TMC7 functions as a suppressor of Piezo2 in primary sensory neurons blunting peripheral mechanotransduction.

The transmembrane channel-like (TMC) protein family comprises eight members, with TMC1 and TMC2 being extensively studied. This study demonstrates substantial co-expression of TMC7 with the mechanosensitive channel Piezo2 in somatosensory neurons. Genetic deletion of TMC7 in primary sensory ganglia neurons in vivo enhances sensitivity in both physiological and pathological mechanosensory transduction. This deletion leads to an increase in proportion of rapidly adapting (RA) currents conducted by Piezo2 in dorsal root ganglion (DRG) neurons and accelerates RA deactivation kinetics. In HEK293 cells expressing both proteins, TMC7 significantly suppresses the current amplitudes of co-expressed Piezo2. Our findings reveal that TMC7 and Piezo2 exhibit physical interactions, and both proteins also physically interact with cytoskeletal ß-actin. We hypothesize that TMC7 functions as an inhibitory modulator of Piezo2 in DRG neurons, either through direct inhibition or by disrupting the transmission of mechanical forces from the cytoskeleton to the channel.

Mechanical activation opens a lipid-lined pore in OSCA ion channels.

OSCA/TMEM63 channels are the largest known family of mechanosensitive channels1-3, playing critical roles in plant4-7 and mammalian8,9 mechanotransduction. Here we determined 44 cryogenic electron microscopy structures of OSCA/TMEM63 channels in different environments to investigate the molecular basis of OSCA/TMEM63 channel mechanosensitivity. In nanodiscs, we mimicked increased membrane tension and observed a dilated pore with membrane access in one of the OSCA1.2 subunits. In liposomes, we captured the fully open structure of OSCA1.2 in the inside-in orientation, in which the pore shows a large lateral opening to the membrane. Unusually for ion channels, structural, functional and computational evidence supports the existence of a 'proteo-lipidic pore' in which lipids act as a wall of the ion permeation pathway. In the less tension-sensitive homologue OSCA3.1, we identified an 'interlocking' lipid tightly bound in the central cleft, keeping the channel closed. Mutation of the lipid-coordinating residues induced OSCA3.1 activation, revealing a conserved open conformation of OSCA channels. Our structures provide a global picture of the OSCA channel gating cycle, uncover the importance of bound lipids and show that each subunit can open independently. This expands both our understanding of channel-mediated mechanotransduction and channel pore formation, with important mechanistic implications for the TMEM16 and TMC protein families.

Intestinal Piezo1 aggravates intestinal barrier dysfunction during sepsis by mediating Ca2+ influx.

INTRODUCTION: Intestinal barrier dysfunction is a pivotal factor in sepsis progression. The mechanosensitive ion channel Piezo1 is associated with barrier function; however, its role in sepsis-induced intestinal barrier dysfunction remains poorly understood. METHODS: The application of cecal ligation and puncture (CLP) modeling was performed on both mice of the wild-type (WT) variety and those with Villin-Piezo1flox/flox genetic makeup to assess the barrier function using in vivo FITC-dextran permeability measurements and immunofluorescence microscopy analysis of tight junctions (TJs) and apoptosis levels. In vitro, Caco-2 monolayers were subjected to TNF-α incubation. Moreover, to modulate Piezo1 activation, GsMTx4 was applied to inhibit Piezo1 activation. The barrier function, intracellular calcium levels, and mitochondrial function were monitored using calcium imaging and immunofluorescence techniques. RESULTS: In the intestinal tissues of CLP-induced septic mice, Piezo1 protein levels were notably elevated compared with those in normal mice. Piezo1 has been implicated in the sepsis-mediated disruption of TJs, apoptosis of intestinal epithelial cells, elevated intestinal mucosal permeability, and systemic inflammation in WT mice, whereas these effects were absent in Villin-Piezo1flox/flox CLP mice. In Caco-2 cells, TNF-α prompted calcium influx, an effect reversed by GsMTx4 treatment. Elevated calcium concentrations are correlated with increased accumulation of reactive oxygen species, diminished mitochondrial membrane potential, and TJ disruption. CONCLUSIONS: Thus, Piezo1 is a potential contributor to sepsis-induced intestinal barrier dysfunction, influencing apoptosis and TJ modification through calcium influx-mediated mitochondrial dysfunction.

