RESUMEN
ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.
Asunto(s)
Channelrhodopsins/química , Channelrhodopsins/metabolismo , Activación del Canal Iónico , Animales , Channelrhodopsins/ultraestructura , Microscopía por Crioelectrón , Femenino , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Modelos Moleculares , Optogenética , Filogenia , Ratas Sprague-Dawley , Bases de Schiff/química , Células Sf9 , Relación Estructura-ActividadRESUMEN
Many transient receptor potential (TRP) channels respond to diverse stimuli and conditionally conduct small and large cations. Such functional plasticity is presumably enabled by a uniquely dynamic ion selectivity filter that is regulated by physiological agents. What is currently missing is a "photo series" of intermediate structural states that directly address this hypothesis and reveal specific mechanisms behind such dynamic channel regulation. Here, we exploit cryoelectron microscopy (cryo-EM) to visualize conformational transitions of the capsaicin receptor, TRPV1, as a model to understand how dynamic transitions of the selectivity filter in response to algogenic agents, including protons, vanilloid agonists, and peptide toxins, permit permeation by small and large organic cations. These structures also reveal mechanisms governing ligand binding substates, as well as allosteric coupling between key sites that are proximal to the selectivity filter and cytoplasmic gate. These insights suggest a general framework for understanding how TRP channels function as polymodal signal integrators.
Asunto(s)
Canales Catiónicos TRPV/química , Canales Catiónicos TRPV/metabolismo , Regulación Alostérica , Permeabilidad de la Membrana Celular/efectos de los fármacos , Microscopía por Crioelectrón , Diterpenos/farmacología , Células HEK293 , Humanos , Activación del Canal Iónico , Lípidos/química , Meglumina/farmacología , Modelos Moleculares , Unión Proteica , Conformación Proteica , Protones , Canales Catiónicos TRPV/agonistasRESUMEN
The heartbeat is initiated by voltage-gated sodium channel NaV1.5, which opens rapidly and triggers the cardiac action potential; however, the structural basis for pore opening remains unknown. Here, we blocked fast inactivation with a mutation and captured the elusive open-state structure. The fast inactivation gate moves away from its receptor, allowing asymmetric opening of pore-lining S6 segments, which bend and rotate at their intracellular ends to dilate the activation gate to â¼10 Å diameter. Molecular dynamics analyses predict physiological rates of Na+ conductance. The open-state pore blocker propafenone binds in a high-affinity pose, and drug-access pathways are revealed through the open activation gate and fenestrations. Comparison with mutagenesis results provides a structural map of arrhythmia mutations that target the activation and fast inactivation gates. These results give atomic-level insights into molecular events that underlie generation of the action potential, open-state drug block, and fast inactivation of cardiac sodium channels, which initiate the heartbeat.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.5/química , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Animales , Arritmias Cardíacas/genética , Microscopía por Crioelectrón , Células HEK293 , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Activación del Canal Iónico , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación/genética , Miocardio , Canal de Sodio Activado por Voltaje NAV1.5/aislamiento & purificación , Canal de Sodio Activado por Voltaje NAV1.5/ultraestructura , Propafenona/farmacología , Conformación Proteica , Ratas , Sodio/metabolismo , Factores de Tiempo , Agua/químicaRESUMEN
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/metabolismoRESUMEN
KCNQ1, also known as Kv7.1, is a voltage-dependent K+ channel that regulates gastric acid secretion, salt and glucose homeostasis, and heart rhythm. Its functional properties are regulated in a tissue-specific manner through co-assembly with beta subunits KCNE1-5. In non-excitable cells, KCNQ1 forms a complex with KCNE3, which suppresses channel closure at negative membrane voltages that otherwise would close it. Pore opening is regulated by the signaling lipid PIP2. Using cryoelectron microscopy (cryo-EM), we show that KCNE3 tucks its single-membrane-spanning helix against KCNQ1, at a location that appears to lock the voltage sensor in its depolarized conformation. Without PIP2, the pore remains closed. Upon addition, PIP2 occupies a site on KCNQ1 within the inner membrane leaflet, which triggers a large conformational change that leads to dilation of the pore's gate. It is likely that this mechanism of PIP2 activation is conserved among Kv7 channels.
