Cation-selective Ion Channels Research Articles

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.

The open gate of the AMPA receptor forms a Ca2+ binding site critical in regulating ion transport.

Alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptors (AMPARs) are cation-selective ion channels that mediate most fast excitatory neurotransmission in the brain. Although their gating mechanism has been studied extensively, understanding how cations traverse the pore has remained elusive. Here we investigated putative ion and water densities in the open pore of Ca2+-permeable AMPARs (rat GRIA2 flip-Q isoform) at 2.3-2.6 Å resolution. We show that the ion permeation pathway attains an extracellular Ca2+ binding site (site-G) when the channel gate moves into the open configuration. Site-G is highly selective for Ca2+ over Na+, favoring the movement of Ca2+ into the selectivity filter of the pore. Seizure-related N619K mutation, adjacent to site-G, promotes channel opening but attenuates Ca2+ binding and thus diminishes Ca2+ permeability. Our work identifies the importance of site-G, which coordinates with the Q/R site of the selectivity filter to ensure the preferential transport of Ca2+ through the channel pore.

Symmetrical Bispyridinium Compounds Act as Open Channel Blockers of Cation-Selective Ion Channels.

Current treatments against organophosphate poisoning (OPP) do not directly address effects mediated by the overstimulation of nicotinic acetylcholine receptors (nAChR). Non-oxime bispyridinium compounds (BPC) promote acetylcholine esterase-independent recovery of organophosphate-induced paralysis. Here, we test the hypothesis that they act by positive modulatory action on nAChRs. Using two-electrode voltage clamp analysis in combination with mutagenesis and molecular docking analysis, the potency and molecular mode of action of a series of nine BPCs was investigated on human α7 and muscle-type nAChRs expressed in Xenopus laevis oocytes. The investigated BPCs inhibited α7 and/or muscle-type nAChRs with IC50 values in the high nanomolar to high micromolar range. Further analysis of the most potent analogues revealed a noncompetitive, voltage-dependent inhibition. Co-application with the α7-selective positive allosteric modulator PNU120596 and generation of α7/5HT3 receptor chimeras excluded direct interaction with the PNU120596 binding site and binding to the extracellular domain of the α7 nAChR, suggesting that they act as open channel blockers (OCBs). Molecular docking supported by mutagenesis localized the BPC binding area in the outer channel vestibule between the extracellular and transmembrane domains. Analysis of BPC action on other cation-selective channels suggests a rather nonspecific inhibition of pentameric cation channels. BPCs have been shown to ameliorate organophosphate-induced paralysis in vitro and in vivo. Our data support molecular action as OCBs at α7 and muscle-type nAChRs and suggest that their positive physiological effects are more complex than anticipated and require further investigation.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.2

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.2

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.1

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.1

High-speed AFM reveals a rare structural state for TRPV3.

TRPV3 is a cation selective ion channel belonging to the vanilloid-subfamily of temperature sensitive TRP channels. TRPV3 function is implicated in cutaneous pain, itch, and temperature-sensation, as well as in skin barrier formation, wound healing, and hair growth, while its dysfunction is associated with many diseases such as the Olmsted syndrome, atopic dermatitis, and rosacea. TRPV3 is activated and sensitized upon repeated stimulation by warm temperatures as well as by numerous chemicals, including plant extracts, lipid metabolites, and synthetic small molecules such as 2-aminoethoxydiphenyl borate (2-APB) and diphenylboronic anhydride (DPBA). TrpV3, like all other TRP channels, displays a homo-tetrameric structure, and possesses a similar architecture to those of the other thermo-sensitive TRPV channels. Multiple Cryo-EM structures of TRPV3 have been solved, revealing various conformations assigned as open, closed, and sensitized states. However, much information is still lacking regarding the full molecular-level mechanism of TrpV3 sensitization and gating, and it appears that more structural states have yet to be determined. Using high-speed atomic force microscopy (HS-AFM), which allows the direct imaging of membrane proteins at the single-molecule level in membrane and in buffer solution at physiological temperature, we reveal a hitherto uncharacterized and rare structural state. Interestingly, we demonstrate that the prevalence of this state is significantly increased upon the addition of DPBA, which has been shown in the past to lead to a secondary and enhanced TRPV3 current. With the ability of HS-AFM to visualize dynamic processes in real-time, we demonstrate the transience and reversibility of this state, providing an explanation as to why this state has so far eluded structural determination by cryo-EM. Overall, our results provide crucial structural information regarding TRPV3, and enable a more complete understanding of its full structural gating mechanism.

DNA-Based Optical Quantification of Ion Transport across Giant Vesicles.