Electrophysiology of fluoride channels in the yeasts Saccharomyces cerevisiae and Candida albicans.

Tight regulation of molecules moving through the cell membrane is particularly important for free-living microorganisms because of their small cell volumes and frequent changes in the chemical composition of the extracellular environment. This is true for nutrients, but even more so for toxic molecules. Traditionally, the transport of these diverse molecules in microorganisms has been studied on cell populations rather than on single cells, mainly because of technical difficulties. The goal of this chapter is to make available a detailed method to prepare yeast spheroplasts to study the movement of fluoride ions across the plasma membrane of single cells by the patch-clamp technique. In this procedure, three steps are critical to achieve high resistance (GΩ) seals between the membrane and the glass electrode: (1) appropriate removal of the cell wall by enzymatic treatment; (2) balance between the osmotic strength of sealing solutions and cell membrane turgor; and (3) meticulous morphological inspection of spheroplasts suitable for gigaseal formation. We show now that this method, originally developed for Saccharomyces cerevisiae, can also be applied to Candida albicans, an opportunistic human pathogen.

Roles of TRP and PIEZO receptors in autoimmune diseases.

Autoimmune diseases are pathological autoimmune reactions in the body caused by various factors, which can lead to tissue damage and organ dysfunction. They can be divided into organ-specific and systemic autoimmune diseases. These diseases usually involve various body systems, including the blood, muscles, bones, joints and soft tissues. The transient receptor potential (TRP) and PIEZO receptors, which resulted in David Julius and Ardem Patapoutian winning the Nobel Prize in Physiology or Medicine in 2021, attracted people's attention. Most current studies on TRP and PIEZO receptors in autoimmune diseases have been carried out on animal model, only few clinical studies have been conducted. Therefore, this study aimed to review existing studies on TRP and PIEZO to understand the roles of these receptors in autoimmune diseases, which may help elucidate novel treatment strategies.

Expression patterns of mechanosensitive ion channel PIEZOs in irreversible pulpitis.

BACKGROUND: Mechanosensitive ion channel PIEZOs have been widely reported to involve inflammation and pain. This study aimed to clarify expression patterns of PIEZOs and their potential relations to irreversible pulpitis. MATERIALS AND METHODS: Normal pulp tissues (n = 29) from patients with impacted third molars and inflamed pulp tissues (n = 23) from patients with irreversible pulpitis were collected. Pain levels were assessed using a numerical rating scale. PIEZO expressions were measured using real-time PCR and then confirmed using GEO datasets GSE77459, immunoblot, and immunohistochemistry staining. Correlations of PIEZO mRNA expression with inflammatory markers, pain markers, or clinical pain levels were evaluated using Spearman's correlation analysis. Univariate analysis was conducted to analyze PIEZO expressions based on pain description and clinical examinations of cold test, percussion, palpation, and bite test. RESULTS: Compared with normal pulp tissues, mRNA expression levels of PIEZO1 were significantly increased in inflamed pulp tissues, while PIEZO2 was significantly decreased, which was further confirmed in GSE77459 and on a protein and histological level. The positive correlation of the mRNA expression levels between PIEZO1 and inflammatory markers, as well as between PIEZO2 and pain markers, was verified. PIEZO2 expression was also positively correlated with pain levels. Besides, irreversible pulpitis patients who reported continuous pain and who detected a positive response to cold stimulus exhibited a higher expression level of PIEZO2 in the inflamed pulp tissues. By contrast, patients reporting pain duration of more than one week showed a higher expression level of PIEZO1. CONCLUSIONS: This study demonstrated the upregulation of PIEZO1 and the downregulation of PIEZO2 in irreversible pulpitis and revealed the potential relation of PIEZO1 and PIEZO2 to inflammation and pain. These findings suggested that PIEZOs might play critical roles in the progression of irreversible pulpitis and paved the way for further investigations aimed at novel therapies of irreversible pulpitis by targeting PIEZOs.

Inorganic polyphosphate and ion transport across biological membranes.

