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1.
Cell ; 184(18): 4669-4679.e13, 2021 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-34390643

RESUMEN

Hearing involves two fundamental processes: mechano-electrical transduction and signal amplification. Despite decades of studies, the molecular bases for both remain elusive. Here, we show how prestin, the electromotive molecule of outer hair cells (OHCs) that senses both voltage and membrane tension, mediates signal amplification by coupling conformational changes to alterations in membrane surface area. Cryoelectron microscopy (cryo-EM) structures of human prestin bound with chloride or salicylate at a common "anion site" adopt contracted or expanded states, respectively. Prestin is ensconced within a perimeter of well-ordered lipids, through which it induces dramatic deformation in the membrane and couples protein conformational changes to the bulk membrane. Together with computational studies, we illustrate how the anion site is allosterically coupled to changes in the transmembrane domain cross-sectional area and the surrounding membrane. These studies provide insight into OHC electromotility by providing a structure-based mechanism of the membrane motor prestin.


Asunto(s)
Fenómenos Electrofisiológicos , Transportadores de Sulfato/metabolismo , Aniones , Sitios de Unión , Cloruros/metabolismo , Microscopía por Crioelectrón , Células HEK293 , Humanos , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Dominios Proteicos , Multimerización de Proteína , Estabilidad Proteica , Ácido Salicílico/metabolismo , Homología Estructural de Proteína , Transportadores de Sulfato/química , Transportadores de Sulfato/ultraestructura
2.
Cell ; 184(4): 957-968.e21, 2021 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-33567265

RESUMEN

Ligand-gated ion channels mediate signal transduction at chemical synapses and transition between resting, open, and desensitized states in response to neurotransmitter binding. Neurotransmitters that produce maximum open channel probabilities (Po) are full agonists, whereas those that yield lower than maximum Po are partial agonists. Cys-loop receptors are an important class of neurotransmitter receptors, yet a structure-based understanding of the mechanism of partial agonist action has proven elusive. Here, we study the glycine receptor with the full agonist glycine and the partial agonists taurine and γ-amino butyric acid (GABA). We use electrophysiology to show how partial agonists populate agonist-bound, closed channel states and cryo-EM reconstructions to illuminate the structures of intermediate, pre-open states, providing insights into previously unseen conformational states along the receptor reaction pathway. We further correlate agonist-induced conformational changes to Po across members of the receptor family, providing a hypothetical mechanism for partial and full agonist action at Cys-loop receptors.


Asunto(s)
Activación del Canal Iónico , Receptores de Glicina/agonistas , Receptores de Glicina/metabolismo , Animales , Sitios de Unión , Línea Celular , Microscopía por Crioelectrón , Glicina , Células HEK293 , Humanos , Imagenología Tridimensional , Maleatos/química , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Neurotransmisores/metabolismo , Dominios Proteicos , Receptores de Glicina/genética , Receptores de Glicina/ultraestructura , Estireno/química , Pez Cebra , Ácido gamma-Aminobutírico/metabolismo
3.
Cell ; 175(6): 1520-1532.e15, 2018 11 29.
Artículo en Inglés | MEDLINE | ID: mdl-30500536

RESUMEN

N-methyl-D-aspartate receptors (NMDARs) play essential roles in memory formation, neuronal plasticity, and brain development, with their dysfunction linked to a range of disorders from ischemia to schizophrenia. Zinc and pH are physiological allosteric modulators of NMDARs, with GluN2A-containing receptors inhibited by nanomolar concentrations of divalent zinc and by excursions to low pH. Despite the widespread importance of zinc and proton modulation of NMDARs, the molecular mechanism by which these ions modulate receptor activity has proven elusive. Here, we use cryoelectron microscopy to elucidate the structure of the GluN1/GluN2A NMDAR in a large ensemble of conformations under a range of physiologically relevant zinc and proton concentrations. We show how zinc binding to the amino terminal domain elicits structural changes that are transduced though the ligand-binding domain and result in constriction of the ion channel gate.


