Structure of tetrameric forms of the serotonin-gated 5-HT3A receptor ion channel.

Multimeric membrane proteins are produced in the endoplasmic reticulum and transported to their target membranes which, for ion channels, is typically the plasma membrane. Despite the availability of many fully assembled channel structures, our understanding of assembly intermediates, multimer assembly mechanisms, and potential functions of non-standard assemblies is limited. We demonstrate that the pentameric ligand-gated serotonin 5-HT3A receptor (5-HT3AR) can assemble to tetrameric forms and report the structures of the tetramers in plasma membranes of cell-derived microvesicles and in membrane memetics using cryo-electron microscopy and tomography. The tetrameric structures have near-symmetric transmembrane domains, and asymmetric extracellular domains, and can bind serotonin molecules. Computer simulations, based on our cryo-EM structures, were used to decipher the assembly pathway of pentameric 5-HT3R and suggest a potential functional role for the tetrameric receptors.

Key role of the TM2-TM3 loop in calcium potentiation of the α9α10 nicotinic acetylcholine receptor.

The α9α10 nicotinic cholinergic receptor (nAChR) is a ligand-gated pentameric cation-permeable ion channel that mediates synaptic transmission between descending efferent neurons and mechanosensory inner ear hair cells. When expressed in heterologous systems, α9 and α10 subunits can assemble into functional homomeric α9 and heteromeric α9α10 receptors. One of the differential properties between these nAChRs is the modulation of their ACh-evoked responses by extracellular calcium (Ca2+). While α9 nAChRs responses are blocked by Ca2+, ACh-evoked currents through α9α10 nAChRs are potentiated by Ca2+ in the micromolar range and blocked at millimolar concentrations. Using chimeric and mutant subunits, together with electrophysiological recordings under two-electrode voltage-clamp, we show that the TM2-TM3 loop of the rat α10 subunit contains key structural determinants responsible for the potentiation of the α9α10 nAChR by extracellular Ca2+. Moreover, molecular dynamics simulations reveal that the TM2-TM3 loop of α10 does not contribute to the Ca2+ potentiation phenotype through the formation of novel Ca2+ binding sites not present in the α9 receptor. These results suggest that the TM2-TM3 loop of α10 might act as a control element that facilitates the intramolecular rearrangements that follow ACh-evoked α9α10 nAChRs gating in response to local and transient changes of extracellular Ca2+ concentration. This finding might pave the way for the future rational design of drugs that target α9α10 nAChRs as otoprotectants.

The Role of Alpha-7 Nicotinic Acetylcholine Receptors in Pain: Potential Therapeutic Implications.

Chronic pain represents a prevalent and costly medical challenge globally. Nicotinic acetylcholine receptors (nAChRs), one type of ligand-gated ion channels found extensively in both the central and peripheral nervous systems, have emerged as promising therapeutic targets for chronic pain. Although there are currently no FDA-approved analgesics specifically targeting nAChRs, accumulating preclinical and clinical evidence suggest that selective ligands for alpha 7 (α7) nAChRs show potential for treating chronic pain, boasting a reduced incidence of side effects compared with other nicotinic receptor types. The recent structural resolution of human α7 nAChRs has confirmed their negative association with heightened pain, providing a valuable foundation for the development of targeted medications. This review presents a comprehensive overview, encompassing insights into the roles of α7 nAChRs derived from structural and functional studies, recent advancements in pharmacology, and investigations into their involvement in the pathophysiology of chronic pain. Moreover, the review addresses the variability in analgesic effects based on the type of receptor agonist and highlights the current research limitations. As such, this review offers potential therapeutic approaches for the development of innovative strategies for chronic pain management.

Phytochemical Modulation of Ion Channels in Oncologic Symptomatology and Treatment.

