RESUMEN
The L-type voltage-gated Ca2+ (Cav) channels are modulated by various compounds exemplified by 1,4-dihydropyridines (DHP), benzothiazepines (BTZ), and phenylalkylamines (PAA), many of which have been used for characterizing channel properties and for treatment of hypertension and other disorders. Here, we report the cryoelectron microscopy (cryo-EM) structures of Cav1.1 in complex with archetypal antagonistic drugs, nifedipine, diltiazem, and verapamil, at resolutions of 2.9 Å, 3.0 Å, and 2.7 Å, respectively, and with a DHP agonist Bay K 8644 at 2.8 Å. Diltiazem and verapamil traverse the central cavity of the pore domain, directly blocking ion permeation. Although nifedipine and Bay K 8644 occupy the same fenestration site at the interface of repeats III and IV, the coordination details support previous functional observations that Bay K 8644 is less favored in the inactivated state. These structures elucidate the modes of action of different Cav ligands and establish a framework for structure-guided drug discovery.
Asunto(s)
Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/ultraestructura , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico , Secuencia de Aminoácidos , Animales , Sitios de Unión , Canales de Calcio/metabolismo , Canales de Calcio/fisiología , Canales de Calcio/ultraestructura , Canales de Calcio Tipo L/fisiología , Microscopía por Crioelectrón , Diltiazem , Ligandos , Masculino , Modelos Moleculares , Nifedipino , Conejos , VerapamiloRESUMEN
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
AMPA receptors (AMPARs) mediate the majority of excitatory transmission in the brain and enable the synaptic plasticity that underlies learning1. A diverse array of AMPAR signalling complexes are established by receptor auxiliary subunits, which associate with the AMPAR in various combinations to modulate trafficking, gating and synaptic strength2. However, their mechanisms of action are poorly understood. Here we determine cryo-electron microscopy structures of the heteromeric GluA1-GluA2 receptor assembled with both TARP-γ8 and CNIH2, the predominant AMPAR complex in the forebrain, in both resting and active states. Two TARP-γ8 and two CNIH2 subunits insert at distinct sites beneath the ligand-binding domains of the receptor, with site-specific lipids shaping each interaction and affecting the gating regulation of the AMPARs. Activation of the receptor leads to asymmetry between GluA1 and GluA2 along the ion conduction path and an outward expansion of the channel triggers counter-rotations of both auxiliary subunit pairs, promoting the active-state conformation. In addition, both TARP-γ8 and CNIH2 pivot towards the pore exit upon activation, extending their reach for cytoplasmic receptor elements. CNIH2 achieves this through its uniquely extended M2 helix, which has transformed this endoplasmic reticulum-export factor into a powerful AMPAR modulator that is capable of providing hippocampal pyramidal neurons with their integrative synaptic properties.
Asunto(s)
Microscopía por Crioelectrón , Activación del Canal Iónico , Multimerización de Proteína , Receptores AMPA/metabolismo , Receptores AMPA/ultraestructura , Secuencia de Aminoácidos , Animales , Canales de Calcio/química , Canales de Calcio/metabolismo , Canales de Calcio/ultraestructura , Hipocampo , Metabolismo de los Lípidos , Lípidos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Células Piramidales/metabolismo , Receptores AMPA/química , RotaciónRESUMEN
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/ultraestructuraRESUMEN
Mitochondria take up Ca2+ through the mitochondrial calcium uniporter complex to regulate energy production, cytosolic Ca2+ signalling and cell death1,2. In mammals, the uniporter complex (uniplex) contains four core components: the pore-forming MCU protein, the gatekeepers MICU1 and MICU2, and an auxiliary subunit, EMRE, essential for Ca2+ transport3-8. To prevent detrimental Ca2+ overload, the activity of MCU must be tightly regulated by MICUs, which sense changes in cytosolic Ca2+ concentrations to switch MCU on and off9,10. Here we report cryo-electron microscopic structures of the human mitochondrial calcium uniporter holocomplex in inhibited and Ca2+-activated states. These structures define the architecture of this multicomponent Ca2+-uptake machinery and reveal the gating mechanism by which MICUs control uniporter activity. Our work provides a framework for understanding regulated Ca2+ uptake in mitochondria, and could suggest ways of modulating uniporter activity to treat diseases related to mitochondrial Ca2+ overload.
