RESUMO
Delta receptors (GluD1 and GluD2), members of the large ionotropic glutamate receptor (iGluR) family, play a central role in numerous neurodevelopmental and psychiatric disorders. The amino-terminal domain (ATD) of GluD orchestrates synapse formation and maturation processes through its interaction with the Cbln family of synaptic organizers and neurexin (Nrxn). The transsynaptic triad of Nrxn-Cbln-GluD also serves as a potent regulator of synaptic plasticity, at both excitatory and inhibitory synapses. Despite these recognized functions, there is still debate as to whether GluD functions as a "canonical" ion channel, similar to other iGluRs. A recent report proposes that the ATD of GluD2 imposes conformational constraints on channel activity; removal of this constraint by binding to Cbln1 and Nrxn, or removal of the ATD, reveals channel activity in GluD2 upon administration of glycine (Gly) and d-serine (d-Ser), two GluD ligands. We were able to reproduce currents when Gly or d-Ser was administered to clusters of heterologous human embryonic kidney 293 (HEK293) cells expressing Cbln1, GluD2 (or GluD1), and Nrxn. However, Gly or d-Ser, but also l-glutamate (l-Glu), evoked similar currents in naive (i.e., untransfected) HEK293 cells and in GluD2-null Purkinje neurons. Furthermore, no current was detected in isolated HEK293 cells expressing GluD2 lacking the ATD upon administration of Gly. Taken together, these results cast doubt on the previously proposed hypothesis that extracellular ligands directly gate wild-type GluD channels.
Assuntos
Ativação do Canal Iônico , Receptores de Glutamato , Animais , Humanos , Camundongos , Glicina/metabolismo , Células HEK293 , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Canais Iônicos de Abertura Ativada por Ligante/genética , Ligantes , Receptores de Glutamato/metabolismo , Serina/metabolismoRESUMO
Pentameric ligand-gated ion channels provide rapid chemical-electrical signal transmission between cells in the central and peripheral nervous system. Their dysfunction is associated with many nervous system disorders. They are composed of five identical (homomeric receptors) or homologous (heteromeric receptors) subunits. VHH nanobodies, or single-chain antibodies, are the variable domain, VHH, of antibodies that are composed of the heavy chain only from camelids. Their unique structure results in many specific biochemical and biophysical properties that make them an excellent alternative to conventional antibodies. This Perspective explores the published VHH nanobodies which have been isolated against pentameric ligand-gated ion channel subfamilies. It outlines the genetic and chemical modifications available to alter nanobody function. An assessment of the available functional and structural data indicate that it is feasible to create therapeutic agents and impart, through their modification, a given desired modulatory effect of its target receptor for current stoichiometric-specific VHH nanobodies.
Assuntos
Anticorpos de Domínio Único , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/farmacologia , Humanos , Animais , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Canais Iônicos de Abertura Ativada por Ligante/imunologia , Canais Iônicos de Abertura Ativada por Ligante/química , Modelos MolecularesRESUMO
In this report we present seven lines of bioinformatic evidence supporting the conclusion that the Pentameric Ligand-gated Ion Channel (pLIC) Family is a member of the Voltage-gated Ion Channel (VIC) Superfamily. In our approach, we used the Transporter Classification Database (TCDB) as a reference and applied a series of bioinformatic methods to search for similarities between the pLIC family and members of the VIC superfamily. These include: (1) sequence similarity, (2) compatibility of topology and hydropathy profiles, (3) shared domains, (4) conserved motifs, (5) similarity of Hidden Markov Model profiles between families, (6) common 3D structural folds, and (7) clustering analysis of all families. Furthermore, sequence and structural comparisons as well as the identification of a 3-TMS repeat unit in the VIC superfamily suggests that the sixth transmembrane segment evolved into a re-entrant loop. This evidence suggests that the voltage-sensor domain and the channel domain have a common origin. The classification of the pLIC family within the VIC superfamily sheds light onto the topological origins of this family and its evolution, which will facilitate experimental verification and further research into this superfamily by the scientific community.