Modern chemotherapies offer a broad approach to cancer treatment but eliminate both cancer and non-cancer cells indiscriminately and, thus, are associated with a host of side effects. Advances in precision oncology have brought about new targeted therapeutics, albeit mostly limited to a subset of patients with an actionable mutation. They too come with side effects and, ultimately, 'self-resistance' to the treatment. There is recent interest in the modulation of ion channels, transmembrane proteins that regulate the flow of electrically charged molecules in and out of cells, as an approach to aid treatment of cancer. Phytochemicals have been shown to act on ion channels with high specificity regardless of the tumor's genetic profile. This paper explores the use of phytochemicals in cancer symptom management and treatment.

Constitutive activity of ionotropic glutamate receptors via hydrophobic substitutions in the ligand-binding domain.

Neurotransmitter ligands electrically excite neurons by activating ionotropic glutamate receptor (iGluR) ion channels. Knowledge of the iGluR amino acid residues that dominate ligand-induced activation would enable the prediction of function from sequence. We therefore explored the molecular determinants of activity in rat N-methyl-D-aspartate (NMDA)-type iGluRs (NMDA receptors), complex heteromeric iGluRs comprising two glycine-binding GluN1 and two glutamate-binding GluN2 subunits, using amino acid sequence analysis, mutagenesis, and electrophysiology. We find that a broadly conserved aspartate residue controls both ligand potency and channel activity, to the extent that certain substitutions at this position bypass the need for ligand binding in GluN1 subunits, generating NMDA receptors activated solely by glutamate. Furthermore, we identify a homomeric iGluR from the placozoan Trichoplax adhaerens that has utilized native mutations of this crucial residue to evolve into a leak channel that is inhibited by neurotransmitter binding, pointing to a dominant role of this residue throughout the iGluR superfamily.

Progress on functions of intracellular domain of trimeric ligand-gated ion channels. / ä¸‰èšä½“é ä½“é—¨æŽ§ç¦»å­é€šé“èƒžå† åŒºåŸŸçš„åŠŸèƒ½ç ”ç©¶è¿›å±•.

Ligand-gated ion channels are a large category of essential ion channels, modulating their state by binding to specific ligands to allow ions to pass through the cell membrane. Purinergic ligand-gated ion channel receptors (P2XRs) and acid-sensitive ion channels (ASICs) are representative members of trimeric ligand-gated ion channel. Recent studies have shown that structural differences in the intracellular domain of P2XRs may determine the desensitization process. The lateral fenestrations of P2XRs potentially serve as a pathway for ion conductance and play a decisive role in ion selectivity. Phosphorylation of numerous amino acid residues in the P2XRs are involved in regulating the activity of ion channels. Additionally, the P2XRs interact with other ligand-gated ion channels including N-methyl-D-aspartate receptors, γ-aminobutyric acid receptors, 5-hydroxytryptamin receptors and nicotinic acetylcholine receptors, mediating physiological processes such as synaptic plasticity. Conformational changes in the intracellular domain of the ASICs expose binding sites of intracellular signal partners, facilitating metabolic signal transduction. Amino acids such as Val16, Ser17, Ile18, Gln19 and Ala20 in the ASICs participate in channel opening and membrane expression. ASICs can also bind to intracellular proteins, such as CIPP and p11, to regulate channel function. Many phosphorylation sites at the C-terminus and N-terminus of ASICs are involved in the regulation of receptors. Furthermore, ASICs are involved in various physiological and pathophysiological processes, which include pain, ischemic stroke, psychiatric disorders, and neurodegenerative disease. In this article, we review the roles of the intracellular domains of these trimeric ligand-gated ion channels in channel gating as well as their physiological and pathological functions, in order to provide new insights into the discovery of related drugs.

Advancements in the use of xenopus oocytes for modelling neurological disease for novel drug discovery.