Inorganic polyphosphate (polyP) is widely recognized for playing important roles and processes involved in energy and phosphate storage, regulation of gene expression, and calcium signaling. The less well-known role of polyP is as a direct mediator of ion transport across biological membranes. Here, we will briefly summarize current knowledge of the molecular mechanisms of how polyP can be involved in membrane ion transport. We discuss three types of mechanisms that might involve polyP: (1) formation of non-protein channel complex that includes calcium, polyP, and polyhydroxybutyrate (PHB); (2) modulation of the channel activity of PHBlated protein channels; and (3) direct effects of polyP on the function of the voltage-gated ion channels in the process that do not involve PHB.

Cellular geometry and epithelial-mesenchymal plasticity intersect with PIEZO1 in breast cancer cells.

Differences in shape can be a distinguishing feature between different cell types, but the shape of a cell can also be dynamic. Changes in cell shape are critical when cancer cells escape from the primary tumor and undergo major morphological changes that allow them to squeeze between endothelial cells, enter the vasculature, and metastasize to other areas of the body. A shift from rounded to spindly cellular geometry is a consequence of epithelial-mesenchymal plasticity, which is also associated with changes in gene expression, increased invasiveness, and therapeutic resistance. However, the consequences and functional impacts of cell shape changes and the mechanisms through which they occur are still poorly understood. Here, we demonstrate that altering the morphology of a cell produces a remodeling of calcium influx via the ion channel PIEZO1 and identify PIEZO1 as an inducer of features of epithelial-to-mesenchymal plasticity. Combining automated epifluorescence microscopy and a genetically encoded calcium indicator, we demonstrate that activation of the PIEZO1 force channel with the PIEZO1 agonist, YODA 1, induces features of epithelial-to-mesenchymal plasticity in breast cancer cells. These findings suggest that PIEZO1 is a critical point of convergence between shape-induced changes in cellular signaling and epithelial-mesenchymal plasticity in breast cancer cells.

Computational Insights into the Conformational Dynamics of HIV-1 Vpr in a Lipid Bilayer for Ion Channel Modeling.

HIV-1 Vpr is a multifunctional accessory protein consisting of 96 amino acids that play a critical role in viral pathogenesis. Among its diverse range of activities, Vpr can create a cation-selective ion channel within the plasma membrane. However, the oligomeric state of this channel has not yet been elucidated. In this study, we investigated the conformational dynamics of Vpr helices to model the ion channel topology. First, we employed a series of multiscale simulations to investigate the specific structure of monomeric Vpr in a membrane model. During the lipid bilayer self-assembly coarse grain simulation, the C-terminal helix (residues 56-77) effectively formed the transmembrane region, while the N-terminal helix exhibited an amphipathic nature by associating horizontally with a single leaflet. All-atom molecular dynamics (MD) simulations of full-length Vpr inside a phospholipid bilayer show that the C-terminal helix remains very stable inside the bilayer core in a vertical orientation. Subsequently, using the predicted C-terminal helix orientation and conformation, various oligomeric states (ranging from tetramer to heptamer) possibly forming the Vpr ion channel were built and further evaluated. Among these models, the pentameric form exhibited consistent stability in MD simulations and displayed a compatible conformation for a water-assisted ion transport mechanism. This study provides structural insights into the ion channel activity of the Vpr protein and the foundation for developing therapeutics against HIV-1 Vpr-related conditions.

Expression, Purification, and Nanodisc Reconstitution of Connexin-43 Hemichannels for Structural Characterization by Cryo-Electron Microscopy.

Connexins are polytopic domain membrane proteins that form hexameric hemichannels (HCs) which can assemble into gap junction channels (GJCs) at the interface of two neighboring cells. The HCs may be involved in ion and small-molecule transport across the cellular plasma membrane in response to various stimuli. Despite their importance, relatively few structures of connexin HCs are available to date, compared to the structures of the GJCs. Here, we describe a protocol for expression, purification, and nanodisc reconstitution of connexin-43 (Cx43) HCs, which we have recently structurally characterized using cryo-EM analysis. Application of similar protocols to other connexin family members will lead to breakthroughs in the understanding of the structure and function of connexin HCs.

Spatial and Temporal Localization of Connexins in Cells Using Confocal Microscopy.

