the maturation of newborn BrdU (+) cells continued until 28 day-post-injection (dpi) of ATP. The excitability and synaptic E/I balance of neurons and the inward and outward membrane currents of microglia were increased at 3 dpi, when expressions of tumor necrosis factor (TNF)-α/interleukin (IL)-1β and IL-10/IL-4 were also enhanced. These changes of both cells at 3 dpi were mostly decayed at 7 dpi and were suppressed by any of IL-10, IL-4, suramin (P2 receptor inhibitor) and 4-AP (K + channel blocker). Acute ATP application alone induced only small effects from both naïve neurons and microglial cells in brain slice. However, TNF-α alone effectively increased the excitability of naïve neurons, which was blocked by suramin or 4-AP. TNF-α and IL-1β increased and decreased membrane currents of naïve microglia, respectively. Our results suggest that ATP and TNF-α dominantly induce the physiological activities of 3 dpi neurons and microglia, and IL-10 effectively suppresses such changes of both activated cells in K + channel- and P2 receptor-dependent manner, while IL-4 suppresses neurons preferentially.

Late sodium current (late INa) inhibition has been proposed to suppress the incidence of arrhythmias generated by pathological states or induced by drugs. However, the role of late INa in the human heart is still poorly understood. We therefore investigated the role of this conductance in arrhythmias using adult primary cardiomyocytes and tissues from donor hearts. Potentiation of late INa with ATX-II (anemonia sulcata toxin II) and E-4031 (selective blocker of the hERG channel) slowed the kinetics of action potential repolarization, impaired Ca 2+ homeostasis, increased contractility, and increased the manifestation of arrhythmia markers. These effects could be reversed by late INa inhibitors, ranolazine and GS-967. We also report that atrial tissues from donor hearts affected by atrial fibrillation exhibit arrhythmia markers in the absence of drug treatment and inhibition of late INa with GS-967 leads to a significant reduction in arrhythmic behaviour. These findings reveal a critical role for the late INa in cardiac arrhythmias and suggest that inhibition of this conductance could provide an effective therapeutic strategy. Finally, this study highlights the utility of human ex-vivo heart models for advancing cardiac translational sciences.

Sound signals are acquired and digitized in the cochlea by the hair cells that further transmit the coded information to the central auditory pathways. Any defect in hair cell function may induce problems in the auditory system and hearing-based brain function. In the past 2 decades, our understanding of auditory transduction has been substantially deepened because of advances in molecular, structural, and functional studies. Results from these experiments can be perfectly embedded in the previously established profile from anatomical, histological, genetic, and biophysical research. This review aims to summarize the progress on the molecular and cellular mechanisms of the mechano-electrical transduction (MET) channel in the cochlear hair cells, which is involved in the acquisition of sound frequency and intensity-the two major parameters of an acoustic cue. We also discuss recent studies on TMC1, the molecule likely to form the MET channel pore.

Sperm capacitation is essential to gain fertilizing capacity. During this process, a series of biochemical and physiological modifications occur that allow sperm to undergo acrosomal exocytosis (AE). At the molecular level, hyperpolarization of the sperm membrane potential (Em) takes place during capacitation. This study shows that human sperm incubated under conditions that do not support capacitation (NC) can become ready for an agonist stimulated AE by pharmacologically inducing Em hyperpolarization with Valinomycin or Amiloride. To investigate how Em hyperpolarization promotes human sperm's ability to undergo AE, live single-cell imaging experiments were performed to simultaneously monitor changes in [Ca 2+ ] i and the occurrence of AE. Em hyperpolarization turned [Ca 2+ ] i dynamics in NC sperm from spontaneously oscillating into a sustained slow [Ca 2+ ] i increase. The addition of progesterone (P4) or K + to Valinomycin-treated sperm promoted that a significant number of cells displayed a transitory rise in [Ca 2+ ] i which then underwent AE. Altogether, our results demonstrate that Em hyperpolarization is necessary and sufficient to prepare human sperm for the AE. Furthermore, this Em change decreased Ca 2+ oscillations that block the occurrence of AE, providing strong experimental evidence of the molecular mechanism that drives the acquisition of acrosomal responsiveness., (© 2021 Federation of American Societies for Experimental Biology.)