Asunto(s)
Canal de Potasio KCNQ1/metabolismo , Canal de Potasio KCNQ1/ultraestructura , Microscopía por Crioelectrón , Humanos , Activación del Canal Iónico/fisiología , Canal de Potasio KCNQ1/química , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/metabolismo , Canales de Potasio con Entrada de Voltaje/ultraestructuraRESUMEN
Voltage-gated sodium channel Nav1.5 generates cardiac action potentials and initiates the heartbeat. Here, we report structures of NaV1.5 at 3.2-3.5 Å resolution. NaV1.5 is distinguished from other sodium channels by a unique glycosyl moiety and loss of disulfide-bonding capability at the NaVß subunit-interaction sites. The antiarrhythmic drug flecainide specifically targets the central cavity of the pore. The voltage sensors are partially activated, and the fast-inactivation gate is partially closed. Activation of the voltage sensor of Domain III allows binding of the isoleucine-phenylalanine-methionine (IFM) motif to the inactivation-gate receptor. Asp and Ala, in the selectivity motif DEKA, line the walls of the ion-selectivity filter, whereas Glu and Lys are in positions to accept and release Na+ ions via a charge-delocalization network. Arrhythmia mutation sites undergo large translocations during gating, providing a potential mechanism for pathogenic effects. Our results provide detailed insights into Nav1.5 structure, pharmacology, activation, inactivation, ion selectivity, and arrhythmias.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/ultraestructura , Animales , Línea Celular , Células HEK293 , Corazón/fisiología , Humanos , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp/métodos , Ratas , Sodio/metabolismo , Canales de Sodio/química , Relación Estructura-Actividad , Canales de Sodio Activados por Voltaje/metabolismo , Canales de Sodio Activados por Voltaje/ultraestructuraRESUMEN
Mitochondrial calcium uptake is crucial to the regulation of eukaryotic Ca2+ homeostasis and is mediated by the mitochondrial calcium uniporter (MCU). While MCU alone can transport Ca2+ in primitive eukaryotes, metazoans require an essential single membrane-spanning auxiliary component called EMRE to form functional channels; however, the molecular mechanism of EMRE regulation remains elusive. Here, we present the cryo-EM structure of the human MCU-EMRE complex, which defines the interactions between MCU and EMRE as well as pinpoints the juxtamembrane loop of MCU and extended linker of EMRE as the crucial elements in the EMRE-dependent gating mechanism among metazoan MCUs. The structure also features the dimerization of two MCU-EMRE complexes along an interface at the N-terminal domain (NTD) of human MCU that is a hotspot for post-translational modifications. Thus, the human MCU-EMRE complex, which constitutes the minimal channel components among metazoans, provides a framework for future mechanistic studies on MCU.
Asunto(s)
Canales de Calcio/metabolismo , Activación del Canal Iónico/fisiología , Complejos Multiproteicos/metabolismo , Multimerización de Proteína/fisiología , Canales de Calcio/genética , Células HEK293 , Humanos , Complejos Multiproteicos/genética , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
The hyperpolarization-activated cyclic nucleotide-gated (HCN) channel is a voltage-gated cation channel that mediates neuronal and cardiac pacemaker activity. The HCN channel exhibits reversed voltage dependence, meaning it closes with depolarization and opens with hyperpolarization. Different from Na+, Ca2+, and Kv1-Kv7 channels, the HCN channel does not have domain-swapped voltage sensors. We introduced a reversible, metal-mediated cross bridge into the voltage sensors to create the chemical equivalent of a hyperpolarized conformation and determined the structure using cryoelectron microscopy (cryo-EM). Unlike the depolarized HCN channel, the S4 helix is displaced toward the cytoplasm by two helical turns. Near the cytoplasm, the S4 helix breaks into two helices, one running parallel to the membrane surface, analogous to the S4-S5 linker of domain-swapped voltage-gated channels. These findings suggest a basis for allosteric communication between voltage sensors and the gate in this kind of channel. They also imply that voltage sensor movements are not the same in all voltage-gated channels.