Asunto(s)
Complejos Multiproteicos/química , Protones , Receptores de N-Metil-D-Aspartato/química , Zinc/química , Regulación Alostérica , Animales , Microscopía por Crioelectrón , Concentración de Iones de Hidrógeno , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Dominios Proteicos , Ratas , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Células Sf9 , Spodoptera , Zinc/metabolismo
4.
Cell ; 170(6): 1234-1246.e14, 2017 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-28823560

RESUMEN

AMPA receptors mediate fast excitatory neurotransmission in the mammalian brain and transduce the binding of presynaptically released glutamate to the opening of a transmembrane cation channel. Within the postsynaptic density, however, AMPA receptors coassemble with transmembrane AMPA receptor regulatory proteins (TARPs), yielding a receptor complex with altered gating kinetics, pharmacology, and pore properties. Here, we elucidate structures of the GluA2-TARP γ2 complex in the presence of the partial agonist kainate or the full agonist quisqualate together with a positive allosteric modulator or with quisqualate alone. We show how TARPs sculpt the ligand-binding domain gating ring, enhancing kainate potency and diminishing the ensemble of desensitized states. TARPs encircle the receptor ion channel, stabilizing M2 helices and pore loops, illustrating how TARPs alter receptor pore properties. Structural and computational analysis suggests the full agonist and modulator complex harbors an ion-permeable channel gate, providing the first view of an activated AMPA receptor.


Asunto(s)
Canales de Calcio/química , Receptores AMPA/química , Animales , Microscopía por Crioelectrón , Agonistas de Aminoácidos Excitadores/química , Agonistas de Aminoácidos Excitadores/farmacología , Ácido Kaínico/química , Ácido Kaínico/farmacología , Modelos Moleculares , Ácido Quiscuálico/química , Ácido Quiscuálico/farmacología , Ratas , Receptores AMPA/agonistas
5.
Cell ; 165(3): 704-14, 2016 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-27062927

RESUMEN

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated, calcium-permeable ion channels that mediate synaptic transmission and underpin learning and memory. NMDAR dysfunction is directly implicated in diseases ranging from seizure to ischemia. Despite its fundamental importance, little is known about how the NMDAR transitions between inactive and active states and how small molecules inhibit or activate ion channel gating. Here, we report electron cryo-microscopy structures of the GluN1-GluN2B NMDA receptor in an ensemble of competitive antagonist-bound states, an agonist-bound form, and a state bound with agonists and the allosteric inhibitor Ro25-6981. Together with double electron-electron resonance experiments, we show how competitive antagonists rupture the ligand binding domain (LBD) gating "ring," how agonists retain the ring in a dimer-of-dimers configuration, and how allosteric inhibitors, acting within the amino terminal domain, further stabilize the LBD layer. These studies illuminate how the LBD gating ring is fundamental to signal transduction and gating in NMDARs.


Asunto(s)
Receptores de N-Metil-D-Aspartato/química , Proteínas de Xenopus/química , Animales , Microscopía por Crioelectrón , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Dominios Proteicos , Subunidades de Proteína/química , Receptores de N-Metil-D-Aspartato/agonistas , Xenopus laevis
6.
Cell ; 156(4): 717-29, 2014 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-24507937

RESUMEN

Acid-sensing ion channels (ASICs) detect extracellular protons produced during inflammation or ischemic injury and belong to the superfamily of degenerin/epithelial sodium channels. Here, we determine the cocrystal structure of chicken ASIC1a with MitTx, a pain-inducing toxin from the Texas coral snake, to define the structure of the open state of ASIC1a. In the MitTx-bound open state and in the previously determined low-pH desensitized state, TM2 is a discontinuous α helix in which the Gly-Ala-Ser selectivity filter adopts an extended, belt-like conformation, swapping the cytoplasmic one-third of TM2 with an adjacent subunit. Gly 443 residues of the selectivity filter provide a ring of three carbonyl oxygen atoms with a radius of ∼3.6 Å, presenting an energetic barrier for hydrated ions. The ASIC1a-MitTx complex illuminates the mechanism of MitTx action, defines the structure of the selectivity filter of voltage-independent, sodium-selective ion channels, and captures the open state of an ASIC.