Membrane potential (V mem ) is a fundamental biophysical signal present in all cells. V mem signals range in time from milliseconds to days, and they span lengths from microns to centimeters. V mem affects many cellular processes, ranging from neurotransmitter release to cell cycle control to tissue patterning. However, existing tools are not suitable for V mem quantification in many of these areas. In this review, we outline the diverse biology of V mem , drafting a wish list of features for a V mem sensing platform. We then use these guidelines to discuss electrode-based and optical platforms for interrogating V mem . On the one hand, electrode-based strategies exhibit excellent quantification but are most effective in short-term, cellular recordings. On the other hand, optical strategies provide easier access to diverse samples but generally only detect relative changes in V mem . By combining the respective strengths of these technologies, recent advances in optical quantification of absolute V mem enable new inquiries into V mem biology.

Mas-related G-protein-coupled receptor X1 (MrgprX1) is a human-specific Mrgpr and its expression is restricted to primary sensory neurons. However, its role in nociception and pain signaling pathways is largely unknown. This study aims to investigate a role for MrgprX1 in nociception via interaction with the pain receptor, Transient Receptor Potential Ankyrin 1 (TRPA1), using in-vitro and in-vivo human neuronal models. MrgprX1 protein expression in human trigeminal nociceptors was investigated by the immunolabeling of the dental pulp and cultured peripheral neuronal equivalent (PNE) cells. MrgprX1 receptor signaling was monitored by Fura-2-based Ca 2+ imaging using PNEs and membrane potential responses were measured using FluoVolt TM . Immunofluorescent staining revealed MrgprX1 expression in-vivo in dental afferents, which was more intense in inflamed compared to healthy dental pulps. Endogenous MrgprX1 protein expression was confirmed in the in-vitro human PNE model. MrgprX1 receptor signaling and the mechanisms through which it couples to TRPA1 were studied by Ca 2+ imaging. Results showed that MrgprX1 activates TRPA1 and induces membrane depolarization in a TRPA1 dependent manner. In addition, MrgprX1 sensitizes TRPA1 to agonist stimulation via Protein Kinase C (PKC). The activation and sensitization of TRPA1 by MrgprX1 in a model of human nerves suggests an important role for this receptor in the modulation of nociception., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

When plants are exposed to hypoxic conditions, the level of γ-aminobutyric acid (GABA) in plant tissues increases by several orders of magnitude. The physiological rationale behind this elevation remains largely unanswered. By combining genetic and electrophysiological approach, in this work we show that hypoxia-induced increase in GABA content is essential for restoration of membrane potential and preventing ROS-induced disturbance to cytosolic K + homeostasis and Ca 2+ signaling. We show that reduced O 2 availability affects H + -ATPase pumping activity, leading to membrane depolarization and K + loss via outward-rectifying GORK channels. Hypoxia stress also results in H 2 O 2 accumulation in the cell that activates ROS-inducible Ca 2+ uptake channels and triggers self-amplifying "ROS-Ca hub," further exacerbating K + loss via non-selective cation channels that results in the loss of the cell's viability. Hypoxia-induced elevation in the GABA level may restore membrane potential by pH-dependent regulation of H + -ATPase and/or by generating more energy through the activation of the GABA shunt pathway and TCA cycle. Elevated GABA can also provide better control of the ROS-Ca 2+ hub by transcriptional control of RBOH genes thus preventing over-excessive H 2 O 2 accumulation. Finally, GABA can operate as a ligand directly controlling the open probability and conductance of K + efflux GORK channels, thus enabling plants adaptation to hypoxic conditions., (© 2021 The Author(s).)