INTRODUCTION: Introduced about 50 years ago, the model of Xenopus oocytes for the expression of recombinant proteins has gained a broad spectrum of applications. The authors herein review the benefits brought from using this model system, with a focus on modeling neurological disease mechanisms and application to drug discovery. AREAS COVERED: Using multiple examples spanning from ligand gated ion channels to transporters, this review presents, in the light of the latest publications, the benefits offered from using Xenopus oocytes. Studies range from the characterization of gene mutations to the discovery of novel treatments for disorders of the central nervous system (CNS). EXPERT OPINION: Development of new drugs targeting CNS disorders has been marked by failures in the translation from preclinical to clinical studies. As progress in genetics and molecular biology highlights large functional differences arising from a single to a few amino acid exchanges, the need for drug screening and functional testing against human proteins is increasing. The use of Xenopus oocytes to enable precise modeling and characterization of clinically relevant genetic variants constitutes a powerful model system that can be used to inform various aspects of CNS drug discovery and development.

Protein semisynthesis underscores the role of a conserved lysine in activation and desensitization of acid-sensing ion channels.

Acid-sensing ion channels (ASICs) are trimeric ion channels that open a cation-conducting pore in response to proton binding. Excessive ASIC activation during prolonged acidosis in conditions such as inflammation and ischemia is linked to pain and stroke. A conserved lysine in the extracellular domain (Lys211 in mASIC1a) is suggested to play a key role in ASIC function. However, the precise contributions are difficult to dissect with conventional mutagenesis, as replacement of Lys211 with naturally occurring amino acids invariably changes multiple physico-chemical parameters. Here, we study the contribution of Lys211 to mASIC1a function using tandem protein trans-splicing (tPTS) to incorporate non-canonical lysine analogs. We conduct optimization efforts to improve splicing and functionally interrogate semisynthetic mASIC1a. In combination with molecular modeling, we show that Lys211 charge and side-chain length are crucial to activation and desensitization, thus emphasizing that tPTS can enable atomic-scale interrogations of membrane proteins in live cells.

The diverse family of Cys-loop receptors in Caenorhabditis elegans: insights from electrophysiological studies.

Cys-loop receptors integrate a large family of pentameric ligand-gated ion channels that mediate fast ionotropic responses in vertebrates and invertebrates. Their vital role in converting neurotransmitter recognition into an electrical impulse makes these receptors essential for a great variety of physiological processes. In vertebrates, the Cys-loop receptor family includes the cation-selective channels, nicotinic acetylcholine and 5-hydroxytryptamine type 3 receptors, and the anion-selective channels, GABAA and glycine receptors, whereas in invertebrates, the repertoire is significantly larger. The free-living nematode Caenorhabditis elegans has the largest known Cys-loop receptor family as well as unique receptors that are absent in vertebrates and constitute attractive targets for anthelmintic drugs. Given the large number and variety of Cys-loop receptor subunits and the multiple possible ways of subunit assembly, C. elegans offers a large diversity of receptors although only a limited number of them have been characterized to date. C. elegans has emerged as a powerful model for the study of the nervous system and human diseases as well as a model for antiparasitic drug discovery. This nematode has also shown promise in the pharmaceutical industry search for new therapeutic compounds. C. elegans is therefore a powerful model organism to explore the biology and pharmacology of Cys-loop receptors and their potential as targets for novel therapeutic interventions. In this review, we provide a comprehensive overview of what is known about the function of C. elegans Cys-loop receptors from an electrophysiological perspective.

Two residues determine nicotinic acetylcholine receptor requirement for RIC-3.

Nicotinic acetylcholine receptors (N-AChRs) mediate fast synaptic signaling and are members of the pentameric ligand-gated ion channel (pLGIC) family. They rely on a network of accessory proteins in vivo for correct formation and transport to the cell surface. Resistance to cholinesterase 3 (RIC-3) is an endoplasmic reticulum protein that physically interacts with nascent pLGIC subunits and promotes their oligomerization. It is not known why some N-AChRs require RIC-3 in heterologous expression systems, whereas others do not. Previously we reported that the ACR-16 N-AChR from the parasitic nematode Dracunculus medinensis does not require RIC-3 in Xenopus laevis oocytes. This is unusual because all other nematode ACR-16, like the closely related Ascaris suum ACR-16, require RIC-3. Their high sequence similarity limits the number of amino acids that may be responsible, and the goal of this study was to identify them. A series of chimeras and point mutations between A. suum and D. medinensis ACR-16, followed by functional characterization with electrophysiology, identified two residues that account for a majority of the receptor requirement for RIC-3. ACR-16 with R/K159 in the cys-loop and I504 in the C-terminal tail did not require RIC-3 for functional expression. Mutating either of these to R/K159E or I504T, residues found in other nematode ACR-16, conferred a RIC-3 requirement. Our results agree with previous studies showing that these regions interact and are involved in receptor synthesis. Although it is currently unclear what precise mechanism they regulate, these residues may be critical during specific subunit folding and/or assembly cascades that RIC-3 may promote.