Asunto(s)
Canales Iónicos Sensibles al Ácido/química , Proteínas Aviares/química , Pollos , Venenos Elapídicos/química , Elapidae , Canales Iónicos Sensibles al Ácido/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Aviares/metabolismo , Cristalografía por Rayos X , Venenos Elapídicos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Canales de Sodio/química
7.
Cell ; 158(4): 778-792, 2014 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-25109876

RESUMEN

Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory signaling in the nervous system. Despite the profound importance of iGluRs to neurotransmission, little is known about the structures and dynamics of intact receptors in distinct functional states. Here, we elucidate the structures of the intact GluA2 AMPA receptor in an apo resting/closed state, in an activated/pre-open state bound with partial agonists and a positive allosteric modulator, and in a desensitized/closed state in complex with fluorowilliardiine. To probe the conformational properties of these states, we carried out double electron-electron resonance experiments on cysteine mutants and cryoelectron microscopy studies. We show how agonist binding modulates the conformation of the ligand-binding domain "layer" of the intact receptors and how, upon desensitization, the receptor undergoes large conformational rearrangements of the amino-terminal and ligand-binding domains. We define mechanistic principles by which to understand antagonism, activation, and desensitization in AMPA iGluRs.


Asunto(s)
Receptores AMPA/química , Receptores AMPA/metabolismo , Animales , Microscopía por Crioelectrón , Cristalografía por Rayos X , Fluorouracilo/análogos & derivados , Fluorouracilo/metabolismo , Técnicas de Inactivación de Genes , Ácido Kaínico/metabolismo , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Ratas , Receptores AMPA/agonistas , Receptores AMPA/genética
8.
Nature ; 622(7981): 195-201, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37730991

RESUMEN

Type A γ-aminobutyric acid receptors (GABAARs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including anaesthetics, sedatives, hypnotics and antidepressants1-3. However, our understanding of GABAAR pharmacology has been hindered by the vast number of pentameric assemblies that can be derived from 19 different subunits4 and the lack of structural knowledge of clinically relevant receptors. Here, we isolate native murine GABAAR assemblies containing the widely expressed α1 subunit and elucidate their structures in complex with drugs used to treat insomnia (zolpidem (ZOL) and flurazepam) and postpartum depression (the neurosteroid allopregnanolone (APG)). Using cryo-electron microscopy (cryo-EM) analysis and single-molecule photobleaching experiments, we uncover three major structural populations in the brain: the canonical α1ß2γ2 receptor containing two α1 subunits, and two assemblies containing one α1 and either an α2 or α3 subunit, in which the single α1-containing receptors feature a more compact arrangement between the transmembrane and extracellular domains. Interestingly, APG is bound at the transmembrane α/ß subunit interface, even when not added to the sample, revealing an important role for endogenous neurosteroids in modulating native GABAARs. Together with structurally engaged lipids, neurosteroids produce global conformational changes throughout the receptor that modify the ion channel pore and the binding sites for GABA and insomnia medications. Our data reveal the major α1-containing GABAAR assemblies, bound with endogenous neurosteroid, thus defining a structural landscape from which subtype-specific drugs can be developed.


Asunto(s)
Microscopía por Crioelectrón , Neuroesteroides , Receptores de GABA-A , Ácido gamma-Aminobutírico , Animales , Ratones , Sitios de Unión/efectos de los fármacos , Depresión Posparto/tratamiento farmacológico , Flurazepam/farmacología , Ácido gamma-Aminobutírico/metabolismo , Hipnóticos y Sedantes/farmacología , Activación del Canal Iónico/efectos de los fármacos , Neuroesteroides/metabolismo , Neuroesteroides/farmacología , Fotoblanqueo , Pregnanolona/farmacología , Conformación Proteica/efectos de los fármacos , Subunidades de Proteína/química , Subunidades de Proteína/efectos de los fármacos , Subunidades de Proteína/metabolismo , Receptores de GABA-A/química , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/metabolismo , Receptores de GABA-A/ultraestructura , Trastornos del Inicio y del Mantenimiento del Sueño/tratamiento farmacológico , Zolpidem/farmacología
9.
Nature ; 610(7933): 796-803, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-36224384

