RESUMO
Glutamate is traditionally viewed as the first messenger to activate NMDAR (N-methyl-D-aspartate receptor)-dependent cell death pathways in stroke1,2, but unsuccessful clinical trials with NMDAR antagonists implicate the engagement of other mechanisms3-7. Here we show that glutamate and its structural analogues, including NMDAR antagonist L-AP5 (also known as APV), robustly potentiate currents mediated by acid-sensing ion channels (ASICs) associated with acidosis-induced neurotoxicity in stroke4. Glutamate increases the affinity of ASICs for protons and their open probability, aggravating ischaemic neurotoxicity in both in vitro and in vivo models. Site-directed mutagenesis, structure-based modelling and functional assays reveal a bona fide glutamate-binding cavity in the extracellular domain of ASIC1a. Computational drug screening identified a small molecule, LK-2, that binds to this cavity and abolishes glutamate-dependent potentiation of ASIC currents but spares NMDARs. LK-2 reduces the infarct volume and improves sensorimotor recovery in a mouse model of ischaemic stroke, reminiscent of that seen in mice with Asic1a knockout or knockout of other cation channels4-7. We conclude that glutamate functions as a positive allosteric modulator for ASICs to exacerbate neurotoxicity, and preferential targeting of the glutamate-binding site on ASICs over that on NMDARs may be strategized for developing stroke therapeutics lacking the psychotic side effects of NMDAR antagonists.
Assuntos
Canais Iônicos Sensíveis a Ácido , Isquemia Encefálica , Ácido Glutâmico , Animais , Feminino , Humanos , Masculino , Camundongos , 2-Amino-5-fosfonovalerato/efeitos adversos , 2-Amino-5-fosfonovalerato/metabolismo , 2-Amino-5-fosfonovalerato/farmacologia , Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/deficiência , Canais Iônicos Sensíveis a Ácido/efeitos dos fármacos , Canais Iônicos Sensíveis a Ácido/genética , Canais Iônicos Sensíveis a Ácido/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sítios de Ligação/genética , Isquemia Encefálica/induzido quimicamente , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Ácido Glutâmico/análogos & derivados , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Ácido Glutâmico/toxicidade , Camundongos Knockout , Mutagênese Sítio-Dirigida , Prótons , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels that play a role in neurotransmission and pain sensation. The snake venom-derived peptides, mambalgins, exhibit potent analgesic effects in rodents by inhibiting central ASIC1a and peripheral ASIC1b. Despite their distinct species- and subtype-dependent pharmacology, previous structure-function studies have focussed on the mambalgin interaction with ASIC1a. Currently, the specific channel residues responsible for this pharmacological profile, and the mambalgin pharmacophore at ASIC1b remain unknown. Here we identify non-conserved residues at the ASIC1 subunit interface that drive differences in the mambalgin pharmacology from rat ASIC1a to ASIC1b, some of which likely do not make peptide binding interactions. Additionally, an amino acid variation below the core binding site explains potency differences between rat and human ASIC1. Two regions within the palm domain, which contribute to subtype-dependent effects for mambalgins, play key roles in ASIC gating, consistent with subtype-specific differences in the peptides mechanism. Lastly, there is a shared primary mambalgin pharmacophore for ASIC1a and ASIC1b activity, with certain peripheral peptide residues showing variant-specific significance for potency. Through our broad mutagenesis studies across various species and subtype variants, we gain a more comprehensive understanding of the pharmacophore and the intricate molecular interactions that underlie ligand specificity. These insights pave the way for the development of more potent and targeted peptide analogues required to advance our understating of human ASIC1 function and its role in disease.