Accurate measurements of ion permeability through cellular membranes remains challenging due to the lack of suitable ion-selective probes. Here we use giant unilamellar vesicles (GUVs) as membrane models for the direct visualization of mass translocation at the single-vesicle level. Ion transport is indicated with a fluorescently adjustable DNA-based sensor that accurately detects sub-millimolar variations in K+ concentration. In combination with microfluidics, we employed our DNA-based K+ sensor for extraction of the permeation coefficient of potassium ions. We measured K+ permeability coefficients at least 1 order of magnitude larger than previously reported values from bulk experiments and show that permeation rates across the lipid bilayer increase in the presence of octanol. In addition, an analysis of the K+ flux in different concentration gradients allows us to estimate the complementary H+ flux that dissipates the charge imbalance across the GUV membrane. Subsequently, we show that our sensor can quantify the K+ transport across prototypical cation-selective ion channels, gramicidin A and OmpF, revealing their relative H+/K+ selectivity. Our results show that gramicidin A is much more selective to protons than OmpF with a H+/K+ permeability ratio of ∼104.

SARS-CoV-2 Envelope Protein Forms Clustered Pentamers in Lipid Bilayers.

The SARS-CoV-2 envelope (E) protein is a viroporin associated with the acute respiratory symptoms of COVID-19. E forms cation-selective ion channels that assemble in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment. The channel activity of E is linked to the inflammatory response of the host cell to the virus. Like many viroporins, E is thought to oligomerize with a well-defined stoichiometry. However, attempts to determine the E stoichiometry have led to inconclusive results and suggested mixtures of oligomers whose exact nature might vary with the detergent used. Here, we employ 19F solid-state nuclear magnetic resonance and the centerband-only detection of exchange (CODEX) technique to determine the oligomeric number of E’s transmembrane domain (ETM) in lipid bilayers. The CODEX equilibrium value, which corresponds to the inverse of the oligomeric number, indicates that ETM assembles into pentamers in lipid bilayers, without any detectable fraction of low-molecular-weight oligomers. Unexpectedly, at high peptide concentrations and in the presence of the lipid phosphatidylinositol, the CODEX data indicate that more than five 19F spins are within a detectable distance of about 2 nm, suggesting that the ETM pentamers cluster in the lipid bilayer. Monte Carlo simulations that take into account peptide–peptide and peptide–lipid interactions yielded pentamer clusters that reproduced the CODEX data. This supramolecular organization is likely important for E-mediated virus assembly and budding and for the channel function of the protein.

SARS-CoV-2 Viroporins: A Multi-Omics Insight from Nucleotides to Amino Acids

COVID-19 is caused by SARS-CoV-2 which has so far affected more than 500 million people worldwide and killed over 6 million as of 1 May 2022. The approved emergency-use vaccines were lifesaving in such a devastating pandemic. Inflammation-related pathways have been well documented to be upregulated in the case of SARS-CoV-2 in rodents, non-human primates and human samples. We reanalysed a previously published dataset to understand if certain molecular components of inflammation could be higher in infected samples. Mechanistically, viroporins are important players in the life cycle of SARS-CoV-2 and are primary to its pathogenesis. We studied the two prominent viroporins of SARS-CoV-2 (i) Orf3a and (ii) envelope (E) protein from a sequence and structural point of view. Orf3a is a cation-selective viral ion channel which has been shown to disrupt the endosomal pathways. E protein is one of the most conserved proteins among the SARS-CoV proteome which affects the ERGIC-related pathways. The aqueous medium through the viroporins mediates the non-selective translocation of cations, affecting ionic homeostasis in the host cellular compartments. We hypothesize a possible mechanistic approach whereby the ionic imbalance caused by viroporin action could potentially be one of the major pathogenic drivers leading to the increased inflammatory response in the host cell. Our results shed light into the transcriptomic, genomic and structural proteomics aspects of widely studied SARS-CoV-2 viroporins, which can be potentially leveraged for the development of antiviral therapeutics.

Two-Pore Channels Regulate Inter-Organellar Ca2+ Homeostasis in Immune Cells.