The 21-member connexin family found in humans is the building block of both single-membrane spanning channels (hemichannels) and double-membrane spanning intercellular channels. These large-pore channels are dynamic and typically have a short life span of only a few hours. Imaging connexins from the time of synthesis in the endoplasmic reticulum through to their degradation can be challenging given their distinct assembly states and transient residences in many subcellular compartments. Here, we describe how connexins can be effectively imaged on a confocal microscope in living cells when tagged with fluorescent proteins and when immunolabeled with high affinity anti-connexin antibodies in fixed cells. Temporal and spatial localization of multiple connexins and disease-linked connexin mutants at the subcellular level extensively informs on the mechanisms governing connexin regulation in health and disease.

Purification, Reconstitution, and Functional Analysis of Connexin Hemichannels.

Connexins are the proteins that form the gap junction channels that are essential for cell-to-cell communication. These channels are formed by head-to-head docking of hemichannels (each from one of two adjacent cells). Free "undocked" hemichannels at the plasma membrane are mostly closed, although they are still important under physiological conditions. However, abnormal and sustained increase in hemichannel activity due to connexin mutations or acquired conditions can produce or contribute to cell damage. For example, mutations of Cx26, a connexin isoform, can increase hemichannel activity and cause deafness. Studies using purified isolated systems under well-controlled conditions are essential for a full understanding of molecular mechanisms of hemichannel function under normal conditions and in disease, and here, we present methodology for the expression, purification, and functional analysis of hemichannels formed by Cx26.

Enhanced Methodologies for Investigating the Electrophysiological Characteristics of Endogenous Pannexin 1 Intercellular Cell-Cell Channels.

Gap junctions, pivotal intercellular conduits, serve as communication channels between adjacent cells, playing a critical role in modulating membrane potential distribution across cellular networks. The family of Pannexin (Panx) proteins, in particular Pannexin1 (Panx1), are widely expressed in vertebrate cells and exhibit sequence homology with innexins, the invertebrate gap junction channel constituents. Despite being ubiquitously expressed, detailed functional and pharmacological properties of Panx1 intercellular cell-cell channels require further investigation. In this chapter, we introduce optimized cell culture methodologies and electrophysiology protocols to expedite the exploration of endogenous Panx1 cell-cell channels in TC620 cells, a human oligodendroglioma cell line that naturally expresses Panx1. We anticipate these refined protocols will significantly contribute to future characterizations of Panx1-based intercellular cell-cell channels across diverse cell types and offer valuable insights into both normal cellular physiology and pathophysiology.

Ion Channels and Metal Ions in Parkinson's Disease: Historical Perspective to the Current Scenario.

Parkinson's disease (PD) is a neurodegenerative condition linked to the deterioration of motor and cognitive performance. It produces degeneration of the dopaminergic neurons along the nigrostriatal pathway in the central nervous system (CNS), which leads to symptoms such as bradykinesias, tremors, rigidity, and postural instability. There are several medications currently approved for the therapy of PD, but a permanent cure for it remains elusive. With the aging population set to increase, a number of PD cases are expected to shoot up in the coming times. Hence, there is a need to look for new molecular targets that could be investigated both preclinically and clinically for PD treatment. Among these, several ion channels and metal ions are being studied for their effects on PD pathology and the functioning of dopaminergic neurons. Ion channels such as N-methyl-D-aspartate (NMDA), γ-aminobutyric acid A (GABAA), voltage-gated calcium channels, potassium channels, HCN channels, Hv1 proton channels, and voltage-gated sodium channels and metal ions such as mercury, zinc, copper, iron, manganese, calcium, and lead showed prominent involvement in PD. Pharmacological agents have been used to target these ion channels and metal ions to prevent or treat PD. Hence, in the present review, we summarize the pathophysiological events linked to PD with an emphasis on the role of ions and ion channels in PD pathology, and pharmacological agents targeting these ion channels have also been listed.

Single shot detection of alterations across multiple ionic currents from assimilation of cell membrane dynamics.