Anterior pituitary endocrine cells that release hormones such as growth hormone and prolactin are excitable and fire action potentials. In these cells, several studies previously showed that extracellular sodium (Na + ) removal resulted in a negative shift of the resting membrane potential (RMP) and a subsequent inhibition of the spontaneous firing of action potentials, suggesting the contribution of a Na + background conductance. Here, we show that the Na + leak channel NALCN conducts a Ca 2+ - Gd 3+ -sensitive and TTX-resistant Na + background conductance in the GH 3 cell line, a cell model of pituitary endocrine cells. NALCN knockdown hyperpolarized the RMP, altered GH 3 cell electrical properties and inhibited prolactin secretion. Conversely, the overexpression of NALCN depolarized the RMP, also reshaping the electrical properties of GH 3 cells. Overall, our results indicate that NALCN is functional in GH 3 cells and involved in endocrine cell excitability as well as in hormone secretion. Indeed, the GH 3 cell line suitably models native pituitary cells that display a similar Na + background conductance and appears as a proper cellular model to study the role of NALCN in cellular excitability., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Eukaryotic cells are complex systems compartmentalized in membrane-bound organelles. Visualization of organellar electrical activity in living cells requires both a suitable reporter and non-invasive imaging at high spatiotemporal resolution. Here we present hVoS org , an optical method to monitor changes in the membrane potential of subcellular membranes. This method takes advantage of a FRET pair consisting of a membrane-bound voltage-insensitive fluorescent donor and a non-fluorescent voltage-dependent acceptor that rapidly moves across the membrane in response to changes in polarity. Compared to the currently available techniques, hVoS org has advantages including simple and precise subcellular targeting, the ability to record from individual organelles, and the potential for optical multiplexing of organellar activity.

Cardiac excitation-contraction coupling and metabolic and signaling activities are centrally modulated by nitric oxide (NO), which is produced by one of three NO synthases (NOSs). Despite the significant role of NO in cardiac Ca 2+ homeostasis regulation under different pathophysiological conditions, such as Duchenne muscular dystrophy (DMD), no precise method describes the production, source or effect of NO through two NO signaling pathways: soluble guanylate cyclase-protein kinase G (NO-sGC-PKG) and S-nitrosylation (SNO). Using a novel strategy involving isolated murine cardiomyocytes loaded with a copper-based dye highly specific for NO, we observed a single transient NO production signal after each electrical stimulation event. The NO transient signal started 67.5 ms after the beginning of Rhod-2 Ca 2+ transient signal and lasted for approximately 430 ms. Specific NOS isoform blockers or NO scavengers significantly inhibited the NO transient, suggesting that wild-type (WT) cardiomyocytes produce nNOS-dependent NO transients. Conversely, NO transient in mdx cardiomyocyte, a mouse model of DMD, was dependent on inducible NOS (iNOS) and endothelial (eNOS). In a consecutive stimulation protocol, the nNOS-dependent NO transient in WT cardiomyocytes significantly reduced the next Ca 2+ transient via NO-sGC-PKG. In mdx cardiomyocytes, this inhibitory effect was iNOS- and eNOS-dependent and occurred through the SNO pathway. Basal NO production was nNOS- and iNOS-dependent in WT cardiomyocytes and eNOS- and iNOS-dependent in mdx cardiomyocytes. These results showed cardiomyocyte produces NO isoform-dependent transients upon membrane depolarization at the millisecond time scale activating a specific signaling pathway to negatively modulate the subsequent Ca 2+ transient.

Modulation of neuronal excitability is a prominent way of shaping the activity of neuronal networks. Recent studies highlight the role of calcium-activated chloride currents in this context, as they can both increase or decrease excitability. The calcium-activated chloride channel Anoctamin 2 (ANO2 alias TMEM16B) has been described in several regions of the mouse brain, including the olivo-cerebellar system. In inferior olivary neurons, ANO2 was proposed to increase excitability by facilitating the generation of high-threshold calcium spikes. An expression of ANO2 in cerebellar Purkinje cells was suggested, but its role in these neurons remains unclear. In the present study, we confirmed the expression of Ano2 mRNA in Purkinje cells and performed electrophysiological recordings to examine the influence of ANO2-chloride channels on the excitability of Purkinje cells by comparing wildtype mice to mice lacking ANO2. Recordings were performed in acute cerebellar slices of adult mice, which provided the possibility to study the role of ANO2 within the cerebellar cortex. Purkinje cells were uncoupled from climbing fiber input to assess specifically the effect of ANO2 channels on Purkinje cell activity. We identified an attenuating effect of ANO2-mediated chloride currents on the instantaneous simple spike activity both during strong current injections and during current injections close to the simple spike threshold. Moreover, we report a reduction of inhibitory currents from GABAergic interneurons upon depolarization, lasting for several seconds. Together with the role of ANO2-chloride channels in inferior olivary neurons, our data extend the evidence for a role of chloride-dependent modulation in the olivo-cerebellar system that might be important for proper cerebellum-dependent motor coordination and learning., Competing Interests: The authors have declared that no competing interests exist.