Asunto(s)
Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/química , Activación del Canal Iónico , Animales , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Potenciales de la Membrana , Conformación Proteica en Hélice alfa , Células Sf9 , SpodopteraRESUMEN
Voltage-gated sodium (NaV) channels initiate action potentials in nerve, muscle, and other electrically excitable cells. The structural basis of voltage gating is uncertain because the resting state exists only at deeply negative membrane potentials. To stabilize the resting conformation, we inserted voltage-shifting mutations and introduced a disulfide crosslink in the VS of the ancestral bacterial sodium channel NaVAb. Here, we present a cryo-EM structure of the resting state and a complete voltage-dependent gating mechanism. The S4 segment of the VS is drawn intracellularly, with three gating charges passing through the transmembrane electric field. This movement forms an elbow connecting S4 to the S4-S5 linker, tightens the collar around the S6 activation gate, and prevents its opening. Our structure supports the classical "sliding helix" mechanism of voltage sensing and provides a complete gating mechanism for voltage sensor function, pore opening, and activation-gate closure based on high-resolution structures of a single sodium channel protein.
Asunto(s)
Potenciales de Acción/fisiología , Membrana Externa Bacteriana/metabolismo , Escherichia coli/metabolismo , Activación del Canal Iónico/fisiología , Canales de Sodio Activados por Voltaje/metabolismo , Animales , Línea Celular , Microscopía por Crioelectrón , Cristalografía por Rayos X , Mutación , Conformación Proteica en Hélice alfa , Sodio/metabolismo , Spodoptera/citología , Canales de Sodio Activados por Voltaje/químicaRESUMEN
Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and toxin modulation remains ill-defined. Protoxin-II (ProTx2) from the Peruvian green velvet tarantula is an inhibitor cystine-knot peptide and selective antagonist of the human Nav1.7 channel. Here, we visualize ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7 using X-ray crystallography and cryoelectron microscopy. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule to antagonize S4 gating-charge movement through an electrostatic mechanism. ProTx2 has trapped activated and deactivated states of VSD2, revealing a remarkable â¼10 Å translation of the S4 helix, providing a structural framework for activation gating in voltage-gated ion channels. Finally, our results deliver key templates to design selective Nav channel antagonists.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/ultraestructura , Péptidos/metabolismo , Venenos de Araña/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Cricetulus , Microscopía por Crioelectrón/métodos , Cristalografía por Rayos X/métodos , Células HEK293 , Humanos , Activación del Canal Iónico , Péptidos/toxicidad , Dominios Proteicos , Venenos de Araña/toxicidad , Arañas , Bloqueadores del Canal de Sodio Activado por Voltaje , Canales de Sodio Activados por Voltaje/metabolismoRESUMEN
Ion channel families are broadly classified into three types according to their major mechanisms of activation. This SnapShot highlights features of these three classes and conveys general modes of channel regulation. To view this SnapShot, open or download the PDF.
Asunto(s)
Activación del Canal Iónico , Canales Iónicos/genética , Canales Iónicos/metabolismo , Animales , Membrana Celular/metabolismo , Regulación de la Expresión Génica , Humanos , Canales Iónicos/químicaRESUMEN
The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca(2+)) release channel required for skeletal muscle contraction. Here, we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca(2+), ATP, and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca(2+) alone induces conformational changes in the cytoplasmic assembly ("priming"), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement, and deformation of the S4-S5 linker and conformational changes in the pseudo-voltage-sensor domain.
Asunto(s)
Agonistas de los Canales de Calcio/química , Activación del Canal Iónico , Contracción Muscular , Canal Liberador de Calcio Receptor de Rianodina/química , Animales , Sitios de Unión , Cafeína/química , Calcio/química , Microscopía por Crioelectrón , Ligandos , Dominios Proteicos , Conejos , Proteínas de Unión a Tacrolimus/químicaRESUMEN
Modulation of large conductance intracellular ligand-activated potassium (BK) channel family (Slo1-3) by auxiliary subunits allows diverse physiological functions in excitable and non-excitable cells. Cryoelectron microscopy (cryo-EM) structures of voltage-gated potassium (Kv) channel complexes have provided insights into how voltage sensitivity is modulated by auxiliary subunits. However, the modulation mechanisms of BK channels, particularly as ligand-activated ion channels, remain unknown. Slo1 is a Ca2+-activated and voltage-gated BK channel and is expressed in neurons, muscle cells, and epithelial cells. Using cryo-EM and electrophysiology, we show that the LRRC26-γ1 subunit modulates not only voltage but also Ca2+ sensitivity of Homo sapiens Slo1. LRRC26 stabilizes the active conformation of voltage-senor domains of Slo1 by an extracellularly S4-locking mechanism. Furthermore, it also stabilizes the active conformation of Ca2+-sensor domains of Slo1 intracellularly, which is functionally equivalent to intracellular Ca2+ in the activation of Slo1. Such a dual allosteric modulatory mechanism may be general in regulating the intracellular ligand-activated BK channel complexes.