Assuntos
Canais Iônicos Sensíveis a Ácido , Venenos Elapídicos , Canais Iônicos Sensíveis a Ácido/metabolismo , Canais Iônicos Sensíveis a Ácido/genética , Canais Iônicos Sensíveis a Ácido/química , Animais , Humanos , Ratos , Venenos Elapídicos/química , Venenos Elapídicos/metabolismo , Venenos Elapídicos/farmacologia , Venenos Elapídicos/genética , Sequência de Aminoácidos , Sítios de Ligação , Modelos Moleculares , Xenopus laevis , PeptídeosRESUMO
Acid-sensing ion channels (ASICs) are neuronal Na+-permeable ion channels activated by extracellular acidification. ASICs are involved in learning, fear sensing, pain sensation and neurodegeneration. Increasing the extracellular Ca2+ concentration decreases the H+ sensitivity of ASIC1a, suggesting a competition for binding sites between H+ and Ca2+ ions. Here, we predicted candidate residues for Ca2+ binding on ASIC1a, based on available structural information and our molecular dynamics simulations. With functional measurements, we identified several residues in cavities previously associated with pH-dependent gating, whose mutation reduced the modulation by extracellular Ca2+ of the ASIC1a pH dependence of activation and desensitization. This occurred likely owing to a disruption of Ca2+ binding. Our results link one of the two predicted Ca2+-binding sites in each ASIC1a acidic pocket to the modulation of channel activation. Mg2+ regulates ASICs in a similar way as does Ca2+. We show that Mg2+ shares some of the binding sites with Ca2+. Finally, we provide evidence that some of the ASIC1a Ca2+-binding sites are functionally conserved in the splice variant ASIC1b. Our identification of divalent cation-binding sites in ASIC1a shows how Ca2+ affects ASIC1a gating, elucidating a regulatory mechanism present in many ion channels.
Assuntos
Canais Iônicos Sensíveis a Ácido , Cálcio , Simulação de Dinâmica Molecular , Canais Iônicos Sensíveis a Ácido/metabolismo , Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/genética , Sítios de Ligação , Cálcio/metabolismo , Animais , Ligação Proteica , Concentração de Íons de Hidrogênio , Magnésio/metabolismo , Humanos , Ativação do Canal Iônico , Mutação , Conformação ProteicaRESUMO
This study carried out to investigate the anti-inflammatory and antinociceptive effect of tropane alkaloid (EB7) isolated from E. bezerrae. It evaluated the toxicity and possible involvement of ion channels in the antinociceptive effect of EB7, as well as its anti-inflammatory effect in adult zebrafish (Zfa). Docking studies with EB7 and COX-1 and 2 were also performed. The tested doses of EB7 (4, 20 and 40â mg/kg) did not show any toxic effect on Zfa during the 96h of analysis (LD50>40â mg/kg). They did not produce any alteration in the locomotor behavior of the animals. Furthermore, EB7 showed promising pharmacological effects as it prevented the nociceptive behavior induced by hypertonic saline, capsaicin, formalin and acid saline. EB7 had its analgesic effect blocked by amiloride involving the neuromodulation of ASICs in Zfa. In evaluating the anti-inflammatory activity, the edema induced by κ-carrageenan 3.5 % was reduced by the dose of 40â mg/kg of EB7 observed after the fourth hour of analysis, indicating an effect similar to that of ibuprofen. Molecular docking results indicated that EB7 exhibited better affinity energy when compared to ibuprofen control against the two evaluated targets binding at different sites in the cocrystallized COX-1 and 2 inhibitors.