Background: Atherosclerosis (AS) is a chronic progressive inflammatory condition with a leading prevalence worldwide. Endothelial dysfunction leads to low-density lipoprotein trafficking into subendothelial space and the subsequent form of oxidized LDL (ox-LDL) within intimal layer, perpetuating the vicious cycle of endothelial dysfunction. K+ exerts beneficial effects in vascular wall by reducing LDL oxidization, vascular smooth muscle cells (VSMCs) proliferation, and free radical generation. K+ also modulates vascular tone through a regulatory effect on cell membrane potential., Materials and Methods: The most relevant papers on the association between 'potassium channels' and 'atherosclerosis' were selected among those deposited on PubMed from 1990 to 2020., Results: Here, we provide a short narrative review that elaborates on the role of K+ in atherosclerosis. This review also update the current knowledge about potential pharmacological agents targeting K+ channels with a special focus on pleiotropic activities of agents such as statins, sulfonylureas and dihydropyridines., Conclusion: In this review, the mechanism of different K+ channels on vascular endothelium will be summarized, mainly focusing on their pathophysiological role in atherosclerosis and potential therapeutic application., (© 2020 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.)

Liposomal technologies are used in order to improve the effectiveness of current therapies or to reduce their negative side effects. However, the liposome-erythrocyte interaction during the intravenous administration of liposomal drug formulations may result in changes within the red blood cells (RBCs). In this study, it was shown that phosphatidylcholine-composed liposomal formulations of Photolon, used as a drug model, significantly influences the transmembrane potential, stiffness, as well as the shape of RBCs. These changes caused decreasing the number of stomatocytes and irregular shapes proportion within the cells exposed to liposomes. Thus, the reduction of anisocytosis was observed. Therefore, some nanodrugs in phosphatidylcholine liposomal formulation may have a beneficial effect on the survival time of erythrocytes.

The transition from quiescence to activation in radial neural stem cells (rNSCs) is a key first step in the process of adult neurogenesis, which can be monitored in the context of circuit activation in live tissue by whole-cell patch-clamp recording of membrane potentials of rNSCs in acute brain slices. However, membrane recordings in small non-neuronal cells such as rNSCs can be challenging. Here, we describe the preparation of materials, recording and stimulation protocols, and analyses necessary to evaluate the efficacy of activity-dependent control of rNSC behavior. For complete details on the use and execution of this protocol, please refer to Asrican et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

The evolution of the eukaryotic cell from the primal endosymbiotic event involved a complex series of adaptations driven primarily by energy optimization. Transfer of genes from endosymbiont to host and concomitant expansion (by infolding) of the endosymbiont's chemiosmotic membrane greatly increased output of adenosine triphosphate (ATP) and placed selective pressure on the membrane at the host-endosymbiont interface to sustain the energy advantage. It is hypothesized that critical functions at this interface (metabolite exchange, polypeptide import, barrier integrity to proteins and DNA) were managed by a precursor β-barrel protein ("pβB") from which the voltage-dependent anion-selective channel (VDAC) descended. VDAC's role as hub for disparate and increasingly complex processes suggests an adaptability that likely springs from a feature inherited from pβB, retained because of important advantages conferred. It is proposed that this property is the remarkable structural flexibility evidenced in VDAC's gating mechanism, a possible origin of which is discussed.

The patch-clamp technique and more recently the high throughput patch-clamp technique have contributed to major advances in the characterization of ion channels. However, the whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that increasing current amplitude profoundly impacts the accuracy of the biophysical analyses of macroscopic ion currents under study. Using mathematical kinetic models of a cardiac voltage-gated sodium channel and a cardiac voltage-gated potassium channel, we demonstrated how large current amplitude and series resistance artefacts induce an undetected alteration in the actual membrane potential and affect the characterization of voltage-dependent activation and inactivation processes. We also computed how dose-response curves are hindered by high current amplitudes. This is of high interest since stable cell lines frequently demonstrating high current amplitudes are used for safety pharmacology using the high throughput patch-clamp technique. It is therefore critical to set experimental limits for current amplitude recordings to prevent inaccuracy in the characterization of channel properties or drug activity, such limits being different from one channel type to another. Based on the predictions generated by the kinetic models, we draw simple guidelines for good practice of whole-cell voltage-clamp recordings.