Ligand-Gated Ion Channels: Prognostic and Therapeutic Implications for Gliomas.

Gliomas are common primary brain malignancies that remain difficult to treat due to their overall aggressiveness and heterogeneity. Although a variety of therapeutic strategies have been employed for the treatment of gliomas, there is increasing evidence that suggests ligand-gated ion channels (LGICs) can serve as a valuable biomarker and diagnostic tool in the pathogenesis of gliomas. Various LGICs, including P2X, SYT16, and PANX2, have the potential to become altered in the pathogenesis of glioma, which can disrupt the homeostatic activity of neurons, microglia, and astrocytes, further exacerbating the symptoms and progression of glioma. Consequently, LGICs, including purinoceptors, glutamate-gated receptors, and Cys-loop receptors, have been targeted in clinical trials for their potential therapeutic benefit in the diagnosis and treatment of gliomas. In this review, we discuss the role of LGICs in the pathogenesis of glioma, including genetic factors and the effect of altered LGIC activity on the biological functioning of neuronal cells. Additionally, we discuss current and emerging investigations regarding the use of LGICs as a clinical target and potential therapeutic for gliomas.

5-HT3 Receptors on Mitochondria Influence Mitochondrial Function.

The 5-hydroxytryptamine 3 (5-HT3) receptor belongs to the pentameric ligand-gated cation channel superfamily. Humans have five different 5-HT3 receptor subunits: A to E. The 5-HT3 receptors are located on the cell membrane, but a previous study suggested that mitochondria could also contain A subunits. In this article, we explored the distribution of 5-HT3 receptor subunits in intracellular and cell-free mitochondria. Organelle prediction software supported the localization of the A and E subunits on the inner membrane of the mitochondria. We transiently transfected HEK293T cells that do not natively express the 5-HT3 receptor with an epitope and fluorescent protein-tagged 5HT3A and 5HT3E subunits. Fluorescence microscopy and cell fractionation indicated that both subunits, A and E, localized to the mitochondria, while transmission electron microscopy revealed the location of the subunits on the mitochondrial inner membrane, where they could form heteromeric complexes. Cell-free mitochondria isolated from cell culture media colocalized with the fluorescent signal for A subunits. The presence of A and E subunits influenced changes in the membrane potential and mitochondrial oxygen consumption rates upon exposure to serotonin; this was inhibited by pre-treatment with ondansetron. Therefore, it is likely that the 5-HT3 receptors present on mitochondria directly impact mitochondrial function and that this may have therapeutic implications.

Desensitization dynamics of the AMPA receptor.

To perform their physiological functions, amino methyl propionic acid receptors (AMPARs) cycle through active, resting, and desensitized states, and dysfunction in AMPAR activity is associated with various neurological disorders. Transitions among AMPAR functional states, however, are largely uncharacterized at atomic resolution and are difficult to examine experimentally. Here, we report long-timescale molecular dynamics simulations of dimerized AMPAR ligand-binding domains (LBDs), whose conformational changes are tightly coupled to changes in AMPAR functional states, in which we observed LBD dimer activation and deactivation upon ligand binding and unbinding at atomic resolution. Importantly, we observed the ligand-bound LBD dimer transition from the active conformation to several other conformations, which may correspond with distinct desensitized conformations. We also identified a linker region whose structural rearrangements heavily affected the transitions to and among these putative desensitized conformations, and confirmed, using electrophysiology experiments, the importance of the linker region in these functional transitions.