Asunto(s)
Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio , Humanos , Calcio/metabolismo , Microscopía por Crioelectrón , Activación del Canal Iónico/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Ligandos , Potasio , Regulación AlostéricaRESUMEN
Temperature profoundly affects macromolecular function, particularly in proteins with temperature sensitivity1,2. However, its impact is often overlooked in biophysical studies that are typically performed at non-physiological temperatures, potentially leading to inaccurate mechanistic and pharmacological insights. Here we demonstrate temperature-dependent changes in the structure and function of TRPM4, a temperature-sensitive Ca2+-activated ion channel3-7. By studying TRPM4 prepared at physiological temperature using single-particle cryo-electron microscopy, we identified a 'warm' conformation that is distinct from those observed at lower temperatures. This conformation is driven by a temperature-dependent Ca2+-binding site in the intracellular domain, and is essential for TRPM4 function in physiological contexts. We demonstrated that ligands, exemplified by decavanadate (a positive modulator)8 and ATP (an inhibitor)9, bind to different locations of TRPM4 at physiological temperatures than at lower temperatures10,11, and that these sites have bona fide functional relevance. We elucidated the TRPM4 gating mechanism by capturing structural snapshots of its different functional states at physiological temperatures, revealing the channel opening that is not observed at lower temperatures. Our study provides an example of temperature-dependent ligand recognition and modulation of an ion channel, underscoring the importance of studying macromolecules at physiological temperatures. It also provides a potential molecular framework for deciphering how thermosensitive TRPM channels perceive temperature changes.
Asunto(s)
Activación del Canal Iónico , Canales Catiónicos TRPM , Temperatura , Humanos , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Sitios de Unión , Calcio/metabolismo , Microscopía por Crioelectrón , Células HEK293 , Activación del Canal Iónico/efectos de los fármacos , Ligandos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Especificidad por Sustrato , Canales Catiónicos TRPM/agonistas , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPM/química , Canales Catiónicos TRPM/metabolismo , Vanadatos/química , Vanadatos/farmacología , Vanadatos/metabolismoRESUMEN
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels1 are essential for pacemaking activity and neural signalling2,3. Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain4 and epileptic seizures5. The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor6 with unknown structural basis. Here, using single-particle cryo-electron microscopy and electrophysiology, we show that propofol inhibits HCN1 by binding to a mechanistic hotspot in a groove between the S5 and S6 transmembrane helices. We found that propofol restored voltage-dependent closing in two HCN1 epilepsy-associated polymorphisms that act by destabilizing the channel closed state: M305L, located in the propofol-binding site in S5, and D401H in S6 (refs. 7,8). To understand the mechanism of propofol inhibition and restoration of voltage-gating, we tracked voltage-sensor movement in spHCN channels and found that propofol inhibition is independent of voltage-sensor conformational changes. Mutations at the homologous methionine in spHCN and an adjacent conserved phenylalanine in S6 similarly destabilize closing without disrupting voltage-sensor movements, indicating that voltage-dependent closure requires this interface intact. We propose a model for voltage-dependent gating in which propofol stabilizes coupling between the voltage sensor and pore at this conserved methionine-phenylalanine interface in HCN channels. These findings unlock potential exploitation of this site to design specific drugs targeting HCN channelopathies.