Assuntos
Canais Iônicos de Abertura Ativada por Ligante , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Canais Iônicos de Abertura Ativada por Ligante/química , Canais Iônicos de Abertura Ativada por Ligante/genética , Humanos , Sequência de Aminoácidos , Biologia Computacional/métodos , Modelos Moleculares , Família Multigênica , Animais , Domínios Proteicos , Filogenia , Cadeias de MarkovRESUMO
The reversal potential refers to the membrane potential at which the net current flow through a channel reverses direction. The reversal potential is determined by transmembrane ion gradients and, in turn, determines how the channel's activity will affect the membrane potential. Traditional investigation into the reversal potential of inhibitory ligand-gated ion channels (EInh) has relied upon the activation of endogenous receptors, such as the GABA-A receptor (GABAAR). There are, however, challenges associated with activating endogenous receptors, including agonist delivery, isolating channel responses, and the effects of receptor saturation and desensitization. Here, we demonstrate the utility of using a light-gated anion channel, stGtACR2, to probe EInh in the rodent brain. Using mice of both sexes, we demonstrate that the properties of this optically activated channel make it a suitable proxy for studying GABAAR receptor-mediated inhibition. We validate this agonist-independent optogenetic strategy in vitro and in vivo and further show how it can accurately capture differences in EInh dynamics following manipulations of endogenous ion fluxes. This allows us to explore distinct resting EInh differences across genetically defined neuronal subpopulations. Using this approach to challenge ion homeostasis mechanisms in neurons, we uncover cell-specific EInh dynamics that are supported by the differential expression of endogenous ion handling mechanisms. Our findings therefore establish an effective optical strategy for revealing novel aspects of inhibitory reversal potentials and thereby expand the repertoire of optogenetics.
Assuntos
Potenciais da Membrana , Optogenética , Animais , Optogenética/métodos , Camundongos , Masculino , Feminino , Potenciais da Membrana/fisiologia , Receptores de GABA-A/metabolismo , Receptores de GABA-A/genética , Neurônios/fisiologia , Neurônios/metabolismo , Camundongos Endogâmicos C57BL , Inibição Neural/fisiologia , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Canais Iônicos de Abertura Ativada por Ligante/genética , Camundongos TransgênicosRESUMO
GLIC, a proton-activated prokaryotic ligand-gated ion channel, served as a model system for understanding the eukaryotic counterparts due to their structural and functional similarities. Despite extensive studies conducted on GLIC, the molecular mechanism of channel gating in the lipid environment requires further investigation. Here, we present the cryo-EM structures of nanodisc-reconstituted GLIC at neutral and acidic pH in the resolution range of 2.6 - 3.4 Å. In our apo state at pH 7.5, the extracellular domain (ECD) displays conformational variations compared to the existing apo structures. At pH 4.0, three distinct conformational states (C1, C2 and O states) are identified. The protonated structures exhibit a compacted and counter-clockwise rotated ECD compared with our apo state. A gradual widening of the pore in the TMD is observed upon reducing the pH, with the widest pore in O state, accompanied by several layers of water pentagons. The pore radius and molecular dynamics (MD) simulations suggest that the O state represents an open conductive state. We also observe state-dependent interactions between several lipids and proteins that may be involved in the regulation of channel gating. Our results provide comprehensive insights into the importance of lipids impact on gating.
Assuntos
Canais Iônicos de Abertura Ativada por Ligante , Canais Iônicos de Abertura Ativada por Ligante/química , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Ativação do Canal Iônico/fisiologia , Microscopia Crioeletrônica , Prótons , Lipídeos , Proteínas de Bactérias/metabolismoRESUMO
Bupropion is an atypical antidepressant and smoking cessation drug that causes adverse effects such as insomnia, irritability, and anxiety. Bupropion inhibits dopamine and norepinephrine reuptake transporters and eukaryotic cation-conducting pentameric ligand-gated ion channels, such as nicotinic acetylcholine and serotonin type 3A receptors, at clinically relevant concentrations. Here, we demonstrate that bupropion also inhibits a prokaryotic homolog of pentameric ligand-gated ion channels, the Gloeobacter violaceus ligand-gated ion channel (GLIC). Using the GLIC as a model, we used molecular docking to predict binding sites for bupropion. Bupropion was found to bind to several sites within the transmembrane domain, with the predominant site being localized to the interface between transmembrane segments M1 and M3 of two adjacent subunits. Residues W213, T214, and W217 in the first transmembrane segment, M1, and F267 and I271 in the third transmembrane segment, M3, most frequently reside within a 4 Šdistance from bupropion. We then used single amino acid substitutions at these positions and two-electrode voltage-clamp recordings to determine their impact on bupropion inhibitory effects. The substitution T214F alters bupropion potency by shifting the half-maximal inhibitory concentration to a 13-fold higher value compared to wild-type GLIC. Residue T214 is found within a previously identified binding pocket for neurosteroids and lipids in the GLIC. This intersubunit binding pocket is structurally conserved and almost identical to a binding pocket described for neurosteroids in γ-aminobutyric acid type A receptors. Our data thus suggest that the T214 that lines a previously identified lipophilic binding pocket in GLIC and γ-aminobutyric acid type A receptors is also a modulatory site for bupropion interaction with the GLIC.