Assuntos
Analgésicos , Simulação de Acoplamento Molecular , Peixe-Zebra , Animais , Analgésicos/farmacologia , Analgésicos/química , Analgésicos/isolamento & purificação , Tropanos/farmacologia , Tropanos/isolamento & purificação , Tropanos/química , Edema/tratamento farmacológico , Edema/induzido quimicamente , Carragenina/farmacologia , Ciclo-Oxigenase 2/metabolismo , Ciclo-Oxigenase 1/metabolismo , Bignoniaceae/química , Relação Dose-Resposta a Droga , Relação Estrutura-Atividade , Alcaloides/farmacologia , Alcaloides/isolamento & purificação , Alcaloides/química , Canais Iônicos Sensíveis a Ácido/metabolismo , Canais Iônicos Sensíveis a Ácido/química , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/isolamento & purificação , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Estrutura MolecularRESUMO
Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to fast synaptic transmission and have roles in fear conditioning and nociception. Apart from activation at low pH, ASIC1a also undergoes several types of desensitization, including acute desensitization, which terminates activation; steady-state desensitization, which occurs at sub-activating proton concentrations and limits subsequent activation; and tachyphylaxis, which results in a progressive decrease in response during a series of activations. Structural insights from a desensitized state of ASIC1 have provided great spatial detail, but dynamic insights into conformational changes in different desensitizing conditions are largely missing. Here, we use electrophysiology and voltage-clamp fluorometry to follow the functional changes of the pore along with conformational changes at several positions in the extracellular and upper transmembrane domain via cysteine-labeled fluorophores. Acute desensitization terminates activation in wild type, but introducing an N414K mutation in the ß11-12 linker of mouse ASIC1a interfered with this process. The mutation also affected steady-state desensitization and led to pronounced tachyphylaxis. Although the extracellular domain of this mutant remained sensitive to pH and underwent pH-dependent conformational changes, these conformational changes did not necessarily lead to desensitization. N414K-containing channels also remained sensitive to a known peptide modulator that increases steady-state desensitization, indicating that the mutation only reduced, but not precluded, desensitization. Together, this study contributes to our understanding of the fundamental properties of ASIC1a desensitization, emphasizing the complex interplay between the conformational changes of the extracellular domain and the pore during channel activation and desensitization.
Assuntos
Canais Iônicos Sensíveis a Ácido , Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/metabolismo , Canais Iônicos Sensíveis a Ácido/genética , Animais , Camundongos , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico , Conformação Proteica , Mutação , Domínios Proteicos , Xenopus laevisRESUMO
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.
Assuntos
Canais Iônicos Sensíveis a Ácido , Lisina , Canais Iônicos Sensíveis a Ácido/metabolismo , Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/genética , Lisina/química , Lisina/metabolismo , Humanos , Animais , Modelos Moleculares , Processamento de ProteínaRESUMO
Acid-sensing ion channels (ASICs) are important players in detecting extracellular acidification throughout the brain and body. ASICs have large extracellular domains containing two regions replete with acidic residues: the acidic pocket, and the palm domain. In the resting state, the acidic pocket is in an expanded conformation but collapses in low pH conditions as the acidic side chains are neutralized. Thus, extracellular acidification has been hypothesized to collapse the acidic pocket that, in turn, ultimately drives channel activation. However, several observations run counter to this idea. To explore how collapse or mobility of the acidic pocket is linked to channel gating, we employed two distinct tools. First, we incorporated the photocrosslinkable noncanonical amino acids (ncAAs) 4-azido-L-phenylalanine (AzF) or 4-benzoyl-L-phenylalanine (BzF) into several positions in the acidic pocket. At both E315 and Y318, AzF incorporation followed by UV irradiation led to right shifts in pH response curves and accelerations of desensitization and deactivation, consistent with restrictions of acidic pocket mobility destabilizing the open state. Second, we reasoned that because Cl- ions are found in the open and desensitized structures but absent in the resting state structures, Cl- substitution would provide insight into how stability of the pocket is linked to gating. Anion substitution resulted in faster deactivation and desensitization, consistent with the acidic pocket regulating the stability of the open state. Taken together, our data support a model where acidic pocket collapse is not essential for channel activation. Rather, collapse of the acidic pocket influences the stability of the open state of the pore.