The importance of ligand gated ion channels in sleep and sleep disorders.

On average, humans spend about 26 years of their life sleeping. Increased sleep duration and quality has been linked to reduced disease risk; however, the cellular and molecular underpinnings of sleep remain open questions. It has been known for some time that pharmacological modulation of neurotransmission in the brain can promote either sleep or wakefulness thereby providing some clues about the molecular mechanisms at play. However, the field of sleep research has developed an increasingly detailed understanding of the requisite neuronal circuitry and key neurotransmitter receptor subtypes, suggesting that it may be possible to identify next generation pharmacological interventions to treat sleep disorders within this same space. The aim of this work is to examine the latest physiological and pharmacological findings highlighting the contribution of ligand gated ion channels including the inhibitory GABAA and glycine receptors and excitatory nicotinic acetylcholine receptors and glutamate receptors in the sleep-wake cycle regulation. Overall, a better understanding of ligand gated ion channels in sleep will help determine if these highly druggable targets could facilitate a better night's sleep.

Transient receptor potential vanilloid 1 (TRPV1)-independent actions of capsaicin on cellular excitability and ion transport.

Capsaicin is a naturally occurring alkaloid derived from chili pepper that is responsible for its hot pungent taste. Capsaicin is known to exert multiple pharmacological actions, including analgesia, anticancer, anti-inflammatory, antiobesity, and antioxidant effects. The transient receptor potential vanilloid subfamily member 1 (TRPV1) is the main receptor mediating the majority of the capsaicin effects. However, numerous studies suggest that the TRPV1 receptor is not the only target for capsaicin. An increasing number of studies indicates that capsaicin, at low to mid µM ranges, not only indirectly through TRPV1-mediated Ca2+ increases, but also directly modulates the functions of voltage-gated Na+ , K+ , and Ca2+ channels, as well as ligand-gated ion channels and other ion transporters and enzymes involved in cellular excitability. These TRPV1-independent effects are mediated by alterations of the biophysical properties of the lipid membrane and subsequent modulation of the functional properties of ion channels and by direct binding of capsaicin to the channels. The present study, for the first time, systematically categorizes this diverse range of non-TRPV1 targets and discusses cellular and molecular mechanisms mediating TRPV1-independent effects of capsaicin in excitable, as well as nonexcitable cells.

Pharmacokinetic and pharmacodynamic considerations for treating sarcoptic mange with cross-relevance to Australian wildlife.

Sarcoptes scabiei is the microscopic burrowing mite responsible for sarcoptic mange, which is reported in approximately 150 mammalian species. In Australia, sarcoptic mange affects a number of native and introduced wildlife species, is particularly severe in bare-nosed wombats (Vombatus ursinus) and an emerging issue in koala and quenda. There are a variety of acaricides available for the treatment of sarcoptic mange which are generally effective in eliminating mites from humans and animals in captivity. In wild populations, effective treatment is challenging, and concerns exist regarding safety, efficacy and the potential emergence of acaricide resistance. There are risks where acaricides are used intensively or inadequately, which could adversely affect treatment success rates as well as animal welfare. While reviews on epidemiology, treatment strategies, and pathogenesis of sarcoptic mange in wildlife are available, there is currently no review evaluating the use of specific acaricides in the context of their pharmacokinetic and pharmacodynamic properties, and subsequent likelihood of emerging drug resistance, particularly for Australian wildlife. This review critically evaluates acaricides that have been utilised to treat sarcoptic mange in wildlife, including dosage forms and routes, pharmacokinetics, mode of action and efficacy. We also highlight the reports of resistance of S. scabiei to acaricides, including clinical and in vitro observations.

Regulation of nicotinic acetylcholine receptors by post-translational modifications.