The dysfunction of ion channels is a causative factor in a variety of neurological diseases, thereby defining the implicated channels as key drug targets. The detection of functional changes in multiple specific ionic currents currently presents a challenge, particularly when the neurological causes are either a priori unknown, or are unexpected. Traditional patch clamp electrophysiology is a powerful tool in this regard but is low throughput. Here, we introduce a single-shot method for detecting alterations amongst a range of ion channel types from subtle changes in membrane voltage in response to a short chaotically driven current clamp protocol. We used data assimilation to estimate the parameters of individual ion channels and from these we reconstructed ionic currents which exhibit significantly lower error than the parameter estimates. Such reconstructed currents thereby become sensitive predictors of functional alterations in biological ion channels. The technique correctly predicted which ionic current was altered, and by approximately how much, following pharmacological blockade of BK, SK, A-type K+ and HCN channels in hippocampal CA1 neurons. We anticipate this assay technique could aid in the detection of functional changes in specific ionic currents during drug screening, as well as in research targeting ion channel dysfunction.

Endophilin A2 controls touch and mechanical allodynia via kinesin-mediated Piezo2 trafficking.

BACKGROUND: Tactile and mechanical pain are crucial to our interaction with the environment, yet the underpinning molecular mechanism is still elusive. Endophilin A2 (EndoA2) is an evolutionarily conserved protein that is documented in the endocytosis pathway. However, the role of EndoA2 in the regulation of mechanical sensitivity and its underlying mechanisms are currently unclear. METHODS: Male and female C57BL/6 mice (8-12 weeks) and male cynomolgus monkeys (7-10 years old) were used in our experiments. Nerve injury-, inflammatory-, and chemotherapy-induced pathological pain models were established for this study. Behavioral tests of touch, mechanical pain, heat pain, and cold pain were performed in mice and nonhuman primates. Western blotting, immunostaining, co-immunoprecipitation, proximity ligation and patch-clamp recordings were performed to gain insight into the mechanisms. RESULTS: The results showed that EndoA2 was primarily distributed in neurofilament-200-positive (NF200+) medium-to-large diameter dorsal root ganglion (DRG) neurons of mice and humans. Loss of EndoA2 in mouse NF200+ DRG neurons selectively impaired the tactile and mechanical allodynia. Furthermore, EndoA2 interacted with the mechanically sensitive ion channel Piezo2 and promoted the membrane trafficking of Piezo2 in DRG neurons. Moreover, as an adaptor protein, EndoA2 also bound to kinesin family member 5B (KIF5B), which was involved in the EndoA2-mediated membrane trafficking process of Piezo2. Loss of EndoA2 in mouse DRG neurons damaged Piezo2-mediated rapidly adapting mechanically activated currents, and re-expression of EndoA2 rescued the MA currents. In addition, interference with EndoA2 also suppressed touch sensitivity and mechanical hypersensitivity in nonhuman primates. CONCLUSIONS: Our data reveal that the KIF5B/EndoA2/Piezo2 complex is essential for Piezo2 trafficking and for sustaining transmission of touch and mechanical hypersensitivity signals. EndoA2 regulates touch and mechanical allodynia via kinesin-mediated Piezo2 trafficking in sensory neurons. Our findings identify a potential new target for the treatment of mechanical pain.

Tracking the Activity and Position of Mitochondrial ß-Barrel Proteins.

Total interference reflection fluorescence (TIRF) microscopy of lipid bilayers is an effective technique for studying the lateral movement and ion channel activity of single integral membrane proteins. Here we describe how to integrate the mitochondrial outer membrane preprotein translocase TOM-CC and its ß-barrel protein-conducting channel Tom40 into supported lipid bilayers to identify possible relationships between movement and channel activity. We propose that our approach can be readily applied to membrane protein channels where transient tethering to either membrane-proximal or intramembrane structures is accompanied by a change in channel permeation.

PIEZO1 acts as a cancer suppressor by regulating the ROS/Wnt/ß-catenin axis.