Assuntos
Canais Iônicos Sensíveis a Ácido , Canais Iônicos Sensíveis a Ácido/genética , Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/metabolismo , Conformação Molecular , Concentração de Íons de HidrogênioRESUMO
Acid-sensing ion channels (ASICs) sense extracellular protons and are involved in synaptic transmission and pain sensation. ASIC1a and ASIC3 are the ASIC subunits with the highest proton sensitivity. ASIC2a in contrast has low proton sensitivity but increases the variability of ASICs by forming heteromers with ASIC1a or ASIC3. ASICs are trimers and for the ASIC1a/2a heteromer it has been shown that subunits randomly assemble with a flexible 1:2/2:1 stoichiometry. Both heteromers have almost identical proton sensitivity intermediate between ASIC1a and ASIC2a. Here, we investigated the stoichiometry of the ASIC2a/3 heteromer. Using electrophysiology, we extensively characterized, first, cells expressing ASIC2a and ASIC3 at different ratios, second, concatemeric channels with a fixed subunit stoichiometry, and, third, channels containing loss-of-functions mutations in specific subunits. Our results conclusively show that only ASIC2a/3 heteromers with a 1:2 stoichiometry had a proton-sensitivity intermediate between ASIC2a and ASIC3. In contrast, the proton sensitivity of ASIC2a/3 heteromers with a 2:1 stoichiometry was strongly acid-shifted by more than one pH unit, which suggests that they are not physiologically relevant. Together, our results reveal that the proton sensitivity of the two ASIC2a/3 heteromers is clearly different and that ASIC3 and ASIC1a make remarkably different contributions to heteromers with ASIC2a.
Assuntos
Canais Iônicos Sensíveis a Ácido , Prótons , Canais Iônicos Sensíveis a Ácido/química , Fenômenos Eletrofisiológicos , Transmissão Sináptica , MutaçãoRESUMO
The gating mechanism of acid-sensitive ion channels (ASICs) remains unclear, despite the availability of atomic-scale structures in various functional states. The collapse of the acidic pocket and structural changes in the low-palm region are assumed to trigger activation. For the acidic pocket, protonation of some residues can minimize repulsion in the collapsed conformation. The relationship between low-palm rearrangements and gating is unknown. In this work, we performed a Monte Carlo energy optimization of known ASIC1a structures and determined the residue-residue interactions in different functional states. For rearrangements in the acidic pocket, our results are consistent with previously proposed mechanisms, although significant complexity was revealed for the residue-residue interactions. The data support the proposal of a gating mechanism in the low-palm region, in which residues E80 and E417 share a proton to activate the channel.
Assuntos
Canais Iônicos Sensíveis a Ácido , Prótons , Concentração de Íons de Hidrogênio , Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/metabolismo , Conformação MolecularRESUMO
The neuropeptide nocistatin (NS) is expressed by the nervous system cells and neutrophils as a part of a precursor protein and can undergo stepwise limited proteolysis. Previously, it was shown that rat NS (rNS) is able to activate acid-sensing ion channels (ASICs) and that this effect correlates with the acidic nature of NS. Here, we investigated changes in the properties of rNS in the course of its proteolytic degradation by comparing the effects of the full-size rNS and its two cleavage fragments on the rat isoform 3 ASICs (ASIC3) expressed in X. laevis oocytes and pain perception in mice. The rNS acted as both positive and negative modulator by lowering the steady-state desensitization of ASIC3 at pH 6.8-7.0 and reducing the channel's response to stimuli at pH 6.0-6.9, respectively. The truncated rNSΔ21 peptide lacking 21 amino acid residues from the N-terminus retained the positive modulatory activity, while the C-terminal pentapeptide (rNSΔ30) acted only as a negative ASIC3 modulator. The effects of the studied peptides were confirmed in animal tests: rNS and rNSΔ21 induced a pain-related behavior, whereas rNSΔ30 showed the analgesic effect. Therefore, we have shown that the mode of rNS action changes during its stepwise degradation, from an algesic molecule through a pain enhancer to a pain reliever (rNSΔ30 pentapeptide), which can be considered as a promising drug candidate.