Asunto(s)
Canales de Calcio/química , Canales de Calcio/metabolismo , Microscopía por Crioelectrón , Sitios de Unión/efectos de los fármacos , Calcio/metabolismo , Calcio/farmacología , Canales de Calcio/ultraestructura , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructuraRESUMEN
The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel and a major route of calcium entry into mitochondria. How the channel catalyses ion permeation and achieves ion selectivity are not well understood, partly because MCU is thought to have a distinct architecture in comparison to other cellular channels. Here we report cryo-electron microscopy reconstructions of MCU channels from zebrafish and Cyphellophora europaea at 8.5 Å and 3.2 Å resolutions, respectively. In contrast to a previous report of pentameric stoichiometry for MCU, both channels are tetramers. The atomic model of C. europaea MCU shows that a conserved WDXXEP signature sequence forms the selectivity filter, in which calcium ions are arranged in single file. Coiled-coil legs connect the pore to N-terminal domains in the mitochondrial matrix. In C. europaea MCU, the N-terminal domains assemble as a dimer of dimers; in zebrafish MCU, they form an asymmetric crescent. The structures define principles that underlie ion permeation and calcium selectivity in this unusual channel.
Asunto(s)
Canales de Calcio/química , Canales de Calcio/ultraestructura , Microscopía por Crioelectrón , Phialophora/química , Pez Cebra , Animales , Caenorhabditis elegans/química , Calcio/metabolismo , Canales de Calcio/metabolismo , Activación del Canal Iónico , Modelos Moleculares , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismoRESUMEN
Calcium-selective transient receptor potential vanilloid subfamily member 6 (TRPV6) channels play a critical role in calcium uptake in epithelial tissues. Altered TRPV6 expression is associated with a variety of human diseases, including cancers. TRPV6 channels are constitutively active and their open probability depends on the lipidic composition of the membrane in which they reside; it increases substantially in the presence of phosphatidylinositol 4,5-bisphosphate. Crystal structures of detergent-solubilized rat TRPV6 in the closed state have previously been solved. Corroborating electrophysiological results, these structures demonstrated that the Ca2+ selectivity of TRPV6 arises from a ring of aspartate side chains in the selectivity filter that binds Ca2+ tightly. However, how TRPV6 channels open and close their pores for ion permeation has remained unclear. Here we present cryo-electron microscopy structures of human TRPV6 in the open and closed states. The channel selectivity filter adopts similar conformations in both states, consistent with its explicit role in ion permeation. The iris-like channel opening is accompanied by an α-to-π-helical transition in the pore-lining transmembrane helix S6 at an alanine hinge just below the selectivity filter. As a result of this transition, the S6 helices bend and rotate, exposing different residues to the ion channel pore in the open and closed states. This gating mechanism, which defines the constitutive activity of TRPV6, is, to our knowledge, unique among tetrameric ion channels and provides structural insights for understanding their diverse roles in physiology and disease.
Asunto(s)
Canales de Calcio/metabolismo , Canales de Calcio/ultraestructura , Microscopía por Crioelectrón , Células Epiteliales/metabolismo , Activación del Canal Iónico , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/ultraestructura , Alanina/metabolismo , Calcio/metabolismo , Canales de Calcio/química , Humanos , Transporte Iónico , Conformación Proteica , Rotación , Canales Catiónicos TRPV/químicaRESUMEN
Mitochondrial calcium uptake is critical for regulating ATP production, intracellular calcium signalling, and cell death. This uptake is mediated by a highly selective calcium channel called the mitochondrial calcium uniporter (MCU). Here, we determined the structures of the pore-forming MCU proteins from two fungi by X-ray crystallography and single-particle cryo-electron microscopy. The stoichiometry, overall architecture, and individual subunit structure differed markedly from those described in the recent nuclear magnetic resonance structure of Caenorhabditis elegans MCU. We observed a dimer-of-dimer architecture across species and chemical environments, which was corroborated by biochemical experiments. Structural analyses and functional characterization uncovered the roles of key residues in the pore. These results reveal a new ion channel architecture, provide insights into calcium coordination, selectivity and conduction, and establish a structural framework for understanding the mechanism of mitochondrial calcium uniporter function.