Asunto(s)
Epilepsia , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Activación del Canal Iónico , Mutación , Canales de Potasio , Propofol , Humanos , Sitios de Unión , Microscopía por Crioelectrón , Electrofisiología , Epilepsia/tratamiento farmacológico , Epilepsia/genética , Epilepsia/metabolismo , Células HEK293 , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/antagonistas & inhibidores , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/química , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/ultraestructura , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/genética , Metionina/genética , Metionina/metabolismo , Modelos Moleculares , Movimiento/efectos de los fármacos , Fenilalanina/genética , Fenilalanina/metabolismo , Polimorfismo Genético , Canales de Potasio/química , Canales de Potasio/genética , Canales de Potasio/metabolismo , Canales de Potasio/ultraestructura , Propofol/farmacología , Propofol/químicaRESUMEN
Glutamate transmission and activation of ionotropic glutamate receptors are the fundamental means by which neurons control their excitability and neuroplasticity1. The N-methyl-D-aspartate receptor (NMDAR) is unique among all ligand-gated channels, requiring two ligands-glutamate and glycine-for activation. These receptors function as heterotetrameric ion channels, with the channel opening dependent on the simultaneous binding of glycine and glutamate to the extracellular ligand-binding domains (LBDs) of the GluN1 and GluN2 subunits, respectively2,3. The exact molecular mechanism for channel gating by the two ligands has been unclear, particularly without structures representing the open channel and apo states. Here we show that the channel gate opening requires tension in the linker connecting the LBD and transmembrane domain (TMD) and rotation of the extracellular domain relative to the TMD. Using electron cryomicroscopy, we captured the structure of the GluN1-GluN2B (GluN1-2B) NMDAR in its open state bound to a positive allosteric modulator. This process rotates and bends the pore-forming helices in GluN1 and GluN2B, altering the symmetry of the TMD channel from pseudofourfold to twofold. Structures of GluN1-2B NMDAR in apo and single-liganded states showed that binding of either glycine or glutamate alone leads to distinct GluN1-2B dimer arrangements but insufficient tension in the LBD-TMD linker for channel opening. This mechanistic framework identifies a key determinant for channel gating and a potential pharmacological strategy for modulating NMDAR activity.
Asunto(s)
Ácido Glutámico , Glicina , Activación del Canal Iónico , Receptores de N-Metil-D-Aspartato , Animales , Ratas , Regulación Alostérica , Microscopía por Crioelectrón , Ácido Glutámico/metabolismo , Glicina/metabolismo , Ligandos , Modelos Moleculares , Oocitos/metabolismo , Dominios Proteicos , Multimerización de Proteína , Subunidades de Proteína/metabolismo , Subunidades de Proteína/química , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/ultraestructura , Rotación , Xenopus laevisRESUMEN
Kainate receptors, a subclass of ionotropic glutamate receptors, are tetrameric ligand-gated ion channels that mediate excitatory neurotransmission1-4. Kainate receptors modulate neuronal circuits and synaptic plasticity during the development and function of the central nervous system and are implicated in various neurological and psychiatric diseases, including epilepsy, depression, schizophrenia, anxiety and autism5-11. Although structures of kainate receptor domains and subunit assemblies are available12-18, the mechanism of kainate receptor gating remains poorly understood. Here we present cryo-electron microscopy structures of the kainate receptor GluK2 in the presence of the agonist glutamate and the positive allosteric modulators lectin concanavalin A and BPAM344. Concanavalin A and BPAM344 inhibit kainate receptor desensitization and prolong activation by acting as a spacer between the amino-terminal and ligand-binding domains and a stabilizer of the ligand-binding domain dimer interface, respectively. Channel opening involves the kinking of all four pore-forming M3 helices. Our structures reveal the molecular basis of kainate receptor gating, which could guide the development of drugs for treatment of neurological disorders.