Assuntos
Canais Iônicos Sensíveis a Ácido , Peptídeos Opioides , Ratos , Camundongos , Animais , Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/metabolismo , Proteólise , Peptídeos Opioides/metabolismo , Dor , Analgésicos/farmacologia , Concentração de Íons de HidrogênioRESUMO
The extracellular Ca2+ concentration changes locally under certain physiological and pathological conditions. Such variations affect the function of ion channels of the nervous system and consequently also neuronal signalling. We investigated here the mechanisms by which Ca2+ controls the activity of acid-sensing ion channel (ASIC) 3. ASICs are neuronal, H+-gated Na+ channels involved in several physiological and pathological processes, including the expression of fear, learning, pain sensation and neurodegeneration after ischaemic stroke. It was previously shown that Ca2+ negatively modulates the ASIC pH dependence. While protons are default activators of ASIC3, this channel can also be activated at pH7.4 by the removal of the extracellular Ca2+. Two previous studies concluded that low pH opens ASIC3 by displacing Ca2+ ions that block the channel pore at physiological pH. We show here that an acidic residue, distant from the pore, together with pore residues, controls the modulation of ASIC3 by Ca2+. Our study identifies a new regulatory site in ASIC3 and demonstrates that ASIC3 activation involves an allosteric mechanism together with Ca2+ unbinding from the channel pore. We provide a molecular analysis of a regulatory mechanism found in many ion channels.
Assuntos
Isquemia Encefálica , Acidente Vascular Cerebral , Humanos , Canais Iônicos Sensíveis a Ácido/genética , Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/metabolismo , Prótons , Cálcio/metabolismo , Sítio Alostérico , Concentração de Íons de Hidrogênio , ÍonsRESUMO
Acid Sensing Ion Channels (ASICs) are one of the most studied channels of the Epithelial Sodium Channel/Degenerin (ENaC/DEG) superfamily. They are responsible for excitatory responses following acidification of the extracellular medium and are involved in several important physiological roles. The ASIC1 subunit can form a functional homotrimeric channel and its structure is currently the most characterised of the whole ENaC/DEG family. Here we computed the free energy profiles for single ion permeation in two different structures of ASIC1 using both Na+ and Cl- as permeating ions. The first structure is the open structure of the channel from the PDB entry 4NTW, and the second structure is the closed structure with the re-entrant loop which contains the highly conserved 'HG' motif form PDB entry 6VTK. Both structures show cation selective free energy profiles, however the profiles of the permeating Na+ differ significantly between the two structures. Indeed, whereas there is only a small energetically favorable (-0.5 kcal mol-1) location for Na+ in the open channel (4NTW) near the end of the pore, we observed a clear ion binding site (-7.8 kcal mol-1) located in between the 'GAS' belt and the 'HG' loop for the channel containing the re-entrant loop (6VTK). Knowing that the 'GAS' motif was determined as the selectivity filter, our results support previous observations while addressing the importance of the 'HG' motif for the interactions between the pore and the permeating cations.
Assuntos
Canais Iônicos Sensíveis a Ácido , Canais Epiteliais de Sódio , Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/metabolismo , Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/metabolismo , Transporte de Íons , Íons/metabolismo , Sódio/químicaRESUMO
Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels involved in fast synaptic transmission. Pharmacological inhibition of ASIC1a reduces neurotoxicity and stroke infarct volumes, with the cysteine knot toxin psalmotoxin-1 (PcTx1) being one of the most potent and selective inhibitors. PcTx1 binds at the subunit interface in the extracellular domain (ECD), but the mechanism and conformational consequences of the interaction, as well as the number of toxin molecules required for inhibition, remain unknown. Here, we use voltage-clamp fluorometry and subunit concatenation to decipher the mechanism and stoichiometry of PcTx1 inhibition of ASIC1a. Besides the known inhibitory binding mode, we propose PcTx1 to have at least two additional binding modes that are decoupled from the pore. One of these modes induces a long-lived ECD conformation that reduces the activity of an endogenous neuropeptide. This long-lived conformational state is proton-dependent and can be destabilized by a mutation that decreases PcTx1 sensitivity. Lastly, the use of concatemeric channel constructs reveals that disruption of a single PcTx1 binding site is sufficient to destabilize the toxin-induced conformation, while functional inhibition is not impaired until two or more binding sites are mutated. Together, our work provides insight into the mechanism of PcTx1 inhibition of ASICs and uncovers a prolonged conformational change with possible pharmacological implications.