BACKGROUND: PIEZO1 works differently in different cancers and at different stages of development. The objective of the current study was to explore the function and underlying mechanism of PIEZO1 in lung adenocarcinoma (LUAD) cells. METHODS: Different LUAD cell lines were treated with PIEZO1 inhibitor (GsMTx4) and agonist (Yoda1), and the expression of PIEZO1 in LUAD cells was detected using real-time quantitative PCR (RT-qPCR) and western blotting. The effects of PIEZO1 on invasion, migration and epithelial-mesenchymal transition (EMT) markers protein expression of LUAD cells were detected using the MTT assay, flow cytometry, transwell assay, wound-healing assay, and western blotting. Reactive oxygen species (ROS) agonists (BAY 87-2243) and inhibitors (NAC) and Wnt/ß-catenin pathway inhibitors (iCRT3) were selected to treat A549 cells to investigate the mechanism of PIEZO1 on ROS production and Wnt/ß-catenin expression in A549 cells. RESULTS: In A549, NCI-H1395, and NCI-H1975 cells, GsMTx4 promoted cell proliferation, invasion, migration, upregulated EMT-related marker protein expression, and inhibited cell apoptosis, while Yoda1 exerted effects opposite to those of GsMTx4. In A549 cells, GsMTx4 can reduce ROS production, it also inhibited ROS production, apoptosis, and downregulated proapoptotic markers induced by BAY 87-2243. Importantly, BAY 87-2243 blocked the effect of GSMTX4-induced Wnt/ß-catenin overexpression. Similarly, Yoda1 can reduce the effect of NAC. In addition, iCRT3 can block the upregulation of EMT-related marker proteins by GsMTx4, and increase apoptosis and decrease cell invasion and migration. CONCLUSION: In summary, PIEZO1 acts as a cancer suppressor by regulating the ROS/Wnt/ß-catenin axis, providing a new perspective on the role of mechanosensitive channel proteins in cancer.

Viroporins Manipulate Cellular Powerhouses and Modulate Innate Immunity.

Viruses have a wide repertoire of molecular strategies that focus on their replication or the facilitation of different stages of the viral cycle. One of these strategies is mediated by the activity of viroporins, which are multifunctional viral proteins that, upon oligomerization, exhibit ion channel properties with mild ion selectivity. Viroporins facilitate multiple processes, such as the regulation of immune response and inflammasome activation through the induction of pore formation in various cell organelle membranes to facilitate the escape of ions and the alteration of intracellular homeostasis. Viroporins target diverse membranes (such as the cellular membrane), endoplasmic reticulum, and mitochondria. Cumulative data regarding the importance of mitochondria function in multiple processes, such as cellular metabolism, energy production, calcium homeostasis, apoptosis, and mitophagy, have been reported. The direct or indirect interaction of viroporins with mitochondria and how this interaction affects the functioning of mitochondrial cells in the innate immunity of host cells against viruses remains unclear. A better understanding of the viroporin-mitochondria interactions will provide insights into their role in affecting host immune signaling through the mitochondria. Thus, in this review, we mainly focus on descriptions of viroporins and studies that have provided insights into the role of viroporins in hijacked mitochondria.

Ion channels as biomarkers of altered myogenesis in myofiber precursors of Duchenne muscular dystrophy.

Myogenesis is essential for skeletal muscle formation, growth, and regeneration and can be altered in Duchenne muscular dystrophy (DMD), an X-linked disorder due to the absence of the cytoskeletal protein dystrophin. Ion channels play a pivotal role in muscle differentiation and interact with the dystrophin complex. To investigate ion channel involvement in myogenesis in dystrophic settings, we performed electrophysiological characterization of two immortalized mouse cell lines, wild-type (WT) H2K-2B4 and the dystrophic (DYS) H2K-SF1, and measured gene expression of differentiation markers and ion channels. Inward and outward currents/density increased as differentiation progressed in both WT and DYS cells. However, day-11 DYS cells showed higher (27%) inward current density with an increased expression ratio of Scn5a/Scn4a and decreased (48%) barium-sensitive outward current compared to WT. Furthermore, day-11 DYS cells showed more positive resting membrane potential (+10 mV) and lower membrane capacitance (50%) compared to WT. DYS cells also had reduced Myog and Myf5 expression at days 6 and 11. Overall, ion channel profile and myogenesis appeared altered in DYS cells. These results are a first step in validating ion channels as potential drug targets to ameliorate muscle degeneration in DMD settings and as differentiation biomarkers in innovative platforms.