RESUMEN

The initial step in the sensory transduction pathway underpinning hearing and balance in mammals involves the conversion of force into the gating of a mechanosensory transduction channel1. Despite the profound socioeconomic impacts of hearing disorders and the fundamental biological significance of understanding mechanosensory transduction, the composition, structure and mechanism of the mechanosensory transduction complex have remained poorly characterized. Here we report the single-particle cryo-electron microscopy structure of the native transmembrane channel-like protein 1 (TMC-1) mechanosensory transduction complex isolated from Caenorhabditis elegans. The two-fold symmetric complex is composed of two copies each of the pore-forming TMC-1 subunit, the calcium-binding protein CALM-1 and the transmembrane inner ear protein TMIE. CALM-1 makes extensive contacts with the cytoplasmic face of the TMC-1 subunits, whereas the single-pass TMIE subunits reside on the periphery of the complex, poised like the handles of an accordion. A subset of complexes additionally includes a single arrestin-like protein, arrestin domain protein (ARRD-6), bound to a CALM-1 subunit. Single-particle reconstructions and molecular dynamics simulations show how the mechanosensory transduction complex deforms the membrane bilayer and suggest crucial roles for lipid-protein interactions in the mechanism by which mechanical force is transduced to ion channel gating.


Asunto(s)
Caenorhabditis elegans , Microscopía por Crioelectrón , Canales Iónicos , Mecanotransducción Celular , Animales , Arrestinas/química , Arrestinas/metabolismo , Arrestinas/ultraestructura , Caenorhabditis elegans/química , Caenorhabditis elegans/ultraestructura , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/ultraestructura , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/ultraestructura , Activación del Canal Iónico , Canales Iónicos/química , Canales Iónicos/metabolismo , Canales Iónicos/ultraestructura , Lípidos
10.
Nature ; 599(7885): 513-517, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34555840

RESUMEN

Glycine receptors (GlyRs) are pentameric, 'Cys-loop' receptors that form chloride-permeable channels and mediate fast inhibitory signalling throughout the central nervous system1,2. In the spinal cord and brainstem, GlyRs regulate locomotion and cause movement disorders when mutated2,3. However, the stoichiometry of native GlyRs and the mechanism by which they are assembled remain unclear, despite extensive investigation4-8. Here we report cryo-electron microscopy structures of native GlyRs from pig spinal cord and brainstem, revealing structural insights into heteromeric receptors and their predominant subunit stoichiometry of 4α:1ß. Within the heteromeric pentamer, the ß(+)-α(-) interface adopts a structure that is distinct from the α(+)-α(-) and α(+)-ß(-) interfaces. Furthermore, the ß-subunit contains a unique phenylalanine residue that resides within the pore and disrupts the canonical picrotoxin site. These results explain why inclusion of the ß-subunit breaks receptor symmetry and alters ion channel pharmacology. We also find incomplete receptor complexes and, by elucidating their structures, reveal the architectures of partially assembled α-trimers and α-tetramers.


Asunto(s)
Microscopía por Crioelectrón , Receptores de Glicina/química , Receptores de Glicina/metabolismo , Animales , Tronco Encefálico , Modelos Moleculares , Fenilalanina/química , Fenilalanina/metabolismo , Picrotoxina/química , Picrotoxina/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Receptores de Glicina/ultraestructura , Médula Espinal , Porcinos
11.
Nature ; 594(7863): 448-453, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33981040

RESUMEN

AMPA-selective glutamate receptors mediate the transduction of signals between the neuronal circuits of the hippocampus1. The trafficking, localization, kinetics and pharmacology of AMPA receptors are tuned by an ensemble of auxiliary protein subunits, which are integral membrane proteins that associate with the receptor to yield bona fide receptor signalling complexes2. Thus far, extensive studies of recombinant AMPA receptor-auxiliary subunit complexes using engineered protein constructs have not been able to faithfully elucidate the molecular architecture of hippocampal AMPA receptor complexes. Here we obtain mouse hippocampal, calcium-impermeable AMPA receptor complexes using immunoaffinity purification and use single-molecule fluorescence and cryo-electron microscopy experiments to elucidate three major AMPA receptor-auxiliary subunit complexes. The GluA1-GluA2, GluA1-GluA2-GluA3 and GluA2-GluA3 receptors are the predominant assemblies, with the auxiliary subunits TARP-γ8 and CNIH2-SynDIG4 non-stochastically positioned at the B'/D' and A'/C' positions, respectively. We further demonstrate how the receptor-TARP-γ8 stoichiometry explains the mechanism of and submaximal inhibition by a clinically relevant, brain-region-specific allosteric inhibitor.