Assuntos
Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Venenos de Aranha/química , Venenos de Aranha/metabolismo , Animais , Sítios de Ligação , Cisteína/metabolismo , Fluorometria/métodos , Concentração de Íons de Hidrogênio , Conformação Molecular , Mutação , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Peptídeos/genética , Ligação Proteica , Prótons , Venenos de Aranha/genéticaRESUMO
Tissue acidification is associated with a variety of disease states, and acid-sensing ion channels (ASICs) that can sense changes in pH have gained traction as possible pharmaceutical targets. An array of modulators, ranging from small molecules to large biopharmaceuticals, are known to inhibit ASICs. Here, we summarize recent insights from animal studies to assess the therapeutic potential of ASICs in disorders such as ischemic stroke, various pain-related processes, anxiety, and cardiac pathologies. We also review the factors that present a challenge in the pharmacological targeting of ASICs, and which need to be taken into careful consideration when developing potent and selective modulators in the future.
Assuntos
Canais Iônicos Sensíveis a Ácido , Canais Iônicos Sensíveis a Ácido/química , Animais , HumanosRESUMO
Incorporation of noncanonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing human acid-sensing ion channel 1a (hASIC1a) variants in transiently transfected mammalian cells. We introduce 3 different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay, and live-cell crosslinking provides insight into the hASIC1a-psalmotoxin 1 (PcTx1) interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells.
Assuntos
Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/farmacologia , Aminoácidos/química , Canais Iônicos Sensíveis a Ácido/genética , Células HEK293 , Humanos , Peptídeos/química , Venenos de Aranha/química , TransfecçãoRESUMO
Prolonged acidosis, as it occurs during ischemic stroke, induces neuronal death via acid-sensing ion channel 1a (ASIC1a). Concomitantly, it desensitizes ASIC1a, highlighting the pathophysiological significance of modulators of ASIC1a acid sensitivity. One such modulator is the opioid neuropeptide big dynorphin (Big Dyn) which binds to ASIC1a and enhances its activity during prolonged acidosis. The molecular determinants and dynamics of this interaction remain unclear, however. Here, we present a molecular interaction model showing a dynorphin peptide inserting deep into the acidic pocket of ASIC1a. We confirmed experimentally that the interaction is predominantly driven by electrostatic forces, and using noncanonical amino acids as photo-cross-linkers, we identified 16 residues in ASIC1a contributing to Big Dyn binding. Covalently tethering Big Dyn to its ASIC1a binding site dramatically decreased the proton sensitivity of channel activation, suggesting that Big Dyn stabilizes a resting conformation of ASIC1a and dissociates from its binding site during channel opening.
Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Dinorfinas/metabolismo , Prótons , Canais Iônicos Sensíveis a Ácido/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/efeitos dos fármacos , Dinorfinas/química , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Eletricidade Estática , Xenopus laevisRESUMO
Acid-sensing ion channels (ASICs) are trimeric cation-selective channels activated by decreases in extracellular pH. The intracellular N and C terminal tails of ASIC1 influence channel gating, trafficking, and signaling in ischemic cell death. Despite several X-ray and cryo-EM structures of the extracellular and transmembrane segments of ASIC1, these important intracellular tails remain unresolved. Here, we describe the coarse topography of the chicken ASIC1 intracellular domains determined by fluorescence resonance energy transfer (FRET), measured using either fluorescent lifetime imaging or patch clamp fluorometry. We find the C terminal tail projects into the cytosol by approximately 35 Å and that the N and C tails from the same subunits are closer than adjacent subunits. Using pH-insensitive fluorescent proteins, we fail to detect any relative movement between the N and C tails upon extracellular acidification but do observe axial motions of the membrane proximal segments toward the plasma membrane. Taken together, our study furnishes a coarse topographic map of the ASIC intracellular domains while providing directionality and context to intracellular conformational changes induced by extracellular acidification.