Assuntos
Bupropiona , Cianobactérias , Canais Iônicos de Abertura Ativada por Ligante , Bupropiona/farmacologia , Bupropiona/química , Bupropiona/metabolismo , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Canais Iônicos de Abertura Ativada por Ligante/química , Sítios de Ligação , Cianobactérias/metabolismo , Simulação de Acoplamento Molecular , Sequência de AminoácidosRESUMO
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.
Assuntos
Canais Iônicos de Abertura Ativada por Ligante , Animais , Humanos , Canais Iônicos Sensíveis a Ácido/metabolismo , Canais Iônicos de Abertura Ativada por Ligante/química , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Fosforilação , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Nicotínicos/metabolismo , Transdução de SinaisRESUMO
Lipid nanodiscs have become a standard tool for studying membrane proteins, including using single particle cryo-electron microscopy (cryo-EM). We find that reconstituting the pentameric ligand-gated ion channel (pLGIC), Erwinia ligand-gated ion channel (ELIC), in different nanodiscs produces distinct structures by cryo-EM. The effect of the nanodisc on ELIC structure extends to the extracellular domain and agonist binding site. Additionally, molecular dynamic simulations indicate that nanodiscs of different size impact ELIC structure and that the nanodisc scaffold directly interacts with ELIC. These findings suggest that the nanodisc plays a crucial role in determining the structure of pLGICs, and that reconstitution of ion channels in larger nanodiscs may better approximate a lipid membrane environment.
Assuntos
Canais Iônicos de Abertura Ativada por Ligante , Canais Iônicos de Abertura Ativada por Ligante/química , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Microscopia Crioeletrônica , Modelos Moleculares , Sítios de Ligação , LipídeosRESUMO
Ligand-gated ion channels are formed by three to five subunits that control the opening of the pore in a cooperative fashion. We developed a microfluidic chip-based technique for studying ion currents and fluorescence signals in either excised membrane patches or whole cells to measure activation and deactivation kinetics of the channels as well as ligand binding and unbinding when using confocal patch-clamp fluorometry. We show how this approach produces in a few seconds either unidirectional concentration-activation relationships at or near equilibrium and, moreover, respective time courses of activation and deactivation for a large number of freely designed steps of the ligand concentration. The short measuring period strongly minimizes the contribution of disturbing superimposing effects such as run-down phenomena and desensitization effects. To validate gating mechanisms, complex kinetic schemes are quantified without the requirement to have data at equilibrium. The new method has potential for functionally analyzing any ligand-gated ion channel and, beyond, also for other receptors.
Assuntos
Canais Iônicos de Abertura Ativada por Ligante , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , LigantesRESUMO
Ionotropic receptors are ligand-gated ion channels triggering fast neurotransmitter responses. Among them, P2X and 5-HT3 receptors have been shown to physically interact each other and functionally inducing cross inhibitory responses. Nevertheless, despite the importance of P2X4 and 5-HT3A receptors that mediate for example neuropathic pain and psychosis respectively, complementary evidence has recently started to move forward in the understanding of this interaction. In this review, we discuss current evidence supporting the mechanism of crosstalking between both receptors, from the structural to the transduction pathway level. We expect this work may guide the design of further experiments to obtain a comprehensive view for the neuropharmacological role of these interacting receptors. This article is part of the Special Issue on "The receptor-receptor interaction as a new target for therapy".