Asunto(s)
Hipocampo/metabolismo , Receptores AMPA/química , Receptores AMPA/metabolismo , Regulación Alostérica , Animales , Sitios de Unión , Canales de Calcio/química , Canales de Calcio/metabolismo , Canales de Calcio/ultraestructura , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas Portadoras/ultraestructura , Microscopía por Crioelectrón , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Modelos Moleculares , Receptores AMPA/ultraestructura
12.
Proc Natl Acad Sci U S A ; 121(8): e2314096121, 2024 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-38354260

RESUMEN

Mechanotransduction is the process by which a mechanical force, such as touch, is converted into an electrical signal. Transmembrane channel-like (TMC) proteins are an evolutionarily conserved family of membrane proteins whose function has been linked to a variety of mechanosensory processes, including hearing and balance sensation in vertebrates and locomotion in Drosophila. TMC1 and TMC2 are components of ion channel complexes, but the molecular features that tune these complexes to diverse mechanical stimuli are unknown. Caenorhabditis elegans express two TMC homologs, TMC-1 and TMC-2, both of which are the likely pore-forming subunits of mechanosensitive ion channels but differ in their expression pattern and functional role in the worm. Here, we present the single-particle cryo-electron microscopy structure of the native TMC-2 complex isolated from C. elegans. The complex is composed of two copies of the pore-forming TMC-2 subunit, the calcium and integrin binding protein CALM-1 and the transmembrane inner ear protein TMIE. Comparison of the TMC-2 complex to the recently published cryo-EM structure of the C. elegans TMC-1 complex highlights conserved protein-lipid interactions, as well as a π-helical structural motif in the pore-forming helices, that together suggest a mechanism for TMC-mediated mechanosensory transduction.


Asunto(s)
Proteínas de Caenorhabditis elegans , Mecanotransducción Celular , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Microscopía por Crioelectrón , Canales Iónicos/metabolismo , Lípidos , Mecanotransducción Celular/fisiología , Proteínas de la Membrana/metabolismo
13.
Proc Natl Acad Sci U S A ; 120(29): e2304602120, 2023 07 18.
Artículo en Inglés | MEDLINE | ID: mdl-37436958

RESUMEN

The serotonin transporter (SERT) is a member of the SLC6 neurotransmitter transporter family that mediates serotonin reuptake at presynaptic nerve terminals. SERT is the target of both therapeutic antidepressant drugs and psychostimulant substances such as cocaine and methamphetamines, which are small molecules that perturb normal serotonergic transmission by interfering with serotonin transport. Despite decades of studies, important functional aspects of SERT such as the oligomerization state of native SERT and its interactions with potential proteins remain unresolved. Here, we develop methods to isolate SERT from porcine brain (pSERT) using a mild, nonionic detergent, utilize fluorescence-detection size-exclusion chromatography to investigate its oligomerization state and interactions with other proteins, and employ single-particle cryo-electron microscopy to elucidate the structures of pSERT in complexes with methamphetamine or cocaine, providing structural insights into psychostimulant recognition and accompanying pSERT conformations. Methamphetamine and cocaine both bind to the central site, stabilizing the transporter in an outward open conformation. We also identify densities attributable to multiple cholesterol or cholesteryl hemisuccinate (CHS) molecules, as well as to a detergent molecule bound to the pSERT allosteric site. Under our conditions of isolation, we find that pSERT is best described as a monomeric entity, isolated without interacting proteins, and is ensconced by multiple cholesterol or CHS molecules.


Asunto(s)
Estimulantes del Sistema Nervioso Central , Cocaína , Metanfetamina , Animales , Porcinos , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Microscopía por Crioelectrón , Detergentes , Serotonina , Cocaína/farmacología , Metanfetamina/farmacología
14.
Nature ; 569(7754): 141-145, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-31019304