Assuntos
Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/metabolismo , Motivos de Aminoácidos , Animais , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Galinhas , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Ativação do Canal IônicoRESUMO
ASIC1a is a proton-gated sodium channel involved in modulation of pain, fear, addiction, and ischemia-induced neuronal injury. We report isolation and characterization of alpaca-derived nanobodies (Nbs) that specifically target human ASIC1a. Cryo-electron microscopy of the human ASIC1a channel at pH 7.4 in complex with one of these, Nb.C1, yielded a structure at 2.9 Å resolution. It is revealed that Nb.C1 binds to a site overlapping with that of the Texas coral snake toxin (MitTx1) and the black mamba venom Mambalgin-1; however, the Nb.C1-binding site does not overlap with that of the inhibitory tarantula toxin psalmotoxin-1 (PcTx1). Fusion of Nb.C1 with PcTx1 in a single polypeptide markedly enhances the potency of PcTx1, whereas competition of Nb.C1 and MitTx1 for binding reduces channel activation by the toxin. Thus, Nb.C1 is a molecular tool for biochemical and structural studies of hASIC1a; a potential antidote to the pain-inducing component of coral snake bite; and a candidate to potentiate PcTx1-mediated inhibition of hASIC1a in vivo for therapeutic applications.
Assuntos
Canais Iônicos Sensíveis a Ácido/química , Anticorpos de Domínio Único/química , Canais Iônicos Sensíveis a Ácido/ultraestrutura , Animais , Camelídeos Americanos , Microscopia Crioeletrônica , Ligação Proteica , Anticorpos de Domínio Único/ultraestruturaRESUMO
Acid-sensing ion channels (ASICs) are widely expressed in the nervous system. The intracellular C terminus of ASIC1a has many sites involved in regulating its expression and the opening mechanism, but the role of the intracellular N-terminal domain is poorly understood. Here, we explored the correlation of ASIC1a intracellular N terminus with membrane expression and gate opening. We modified the N-terminal structure of ASICs by deletion/truncation/mutation strategies and transfected the recombinant plasmids into CHO cells. Protein expression was analyzed with immunofluorescence, Western blots, and patch-clamp experiments. Deleting the entire N terminus decreased the membrane expression of channel proteins, and ion channel opening was lost. Deleting sections of the N terminus also decreased membrane expression and suggested that all areas were significant, with no single or group of amino acid residues playing a decisive role in regulating ASIC1a membrane expression. In terms of gate opening, five amino acid (AA) residues from AA 16 to AA 20 participated in gate opening, and isoleucine at AA 18 was the most important. The whole N terminus of ASICs participates in the membrane expression of ASIC1a, and five amino acid residues (AA 16-20) are involved in the gate opening mechanism. SIGNIFICANCE STATEMENT: The whole N terminus of ASICs participates in the membrane expression of ASIC1a, and five amino acid resi-dues (amino acid 16-20) are involved in the gate opening mechanism.
Assuntos
Canais Iônicos Sensíveis a Ácido/química , Canais Iônicos Sensíveis a Ácido/metabolismo , Membrana Celular/metabolismo , Mutação , Canais Iônicos Sensíveis a Ácido/genética , Motivos de Aminoácidos , Animais , Células CHO , Cricetulus , Domínios Proteicos , TransfecçãoRESUMO
Acid-sensitive ion channels (ASICs) are sodium channels partially permeable to Ca2+ ions, listed among putative targets in central nervous system (CNS) diseases in which a pH modification occurs. We targeted novel compounds able to modulate ASIC1 and to reduce the progression of ischemic brain injury. We rationally designed and synthesized several diminazene-inspired diaryl mono- and bis-guanyl hydrazones. A correlation between their predicted docking affinities for the acidic pocket (AcP site) in chicken ASIC1 and their inhibition of homo- and heteromeric hASIC1 channels in HEK-293 cells was found. Their activity on murine ASIC1a currents and their selectivity vs mASIC2a were assessed in engineered CHO-K1 cells, highlighting a limited isoform selectivity. Neuroprotective effects were confirmed in vitro, on primary rat cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation, and in vivo, in ischemic mice. Early lead 3b, showing a good selectivity for hASIC1 in human neurons, was neuroprotective against focal ischemia induced in mice.