Chloride secretion by airway epithelial cells is primordial for water and ion homeostasis and airways surface prevention of infections. This secretion is impaired in several human diseases, including cystic fibrosis, a genetic pathology due to CFTR gene mutations leading to chloride channel defects. A potential therapeutic approach is aiming at increasing chloride secretion either by correcting the mutated CFTR itself or by stimulating non-CFTR chloride channels at the plasma membrane. Here, we studied the role of phospholipase C in regulating the transepithelial chloride secretion in human airway epithelial 16HBE14o- and CFBE cells over-expressing wild type (WT)- or F508del-CFTR. Western blot analysis shows expression of the three endogenous phospholipase C (PLC) isoforms, namely, PLCδ1, PLCγ1, and PLCβ3 in 16HBE14o- cells. In 16HBE14o- cells, we performed Ussing chamber experiments after silencing each of these PLC isoforms or using the PLC inhibitor U73122 or its inactive analogue U73343. Our results show the involvement of PLCβ3 and PLCγ1 in CFTR-dependent short-circuit current activated by forskolin, but not of PLCδ1. In CFBE-WT CFTR and corrected CFBE-F508del CFTR cells, PLCβ3 silencing also inhibits CFTR-dependent current activated by forskolin and UTP-activated calcium-dependent chloride channels (CaCC). Our study supports the importance of PLC in maintaining CFTR-dependent chloride secretion over time, getting maximal CFTR-dependent current and increasing CaCC activation in bronchial epithelial cells.

Plasma membrane (PM) hyperpolarization, increased intracellular pH (pH i ), and changes in intracellular calcium concentration ([Ca 2+ ] i ) are physiological events that occur during human sperm capacitation. These parameters are potential predictors of successful outcomes for men undergoing artificial reproduction techniques (ARTs), but methods currently available for their determination pose various technical challenges and limitations. Here, we developed a novel strategy employing time-lapse flow cytometry (TLFC) to determine capacitation-related membrane potential ( E m ) and pH i changes, and progesterone-induced [Ca 2+ ] i increases. Our results show that TLFC is a robust method to measure absolute E m and pH i values and to qualitatively evaluate [Ca 2+ ] i changes. To support the usefulness of our methodology, we used sperm from two types of normozoospermic donors: known paternity (subjects with self-reported paternity) and no-known paternity (subjects without self-reported paternity and no known fertility problems). We found relevant differences between them. The incidences of membrane hyperpolarization, pH i alkalinization, and increased [Ca 2+ ] i were consistently high among known paternity samples (100%, 100%, and 86%, respectively), while they varied widely among no-known paternity samples (44%, 17%, and 45%, respectively). Our results indicate that TLFC is a powerful tool to analyze key physiological parameters of human sperm, which pending clinical validation, could potentially be employed as fertility predictors.

The adipocyte-derived hormone leptin increases trafficking of K ATP and Kv2.1 channels to the pancreatic β-cell surface, resulting in membrane hyperpolarization and suppression of insulin secretion. We have previously shown that this effect of leptin is mediated by the NMDA subtype of glutamate receptors (NMDARs). It does so by potentiating NMDAR activity, thus enhancing Ca 2+ influx and the ensuing downstream signaling events that drive channel trafficking to the cell surface. However, the molecular mechanism by which leptin potentiates NMDARs in β-cells remains unknown. Here, we report that leptin augments NMDAR function via Src kinase-mediated phosphorylation of the GluN2A subunit. Leptin-induced membrane hyperpolarization diminished upon pharmacological inhibition of GluN2A but not GluN2B, indicating involvement of GluN2A-containing NMDARs. GluN2A harbors tyrosine residues that, when phosphorylated by Src family kinases, potentiate NMDAR activity. We found that leptin increases phosphorylation of Tyr-418 in Src, an indicator of kinase activation. Pharmacological inhibition of Src or overexpression of a kinase-dead Src mutant prevented the effect of leptin, whereas a Src kinase activator peptide mimicked it. Using mutant GluN2A overexpression, we show that Tyr-1292 and Tyr-1387 but not Tyr-1325 are responsible for the effect of leptin. Importantly, β-cells from db/db mice, a type 2 diabetes mouse model lacking functional leptin receptors, or from obese diabetic human donors failed to respond to leptin but hyperpolarized in response to NMDA. Our study reveals a signaling pathway wherein leptin modulates NMDARs via Src to regulate β-cell excitability and suggests NMDARs as a potential target to overcome leptin resistance., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Cochrane et al.)