Assuntos
Canais Iônicos de Abertura Ativada por Ligante , Receptores 5-HT3 de Serotonina , Receptores 5-HT3 de Serotonina/metabolismo , Serotonina/metabolismo , Transporte Proteico , Ligação Proteica/fisiologia , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Receptores Purinérgicos P2X4/metabolismoRESUMO
Neurosteroids are steroids synthesized de novo in the brain from cholesterol in an independent manner from peripheral steroid sources. The term "neuroactive steroid" includes all steroids independent of their origin, and newly synthesized analogs of neurosteroids that modify neuronal activities. In vivo application of neuroactive steroids induces potent anxiolytic, antidepressant, anticonvulsant, sedative, analgesic and amnesic effects, mainly through interaction with the γ-aminobutyric acid type-A receptor (GABAAR). However, neuroactive steroids also act as positive or negative allosteric regulators on several ligand-gated channels including N-methyl-d-aspartate receptors (NMDARs), nicotinic acetylcholine receptors (nAChRs) and ATP-gated purinergic P2X receptors. Seven different P2X subunits (P2X1-7) can assemble to form homotrimeric or heterotrimeric ion channels permeable for monovalent cations and calcium. Among them, P2X2, P2X4, and P2X7 are the most abundant within the brain and can be regulated by neurosteroids. Transmembrane domains are necessary for neurosteroid binding, however, no generic motif of amino acids can accurately predict the neurosteroid binding site for any of the ligand-gated ion channels including P2X. Here, we will review what is currently known about the modulation of rat and human P2X by neuroactive steroids and the possible structural determinants underlying neurosteroid-induced potentiation and inhibition of the P2X2 and P2X4 receptors. This article is part of the Special Issue on "Purinergic Signaling: 50 years".
Assuntos
Canais Iônicos de Abertura Ativada por Ligante , Neuroesteroides , Ratos , Humanos , Animais , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Receptores Purinérgicos P2X/metabolismo , Encéfalo/metabolismo , Sítios de Ligação , Trifosfato de Adenosina/metabolismo , Receptores Purinérgicos P2X2/metabolismoRESUMO
Pentameric ligand-gated ion channels (pLGICs) mediate synaptic transmission and are sensitive to their lipid environment. The mechanism of phospholipid modulation of any pLGIC is not well understood. We demonstrate that the model pLGIC, ELIC (Erwinia ligand-gated ion channel), is positively modulated by the anionic phospholipid, phosphatidylglycerol, from the outer leaflet of the membrane. To explore the mechanism of phosphatidylglycerol modulation, we determine a structure of ELIC in an open-channel conformation. The structure shows a bound phospholipid in an outer leaflet site, and structural changes in the phospholipid binding site unique to the open-channel. In combination with streamlined alchemical free energy perturbation calculations and functional measurements in asymmetric liposomes, the data support a mechanism by which an anionic phospholipid stabilizes the activated, open-channel state of a pLGIC by specific, state-dependent binding to this site.
Assuntos
Canais Iônicos de Abertura Ativada por Ligante , Canais Iônicos de Abertura Ativada por Ligante/química , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Fosfolipídeos , Sítios de Ligação , Fosfatidilgliceróis , LipossomosRESUMO
The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel that modulates neuronal excitability, largely by allowing Ca2+ permeation. Agonist binding promotes transition from a resting state to an activated state, and then rapidly to a desensitized state. Recently, cryogenic electron microscopy (cryo-EM) structures of the human α7 receptor in nanodiscs were reported in multiple conformations. These were selectively stabilized by inhibitory, activating, or potentiating compounds. However, the functional annotation of these structures and their differential interactions with unresolved lipids and ligands remain incomplete. Here, we characterized their ion permeation, membrane interactions, and ligand binding using computational electrophysiology, free-energy calculations, and coarse-grained molecular dynamics. In contrast to nonconductive structures in apparent resting and desensitized states, the structure determined in the presence of the potentiator PNU-120596 was consistent with an activated state permeable to Ca2+. Transition to this state was associated with compression and rearrangement of the membrane, particularly in the vicinity of the peripheral MX helix. An intersubunit transmembrane site was implicated in selective binding of either PNU-120596 in the activated state or cholesterol in the desensitized state. This substantiates functional assignment of all three lipid-embedded α7-receptor structures with ion-permeation simulations. It also proposes testable models of their state-dependent interactions with lipophilic ligands, including a mechanism for allosteric modulation at the transmembrane subunit interface.