RESUMEN

The serotonin transporter (SERT) regulates neurotransmitter homeostasis through the sodium- and chloride-dependent recycling of serotonin into presynaptic neurons1-3. Major depression and anxiety disorders are treated using selective serotonin reuptake inhibitors-small molecules that competitively block substrate binding and thereby prolong neurotransmitter action2,4. The dopamine and noradrenaline transporters, together with SERT, are members of the neurotransmitter sodium symporter (NSS) family. The transport activities of NSSs can be inhibited or modulated by cocaine and amphetamines2,3, and genetic variants of NSSs are associated with several neuropsychiatric disorders including attention deficit hyperactivity disorder, autism and bipolar disorder2,5. Studies of bacterial NSS homologues-including LeuT-have shown how their transmembrane helices (TMs) undergo conformational changes during the transport cycle, exposing a central binding site to either side of the membrane1,6-12. However, the conformational changes associated with transport in NSSs remain unknown. To elucidate structure-based mechanisms for transport in SERT we investigated its complexes with ibogaine, a hallucinogenic natural product with psychoactive and anti-addictive properties13,14. Notably, ibogaine is a non-competitive inhibitor of transport but displays competitive binding towards selective serotonin reuptake inhibitors15,16. Here we report cryo-electron microscopy structures of SERT-ibogaine complexes captured in outward-open, occluded and inward-open conformations. Ibogaine binds to the central binding site, and closure of the extracellular gate largely involves movements of TMs 1b and 6a. Opening of the intracellular gate involves a hinge-like movement of TM1a and the partial unwinding of TM5, which together create a permeation pathway that enables substrate and ion diffusion to the cytoplasm. These structures define the structural rearrangements that occur from the outward-open to inward-open conformations, and provide insight into the mechanism of neurotransmitter transport and ibogaine inhibition.


Asunto(s)
Microscopía por Crioelectrón , Ibogaína/química , Ibogaína/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/ultraestructura , Serotonina/metabolismo , Sitios de Unión/efectos de los fármacos , Unión Competitiva , Transporte Biológico/efectos de los fármacos , Alucinógenos/química , Alucinógenos/farmacología , Humanos , Modelos Moleculares , Conformación Proteica/efectos de los fármacos , Proteínas de Transporte de Serotonina en la Membrana Plasmática/química , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Inhibidores Selectivos de la Recaptación de Serotonina/química , Relación Estructura-Actividad
15.
Nature ; 555(7696): 397-401, 2018 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-29513651

RESUMEN

Acid-sensing ion channels (ASICs) are trimeric, proton-gated and sodium-selective members of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily of ion channels and are expressed throughout vertebrate central and peripheral nervous systems. Gating of ASICs occurs on a millisecond time scale and the mechanism involves three conformational states: high pH resting, low pH open and low pH desensitized. Existing X-ray structures of ASIC1a describe the conformations of the open and desensitized states, but the structure of the high pH resting state and detailed mechanisms of the activation and desensitization of the channel have remained elusive. Here we present structures of the high pH resting state of homotrimeric chicken (Gallus gallus) ASIC1a, determined by X-ray crystallography and single particle cryo-electron microscopy, and present a comprehensive molecular mechanism for proton-dependent gating in ASICs. In the resting state, the position of the thumb domain is further from the three-fold molecular axis, thereby expanding the 'acidic pocket' in comparison to the open and desensitized states. Activation therefore involves 'closure' of the thumb into the acidic pocket, expansion of the lower palm domain and an iris-like opening of the channel gate. Furthermore, we demonstrate how the ß11-ß12 linkers that demarcate the upper and lower palm domains serve as a molecular 'clutch', and undergo a simple rearrangement to permit rapid desensitization.


Asunto(s)
Canales Iónicos Sensibles al Ácido/química , Canales Iónicos Sensibles al Ácido/metabolismo , Microscopía por Crioelectrón , Canales Iónicos Sensibles al Ácido/ultraestructura , Animales , Sitios de Unión , Células CHO , Pollos , Cricetulus , Cristalografía por Rayos X , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Activación del Canal Iónico , Modelos Moleculares , Dominios Proteicos , Protones , Células Sf9 , Spodoptera
16.
Nature ; 556(7702): 515-519, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29670280

RESUMEN

The NMDA (N-methyl-D-aspartate) receptor transduces the binding of glutamate and glycine, coupling it to the opening of a calcium-permeable ion channel 1 . Owing to the lack of high-resolution structural studies of the NMDA receptor, the mechanism by which ion-channel blockers occlude ion permeation is not well understood. Here we show that removal of the amino-terminal domains from the GluN1-GluN2B NMDA receptor yields a functional receptor and crystals with good diffraction properties, allowing us to map the binding site of the NMDA receptor blocker, MK-801. This crystal structure, together with long-timescale molecular dynamics simulations, shows how MK-801 and memantine (a drug approved for the treatment of Alzheimer's disease) bind within the vestibule of the ion channel, promote closure of the ion channel gate and lodge between the M3-helix-bundle crossing and the M2-pore loops, physically blocking ion permeation.