Nicotinic acetylcholine receptors (nAChRs) comprise a family of pentameric ligand-gated ion channels widely distributed in the central and peripheric nervous system and in non-neuronal cells. nAChRs are involved in chemical synapses and are key actors in vital physiological processes throughout the animal kingdom. They mediate skeletal muscle contraction, autonomic responses, contribute to cognitive processes, and regulate behaviors. Dysregulation of nAChRs is associated with neurological, neurodegenerative, inflammatory and motor disorders. In spite of the great advances in the elucidation of nAChR structure and function, our knowledge about the impact of post-translational modifications (PTMs) on nAChR functional activity and cholinergic signaling has lagged behind. PTMs occur at different steps of protein life cycle, modulating in time and space protein folding, localization, function, and protein-protein interactions, and allow fine-tuned responses to changes in the environment. A large body of evidence demonstrates that PTMs regulate all levels of nAChR life cycle, with key roles in receptor expression, membrane stability and function. However, our knowledge is still limited, restricted to a few PTMs, and many important aspects remain largely unknown. There is thus a long way to go to decipher the association of aberrant PTMs with disorders of cholinergic signaling and to target PTM regulation for novel therapeutic interventions. In this review we provide a comprehensive overview of what is known about how different PTMs regulate nAChR.

Vanilloid agonist-mediated activation of TRPV1 channels requires coordinated movement of the S1-S4 bundle rather than a quiescent state.

Transient receptor potential vanilloid1 (TRPV1) channel plays an important role in a wide range of physiological and pathological processes, and a comprehensive understanding of TRPV1 gating will create opportunities for therapeutic intervention. Recent incredible advances in cryo-electron microscopy (cryo-EM) have yielded high-resolution structures of all TRPV subtypes (TRPV1-6) and all of them share highly conserved six transmembrane (TM) domains (S1-S6). As revealed by the open structures of TRPV1 in the presence of a bound vanilloid agonist (capsaicin or resiniferatoxin), TM helicesS1 to S4 form a bundle that remains quiescent during channel activation, highlighting differences in the gating mechanism of TRPV1 and voltage-gated ion channels. Here, however, we argue that the structural dynamics rather than quiescence of S1-S4 domains is necessary for capsaicin-mediated activation of TRPV1. Using fluorescent unnatural amino acid (flUAA) incorporation and voltage-clamp fluorometry (VCF) analysis, we directly observed allostery of the S1-S4 bundle upon capsaicin binding. Covalent occupation of VCF-identified sites, single-channel recording, cell apoptosis analysis, and exploration of the role of PSFL828, a novel non-vanilloid agonist we identified, have collectively confirmed the essential role of this coordinated S1-S4 motility in capsaicin-mediated activation of TRPV1. This study concludes that, in contrast to cryo-EM structural studies, vanilloid agonists are also required for S1-S4 movement during TRPV1 activation. Redefining the gating process of vanilloid agonists and the discovery of new non-vanilloid agonists will allow the evaluation of new strategies aimed at the development of TRPV1 modulators.

Effects of cannabinoids on ligand-gated ion channels.

Phytocannabinoids such as Δ9-tetrahydrocannabinol and cannabidiol, endocannabinoids such as N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol, and synthetic cannabinoids such as CP47,497 and JWH-018 constitute major groups of structurally diverse cannabinoids. Along with these cannabinoids, CB1 and CB2 cannabinoid receptors and enzymes involved in synthesis and degradation of endocannabinoids comprise the major components of the cannabinoid system. Although, cannabinoid receptors are known to be involved in anti-convulsant, anti-nociceptive, anti-psychotic, anti-emetic, and anti-oxidant effects of cannabinoids, in recent years, an increasing number of studies suggest that, at pharmacologically relevant concentrations, these compounds interact with several molecular targets including G-protein coupled receptors, ion channels, and enzymes in a cannabinoid-receptor independent manner. In this report, the direct actions of endo-, phyto-, and synthetic cannabinoids on the functional properties of ligand-gated ion channels and the plausible mechanisms mediating these effects were reviewed and discussed.