Two-pore channels (TPCs) are ligand-gated cation-selective ion channels that are preserved in plant and animal cells. In the latter, TPCs are located in membranes of acidic organelles, such as endosomes, lysosomes, and endolysosomes. Here, we focus on the function of these unique ion channels in mast cells, which are leukocytes that mature from myeloid hematopoietic stem cells. The cytoplasm of these innate immune cells contains a large number of granules that comprise messenger substances, such as histamine and heparin. Mast cells, along with basophil granulocytes, play an essential role in anaphylaxis and allergic reactions by releasing inflammatory mediators. Signaling in mast cells is mainly regulated via the release of Ca2+ from the endoplasmic reticulum as well as from acidic compartments, such as endolysosomes. For the crosstalk of these organelles TPCs seem essential. Allergic reactions and anaphylaxis were previously shown to be associated with the endolysosomal two-pore channel TPC1. The release of histamine, controlled by intracellular Ca2+ signals, was increased upon genetic or pharmacologic TPC1 inhibition. Conversely, stimulation of TPC channel activity by one of its endogenous ligands, namely nicotinic adenine dinucleotide phosphate (NAADP) or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), were found to trigger the release of Ca2+ from the endolysosomes; thereby improving the effect of TPC1 on regulated mast cell degranulation. In this review we discuss the importance of TPC1 for regulating Ca2+ homeostasis in mast cells and the overall potential of TPC1 as a pharmacological target in anti-inflammatory therapy.

Transient Receptor Potential channels (TRP) in GtoPdb v.2022.1

Transient Receptor Potential channels (TRP) in GtoPdb v.2022.1

Computer simulations of Piezo1 channel regulation by membrane and small molecule

PIEZO proteins are homotrimeric cation-selective mechanosensitive ion channels that transduce various forms of mechanical forces into intracellular biochemical signals. Using multi-scale molecular dynamics simulations, we have generated an open state of the PIEOZ1 channel from the cryo-EM structure of a nonconducting conformation that reproduces unitary ionic conductance and selectivity in wild-type and mutants channels (Jiang, et al. Commun. Biol. 2021). This open state further allowed us to validate a putative allosteric agonist binding site of Yoda1 we previously identified using MD simulation and electrophysiology (Botello-Smith, et al.

Identification of positive modulators of TRPM5 channel from a high-throughput screen using a fluorescent membrane potential assay.

Transient Receptor Potential Melastatin 5 (TRPM5) is an intracellular calcium-activated cation-selective ion channel expressed in a variety of cell types. Dysfunction of this channel has recently been implied in a range of disease states including diabetes, enteric infections, inflammatory responses, parasitic infection and other pathologies. However, to date, agonists and positive modulators of this channel with sufficient selectivity to enable target validation studies have not been described, limiting the evaluation of TRPM5 biology and its potential as a drug target. We developed a high-throughput assay using a fluorescent membrane potential dye and a medium- and high-throughput electrophysiology assay using QPatch HTX and SyncroPatch 384PE. By employing these assays, we conducted a primary screening campaign and identified hit compounds as TRPM5 channel positive modulators. An initial selectivity profile confirmed hit selectivity to TRPM5 and is presented here. These small molecule TRPM5 compounds have a high potential both as early tool compounds to enable pharmacological studies of TRPM5 and as starting points for the development of potent, selective TRPM5 openers or positive modulators as novel drugs targeting several pathological states.

Spatial structure and oligomerization of viscotoxin A3 in detergent micelles: Implication for mechanisms of ion channel formation and membrane lysis

Thionins are the family of small (∼5 kDa) cationic cysteine-rich peptides involved in the immune response in plants. Viscotoxin A3 (VtA3) is the thionin from mistletoe (Viscum album) demonstrating antimicrobial and cytotoxic activity against cancer cells in vitro. VtA3 (charge +6) interacts with the membranes containing anionic lipids and forms cation-selective ion channels. Here we studied the VtA3 structure in membrane-mimicking media by NMR spectroscopy. Spatial structure of VtA3, consisting of a helical hairpin and a short β-sheet, was stable and did not undergo significant changes during micelle binding. VtA3 molecule bound with high affinity to the surface of zwitterionic dodecylphosphocholine (DPC) micelle by hydrophobic patch in the helical hairpin. Oligomerization of VtA3 was observed in the anionic micelles of sodium dodecylsulphate (SDS). No direct contacts between the peptide molecules were observed and the possible interfaces of detergent-assisted oligomerization were revealed. The data obtained suggest that the VtA3 membrane activity, depending on the concentration, obeys the ‘toroidal’ pore model or the ‘carpet’ mechanism. The model of the membrane disrupting complex, which explains the ion channel formation in the partially anionic membranes, was proposed.