Asunto(s)
Maleato de Dizocilpina/farmacología , Activación del Canal Iónico/efectos de los fármacos , Memantina/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Sitios de Unión , Cristalografía por Rayos X , Maleato de Dizocilpina/química , Memantina/química , Simulación de Dinámica Molecular , Dominios Proteicos , Receptores AMPA/química , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Especificidad por Sustrato , Xenopus
17.
J Biol Chem ; 297(1): 100863, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34118233

RESUMEN

The serotonin transporter (SERT) shapes serotonergic neurotransmission by retrieving its eponymous substrate from the synaptic cleft. Ligands that discriminate between SERT and its close relative, the dopamine transporter DAT, differ in their association rate constant rather than their dissociation rate. The structural basis for this phenomenon is not known. Here we examined the hypothesis that the extracellular loops 2 (EL2) and 4 (EL4) limit access to the ligand-binding site of SERT. We employed an antibody directed against EL4 (residues 388-400) and the antibody fragments 8B6 scFv (directed against EL2 and EL4) and 15B8 Fab (directed against EL2) and analyzed their effects on the transport cycle of and inhibitor binding to SERT. Electrophysiological recordings showed that the EL4 antibody and 8B6 scFv impeded the initial substrate-induced transition from the outward to the inward-facing conformation but not the forward cycling mode of SERT. In contrast, binding of radiolabeled inhibitors to SERT was enhanced by either EL4- or EL2-directed antibodies. We confirmed this observation by determining the association and dissociation rate of the DAT-selective inhibitor methylphenidate via electrophysiological recordings; occupancy of EL2 with 15B8 Fab enhanced the affinity of SERT for methylphenidate by accelerating its binding. Based on these observations, we conclude that (i) EL4 undergoes a major movement during the transition from the outward to the inward-facing state, and (ii) EL2 and EL4 limit access of inhibitors to the binding of SERT, thus acting as a selectivity filter. This insight has repercussions for drug development.


Asunto(s)
Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Proteínas de Transporte de Membrana/genética , Conformación Proteica/efectos de los fármacos , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Secuencia de Aminoácidos/genética , Animales , Sitios de Unión/efectos de los fármacos , Células COS , Chlorocebus aethiops , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/antagonistas & inhibidores , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/ultraestructura , Células HEK293 , Humanos , Ligandos , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/ultraestructura , Técnicas de Placa-Clamp , Dominios Proteicos/genética , Serotonina/química , Serotonina/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/efectos de los fármacos , Proteínas de Transporte de Serotonina en la Membrana Plasmática/ultraestructura , Inhibidores Selectivos de la Recaptación de Serotonina/química
18.
J Biol Chem ; 296: 100387, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33617876

RESUMEN

Like other pentameric ligand-gated channels, glycine receptors (GlyRs) contain long intracellular domains (ICDs) between transmembrane helices 3 and 4. Structurally characterized GlyRs are generally engineered to have a very short ICD. We show here that for one such construct, zebrafish GlyREM, the agonists glycine, ß-alanine, taurine, and GABA have high efficacy and produce maximum single-channel open probabilities greater than 0.9. In contrast, for full-length human α1 GlyR, taurine and GABA were clearly partial agonists, with maximum open probabilities of 0.46 and 0.09, respectively. We found that the elevated open probabilities in GlyREM are not due to the limited sequence differences between the human and zebrafish orthologs, but rather to replacement of the native ICD with a short tripeptide ICD. Consistent with this interpretation, shortening the ICD in the human GlyR increased the maximum open probability produced by taurine and GABA to 0.90 and 0.70, respectively, but further engineering it to resemble GlyREM (by introducing the zebrafish transmembrane helix 4 and C terminus) had no effect. Furthermore, reinstating the native ICD to GlyREM converted taurine and GABA to partial agonists, with maximum open probabilities of 0.66 and 0.40, respectively. Structural comparison of transmembrane helices 3 and 4 in short- and long-ICD GlyR subunits revealed that ICD shortening does not distort the orientation of these helices within each subunit. This suggests that the effects of shortening the ICD stem from removing a modulatory effect of the native ICD on GlyR gating, revealing a new role for the ICD in pentameric ligand-gated channels.