Assuntos
Canais Iônicos de Abertura Ativada por Ligante , Receptores Nicotínicos , Regulação Alostérica , Colesterol , Humanos , Isoxazóis , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Ligantes , Lipídeos , Compostos de Fenilureia , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Glycine Receptors (GlyRs) are cell-surface transmembrane proteins that belong to the Cysloop ligand-gated ion channels superfamily (Cys-loop LGICs). Functional glycine receptors are conformed only by α-subunits (homomeric channels) or by α- and ß-subunits (heteromeric channels). The role of glycine as a cytoprotective is widely studied. New information about glycine modulation of vascular endothelial cells (ECs) function emerged last year. Glycine and its receptors are recognized to play a role as neurovascular protectors by a mechanism that involves α2GlyRs. Interestingly, the expression of α2GlyRs reduces after stroke injury. However, glycine reverses the inhibition of α2GlyRs by a mechanism involving the VEGF/pSTAT3 signaling. On the other hand, consistent evidence has demonstrated that ECs participate actively in the innate and adaptive immunological response. We recently reported that GlyRs are modulated by interleukin-1ß, suggesting new perspectives to explain the immune modulation of vascular function in pathological conditions such as cerebrovascular stroke. In this work, we distinguish the role of glycine and the allosteric modulation of glycine receptors as a new therapeutic target to confront post-ischemic injury.
Assuntos
Canais Iônicos de Abertura Ativada por Ligante , Receptores de Glicina , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Glicina/metabolismo , Glicina/farmacologia , Glicina/uso terapêutico , Humanos , Interleucina-1beta/metabolismo , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Receptores de Glicina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Pentameric ligand-gated ion channels (pLGICs) play a leading role in synaptic communication, are implicated in a variety of neurological processes, and are important targets for the treatment of neurological and neuromuscular disorders. Endogenous lipids and lipophilic compounds are potent modulators of pLGIC function and may help shape synaptic communication. Increasing structural and biophysical data reveal sites for lipid binding to pLGICs. Here, we update our evolving understanding of pLGIC-lipid interactions highlighting newly identified modes of lipid binding along with the mechanistic understanding derived from the new structural data.
Assuntos
Canais Iônicos de Abertura Ativada por Ligante , Sítios de Ligação , Canais Iônicos de Abertura Ativada por Ligante/química , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , LipídeosRESUMO
Fast synaptic communication requires receptors that respond to the presence of neurotransmitter by opening an ion channel across the post-synaptic membrane. The muscle-type nicotinic acetylcholine receptor from the electric fish, Torpedo, is the prototypic ligand-gated ion channel, yet the structural changes underlying channel activation remain undefined. Here we use cryo-EM to solve apo and agonist-bound structures of the Torpedo nicotinic receptor embedded in a lipid nanodisc. Using both a direct biochemical assay to define the conformational landscape and molecular dynamics simulations to assay flux through the pore, we correlate structures with functional states and elucidate the motions that lead to pore activation of a heteromeric nicotinic receptor. We highlight an underappreciated role for the complementary subunit in channel gating, establish the structural basis for the differential agonist affinities of α/δ versus α /γ sites, and explain why nicotine is less potent at muscle nicotinic receptors compared to neuronal ones.
Assuntos
Canais Iônicos de Abertura Ativada por Ligante , Receptores Nicotínicos , Animais , Sítios de Ligação , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Ligantes , Músculos , Receptores Nicotínicos/metabolismo , Torpedo/metabolismoRESUMO
Polyunsaturated fatty acids (PUFAs) inhibit pentameric ligand-gated ion channels (pLGICs) but the mechanism of inhibition is not well understood. The PUFA, docosahexaenoic acid (DHA), inhibits agonist responses of the pLGIC, ELIC, more effectively than palmitic acid, similar to the effects observed in the GABAA receptor and nicotinic acetylcholine receptor. Using photo-affinity labeling and coarse-grained molecular dynamics simulations, we identified two fatty acid binding sites in the outer transmembrane domain (TMD) of ELIC. Fatty acid binding to the photolabeled sites is selective for DHA over palmitic acid, and specific for an agonist-bound state. Hexadecyl-methanethiosulfonate modification of one of the two fatty acid binding sites in the outer TMD recapitulates the inhibitory effect of PUFAs in ELIC. The results demonstrate that DHA selectively binds to multiple sites in the outer TMD of ELIC, but that state-dependent binding to a single intrasubunit site mediates DHA inhibition of ELIC.