Transient Receptor Potential channels (TRP) in GtoPdb v.2021.3

Transient Receptor Potential channels (TRP) in GtoPdb v.2021.3

Membrane-Mediated Lateral Interactions Regulate the Lifetime of Gramicidin Channels

The lipid matrix of cellular membranes is an elastic liquid crystalline medium. Its deformations regulate the functionality and interactions of membrane proteins,f membrane-bound peptides, lipid and protein-lipid domains. Gramicidin A (gA) is a peptide, which incorporates into membrane leaflets as a monomer and may form a transmembrane dimer. In both configurations, gA deforms the membrane. The transmembrane dimer of gA is a cation-selective ion channel. Its electrical response strongly depends on the elastic properties of the membrane. The gA monomer and dimer deform the membrane differently; therefore, the elastic energy contributes to the activation barriers of the dimerization and dissociation of the conducting state. It is shown experimentally that channel characteristics alter if gA molecules have been located in the vicinity of the conducting dimer. Here, based on the theory of elasticity of lipid membranes, we developed a quantitative theoretical model which allows explaining experimentally observed phenomena under conditions of high surface density of gA or its analogues, i.e., in the regime of strong lateral interactions of gA molecules, mediated by elastic deformations of the membrane. The model would be useful for the analysis and prediction of the gA electrical response in various experimental conditions. This potentially widens the possible applications of gA as a convenient molecular sensor of membrane elasticity.

Transgenic Tarantula Toxin: A novel tool to study mechanosensitive ion channels in Drosophila

The tarantula venom toxin GsMTx4 is the only known specific inhibitor of cation-selective mechanosensitive ion channels (MSCs). Its specificity, potency, and ease of use on isolated tissues and cells have made it a powerful pharmacological tool to identify and probe the physiological function of MSCs. In some contexts, however, it would be desirable to deliver the toxin in a controlled way in vivo. Here we describe a novel tool to allow spatial and temporal control of GsMTx4 delivery in vivo in Drosophila. To test the tool, we targeted MSCs required for mechanical nociception in a specific subset of sensory neurons in intact larvae. Expression of GsMTx4 in these neurons results in robust inhibition of mechanical nociception, demonstrating the toxin is active when expressed in vivo. The tool will be particularly useful to manipulate MSC activity in a spatially and temporally-controlled manner to study their role in development, physiology and behaviour in intact, free moving animals.

Coupling structure with function in acid-sensing ion channels: challenges in pursuit of proton sensors.

Acid-sensing ion channels (ASICs) are a class of trimeric cation-selective ion channels activated by changes in pH within the physiological range. They are widely expressed in the central and peripheral nervous systems where they participate in a range of physiological and pathophysiological situations such as learning and memory, pain sensation, fear and anxiety, substance abuse and cell death. ASICs are localized to cell bodies and dendrites, including the postsynaptic density, and within the last 5years several examples of proton-evoked ASIC excitatory postsynaptic currents have emerged. Thus, ASICs have become bona fide neurotransmitter-gated ion channels, activated by the smallest neurotransmitter possible: protons. Here we review how protons are thought to drive the conformational changes associated with ASIC activation and desensitization. In particular, we weigh the evidence for and against the so-called 'acidic pocket' being a vital proton sensor and discuss the emerging role of the β11-12 linker as a desensitization switch or 'molecular clutch'. We also examine how proton-induced conformational changes pose unique challenges to classical molecular dynamics simulations, as well as some possible solutions. Given the emergence of new methodologies and structures, the coming years will probably see many advances in the study of acid-sensing ion channels.

Identification of the Binding Site of Bupropion on Serotonin Type 3A Receptors

Bupropion is an FDA approved medication marketed as an anti-depressant, smoking-cessation, and weight loss drug. Presently, the known effects of bupropion include inhibition of norepinephrine and dopamine reuptake, as well as inhibition of cation-selective Cys-loop ion channels like nicotinic acetylcholine and serotonin type 3 A (5-HT3A) receptors. The present study focuses on bupropion's inhibitory capacity with regard to the 5-HT3A receptor. 5-HT3R is a member of the pentameric ligand-gated ion channel superfamily, which also includes nicotinic acetylcholine, γ-aminobutyric acid type A (GABAA), and glycine receptors. These channels are made up of five homologous subunits around a central ion channel pore. Each subunit is divided into extracellular, transmembrane, and intracellular domain. Dysfunction within this superfamily has been linked to neurological disorders such as anxiety, depression, epilepsy, Alzheimer's, and Parkinson's Disease. Currently, the 5-HT3receptor is targeted clinically by anti-emetics and irritable bowel syndrome treatments, but the receptor could potentially become a target for the treatment of anxiety, psychosis, bipolar disorder, and several other neurological disorders. We engineered amino acids in the α-helical M2 and M3 transmembrane segments near the M2M3 loop that is located at the interface of transmembrane and extracellular domains. We used docking studies and site-directed mutagenesis to analyze potential binding site residues for bupropion. Two-electrode voltage-clamp recordings were used to examine the effect of mutations on bupropion inhibition of these engineered 5-HT3A channels expressed in Xenopus oocytes.