Asunto(s)
Glicina/farmacología , Receptores de Glicina/agonistas , Taurina/farmacología , beta-Alanina/farmacología , Ácido gamma-Aminobutírico/farmacología , Secuencia de Aminoácidos , Animales , Células Cultivadas , GABAérgicos/farmacología , Glicinérgicos/farmacología , Humanos , Técnicas de Placa-Clamp/métodos , Dominios Proteicos , Receptores de Glicina/metabolismo , Relación Estructura-Actividad , Pez Cebra
19.
Nature ; 532(7599): 334-9, 2016 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-27049939

RESUMEN

The serotonin transporter (SERT) terminates serotonergic signalling through the sodium- and chloride-dependent reuptake of neurotransmitter into presynaptic neurons. SERT is a target for antidepressant and psychostimulant drugs, which block reuptake and prolong neurotransmitter signalling. Here we report X-ray crystallographic structures of human SERT at 3.15 Å resolution bound to the antidepressants (S)-citalopram or paroxetine. Antidepressants lock SERT in an outward-open conformation by lodging in the central binding site, located between transmembrane helices 1, 3, 6, 8 and 10, directly blocking serotonin binding. We further identify the location of an allosteric site in the complex as residing at the periphery of the extracellular vestibule, interposed between extracellular loops 4 and 6 and transmembrane helices 1, 6, 10 and 11. Occupancy of the allosteric site sterically hinders ligand unbinding from the central site, providing an explanation for the action of (S)-citalopram as an allosteric ligand. These structures define the mechanism of antidepressant action in SERT, and provide blueprints for future drug design.


Asunto(s)
Proteínas de Transporte de Serotonina en la Membrana Plasmática/química , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Antidepresivos/química , Antidepresivos/metabolismo , Antidepresivos/farmacología , Citalopram/química , Citalopram/metabolismo , Citalopram/farmacología , Cristalografía por Rayos X , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/química , Diseño de Fármacos , Espacio Extracelular/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Espacio Intracelular/metabolismo , Iones/química , Iones/metabolismo , Ligandos , Modelos Moleculares , Paroxetina/química , Paroxetina/metabolismo , Paroxetina/farmacología , Unión Proteica/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Estabilidad Proteica , Serotonina/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/inmunología , Relación Estructura-Actividad
20.
Nature ; 536(7614): 108-11, 2016 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-27368053

RESUMEN

Fast excitatory neurotransmission in the mammalian central nervous system is largely carried out by AMPA-sensitive ionotropic glutamate receptors. Localized within the postsynaptic density of glutamatergic spines, AMPA receptors are composed of heterotetrameric receptor assemblies associated with auxiliary subunits, the most common of which are transmembrane AMPA receptor regulatory proteins (TARPs). The association of TARPs with AMPA receptors modulates receptor trafficking and the kinetics of receptor gating and pharmacology. Here we report the cryo-electron microscopy (cryo-EM) structure of the homomeric rat GluA2 AMPA receptor saturated with TARP γ2 subunits, which shows how the TARPs are arranged with four-fold symmetry around the ion channel domain and make extensive interactions with the M1, M2 and M4 transmembrane helices. Poised like partially opened 'hands' underneath the two-fold symmetric ligand-binding domain (LBD) 'clamshells', one pair of TARPs is juxtaposed near the LBD dimer interface, whereas the other pair is near the LBD dimer-dimer interface. The extracellular 'domains' of TARP are positioned to not only modulate LBD clamshell closure, but also affect conformational rearrangements of the LBD layer associated with receptor activation and desensitization, while the TARP transmembrane domains buttress the ion channel pore.


Asunto(s)
Canales de Calcio/metabolismo , Canales de Calcio/ultraestructura , Microscopía por Crioelectrón , Receptores AMPA/metabolismo , Receptores AMPA/ultraestructura , Animales , Canales de Calcio/química , Activación del Canal Iónico , Modelos Moleculares , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Ratas , Receptores AMPA/química
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