Assuntos
Ácidos Graxos Insaturados/metabolismo , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Sítios de Ligação , Simulação de Dinâmica Molecular , Domínios ProteicosRESUMO
Ammonia and its amine-containing derivatives are widely found in natural decomposition byproducts. Here, we conducted biased chemoreceptor screening to investigate the mechanisms by which different concentrations of ammonium salt, urea, and putrescine in rotten fruits affect feeding and oviposition behavior. We identified three ionotropic receptors, including the two broadly required IR25a and IR76b receptors, as well as the narrowly tuned IR51b receptor. These three IRs were fundamental in eliciting avoidance against nitrogenous waste products, which is mediated by bitter-sensing gustatory receptor neurons (GRNs). The aversion of nitrogenous wastes was evaluated by the cellular requirement by expressing Kir2.1 and behavioral recoveries of the mutants in bitter-sensing GRNs. Furthermore, by conducting electrophysiology assays, we confirmed that ammonia compounds are aversive in taste as they directly activated bitter-sensing GRNs. Therefore, our findings provide insights into the ecological roles of IRs as a means to detect and avoid toxic nitrogenous waste products in nature.
Assuntos
Células Quimiorreceptoras/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Canais Iônicos de Abertura Ativada por Ligante/genética , Receptores Ionotrópicos de Glutamato/genética , Canais de Sódio/genética , Animais , Aprendizagem da Esquiva , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Fezes/química , Feminino , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Masculino , Receptores Ionotrópicos de Glutamato/metabolismo , Canais de Sódio/metabolismoRESUMO
Subcellular and organellar mechanisms have manifested a prominent importance for a broad variety of processes that maintain cellular life at its most basic level. Mammalian two-pore channels (TPCs) appear to be cornerstones of these processes in endo-lysosomes by controlling delicate ion-concentrations in their interiors. With evolutionary remarkable architecture and one-of-a-kind selectivity filter, TPCs are an extremely attractive topic per se. In the light of the current COVID-19 pandemic, hTPC2 emerges to be more than attractive. As a key regulator of the endocytosis pathway, it is potentially essential for diverse viral infections in humans, as demonstrated. Here, by means of multiscale molecular simulations, we propose a model of sodium transport from the lumen to the cytosol where the central cavity works as a reservoir. Since the inhibition of hTPC2 is proven to stop SARS-CoV2 in vitro, shedding light on the hTPC2 function and mechanism is the first step towards the selection of potential inhibiting candidates.
Assuntos
Ativação do Canal Iônico , Canais Iônicos de Abertura Ativada por Ligante/fisiologia , Sódio/metabolismo , COVID-19 , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Ligantes , SARS-CoV-2/isolamento & purificaçãoRESUMO
Pentameric ligand-gated ion channels (pLGICs) mediate chemical signaling through a succession of allosteric transitions that are yet not completely understood as intermediate states remain poorly characterized by structural approaches. In a previous study on the prototypic bacterial proton-gated channel GLIC, we generated several fluorescent sensors of the protein conformation that report a fast transition to a pre-active state, which precedes the slower process of activation with pore opening. Here, we explored the phenotype of a series of allosteric mutations, using simultaneous steady-state fluorescence and electrophysiological measurements over a broad pH range. Our data, fitted to a three-state Monod-Wyman-Changeux model, show that mutations at the subunit interface in the extracellular domain (ECD) principally alter pre-activation, while mutations in the lower ECD and in the transmembrane domain principally alter activation. We also show that propofol alters both transitions. Data are discussed in the framework of transition pathways generated by normal mode analysis (iModFit). It further supports that pre-activation involves major quaternary compaction of the ECD, and suggests that activation involves principally a reorganization of a 'central gating region' involving a contraction of the ECD ß-sandwich and the tilt of the channel lining M2 helix.