Asunto(s)
Concanavalina A , Microscopía por Crioelectrón , Receptor de Ácido Kaínico GluK2 , Ácido Glutámico , Activación del Canal Iónico , Modelos Moleculares , Dominios Proteicos , Receptores de Ácido Kaínico , Receptores de Ácido Kaínico/química , Receptores de Ácido Kaínico/metabolismo , Receptores de Ácido Kaínico/ultraestructura , Humanos , Ácido Glutámico/metabolismo , Ácido Glutámico/química , Animales , Concanavalina A/química , Concanavalina A/metabolismo , Concanavalina A/farmacología , Ligandos , Regulación Alostérica , Sitios de UniónRESUMEN
OSCA/TMEM63 channels are the largest known family of mechanosensitive channels1-3, playing critical roles in plant4-7 and mammalian8,9 mechanotransduction. Here we determined 44 cryogenic electron microscopy structures of OSCA/TMEM63 channels in different environments to investigate the molecular basis of OSCA/TMEM63 channel mechanosensitivity. In nanodiscs, we mimicked increased membrane tension and observed a dilated pore with membrane access in one of the OSCA1.2 subunits. In liposomes, we captured the fully open structure of OSCA1.2 in the inside-in orientation, in which the pore shows a large lateral opening to the membrane. Unusually for ion channels, structural, functional and computational evidence supports the existence of a 'proteo-lipidic pore' in which lipids act as a wall of the ion permeation pathway. In the less tension-sensitive homologue OSCA3.1, we identified an 'interlocking' lipid tightly bound in the central cleft, keeping the channel closed. Mutation of the lipid-coordinating residues induced OSCA3.1 activation, revealing a conserved open conformation of OSCA channels. Our structures provide a global picture of the OSCA channel gating cycle, uncover the importance of bound lipids and show that each subunit can open independently. This expands both our understanding of channel-mediated mechanotransduction and channel pore formation, with important mechanistic implications for the TMEM16 and TMC protein families.
Asunto(s)
Canales de Calcio , Microscopía por Crioelectrón , Activación del Canal Iónico , Mecanotransducción Celular , Humanos , Anoctaminas/química , Anoctaminas/metabolismo , Canales de Calcio/química , Canales de Calcio/metabolismo , Canales de Calcio/ultraestructura , Lípidos/química , Liposomas/metabolismo , Liposomas/química , Modelos Moleculares , Nanoestructuras/químicaRESUMEN
The voltage-gated ion channel activity depends on both activation (transition from the resting state to the open state) and inactivation. Inactivation is a self-restraint mechanism to limit ion conduction and is as crucial to membrane excitability as activation. Inactivation can occur when the channel is open or closed. Although open-state inactivation is well understood, the molecular basis of closed-state inactivation has remained elusive. We report cryo-EM structures of human KV4.2 channel complexes in inactivated, open, and closed states. Closed-state inactivation of KV4 involves an unprecedented symmetry breakdown for pore closure by only two of the four S4-S5 linkers, distinct from known mechanisms of open-state inactivation. We further capture KV4 in a putative resting state, revealing how voltage sensor movements control the pore. Moreover, our structures provide insights regarding channel modulation by KChIP2 and DPP6 auxiliary subunits. Our findings elucidate mechanisms of closed-state inactivation and voltage-dependent activation of the KV4 channel.
Asunto(s)
Activación del Canal Iónico , Canales de Potasio Shal , Humanos , Activación del Canal Iónico/fisiología , Cinética , Potenciales de la Membrana/fisiología , Canales de Potasio Shal/genética , Canales de Potasio Shal/metabolismoRESUMEN
Ion channels play a central role in the regulation of nearly every cellular process. Dating back to the classic 1952 Hodgkin-Huxley model of the generation of the action potential, ion channels have always been thought of as independent agents. A myriad of recent experimental findings exploiting advances in electrophysiology, structural biology, and imaging techniques, however, have posed a serious challenge to this long-held axiom, as several classes of ion channels appear to open and close in a coordinated, cooperative manner. Ion channel cooperativity ranges from variable-sized oligomeric cooperative gating in voltage-gated, dihydropyridine-sensitive CaV1.2 and CaV1.3 channels to obligatory dimeric assembly and gating of voltage-gated NaV1.5 channels. Potassium channels, transient receptor potential channels, hyperpolarization cyclic nucleotide-activated channels, ryanodine receptors (RyRs), and inositol trisphosphate receptors (IP3Rs) have also been shown to gate cooperatively. The implications of cooperative gating of these ion channels range from fine-tuning excitation-contraction coupling in muscle cells to regulating cardiac function and vascular tone, to modulation of action potential and conduction velocity in neurons and cardiac cells, and to control of pacemaking activity in the heart. In this review, we discuss the mechanisms leading to cooperative gating of ion channels, their physiological consequences, and how alterations in cooperative gating of ion channels may induce a range of clinically significant pathologies.