Asunto(s)
Canales de Calcio/química , Canales de Calcio/ultraestructura , Microscopía por Crioelectrón , Fusarium/química , Metarhizium/química , Animales , Caenorhabditis elegans/química , Calcio/metabolismo , Canales de Calcio/metabolismo , Cristalografía por Rayos X , Activación del Canal Iónico , Modelos Moleculares , Dominios Proteicos , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Reproducibilidad de los Resultados , SolubilidadRESUMEN
The organellar two-pore channel (TPC) functions as a homodimer, in which each subunit contains two homologous Shaker-like six-transmembrane (6-TM)-domain repeats. TPCs belong to the voltage-gated ion channel superfamily and are ubiquitously expressed in animals and plants. Mammalian TPC1 and TPC2 are localized at the endolysosomal membrane, and have critical roles in regulating the physiological functions of these acidic organelles. Here we present electron cryo-microscopy structures of mouse TPC1 (MmTPC1)-a voltage-dependent, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2)-activated Na+-selective channel-in both the apo closed state and ligand-bound open state. Combined with functional analysis, these structures provide comprehensive structural insights into the selectivity and gating mechanisms of mammalian TPC channels. The channel has a coin-slot-shaped ion pathway in the filter that defines the selectivity of mammalian TPCs. Only the voltage-sensing domain from the second 6-TM domain confers voltage dependence on MmTPC1. Endolysosome-specific PtdIns(3,5)P2 binds to the first 6-TM domain and activates the channel under conditions of depolarizing membrane potential. Structural comparisons between the apo and PtdIns(3,5)P2-bound structures show the interplay between voltage and ligand in channel activation. These MmTPC1 structures reveal lipid binding and regulation in a 6-TM voltage-gated channel, which is of interest in light of the emerging recognition of the importance of phosphoinositide regulation of ion channels.
Asunto(s)
Canales de Calcio/metabolismo , Canales de Calcio/ultraestructura , Microscopía por Crioelectrón , Activación del Canal Iónico/efectos de los fármacos , Fosfolípidos/farmacología , Secuencia de Aminoácidos , Animales , Canales de Calcio/química , Canales de Calcio/genética , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Fosfolípidos/química , Fosfolípidos/metabolismo , Dominios Proteicos/efectos de los fármacosRESUMEN
The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel localized to the inner mitochondrial membrane. Here, we describe the structure of an MCU orthologue from the fungus Neosartorya fischeri (NfMCU) determined to 3.8 Å resolution by phase-plate cryo-electron microscopy. The channel is a homotetramer with two-fold symmetry in its amino-terminal domain (NTD) that adopts a similar structure to that of human MCU. The NTD assembles as a dimer of dimers to form a tetrameric ring that connects to the transmembrane domain through an elongated coiled-coil domain. The ion-conducting pore domain maintains four-fold symmetry, with the selectivity filter positioned at the start of the pore-forming TM2 helix. The aspartate and glutamate sidechains of the conserved DIME motif are oriented towards the central axis and separated by one helical turn. The structure of NfMCU offers insights into channel assembly, selective calcium permeation, and inhibitor binding.
Asunto(s)
Canales de Calcio/química , Canales de Calcio/ultraestructura , Microscopía por Crioelectrón , Neosartorya/química , Sitios de Unión , Calcio/metabolismo , Canales de Calcio/metabolismo , Humanos , Activación del Canal Iónico/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Modelos Moleculares , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Compuestos de Rutenio/farmacología , SolubilidadRESUMEN
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/químicaRESUMEN
Sensing and responding to environmental water deficiency and osmotic stresses are essential for the growth, development, and survival of plants. Recently, an osmolality-sensing ion channel called OSCA1 was discovered that functions in sensing hyperosmolality in Arabidopsis Here, we report the cryo-electron microscopy (cryo-EM) structure and function of an OSCA1 homolog from rice (Oryza sativa; OsOSCA1.2), leading to a model of how it could mediate hyperosmolality sensing and transport pathway gating. The structure reveals a dimer; the molecular architecture of each subunit consists of 11 transmembrane (TM) helices and a cytosolic soluble domain that has homology to RNA recognition proteins. The TM domain is structurally related to the TMEM16 family of calcium-dependent ion channels and lipid scramblases. The cytosolic soluble domain possesses a distinct structural feature in the form of extended intracellular helical arms that are parallel to the plasma membrane. These helical arms are well positioned to potentially sense lateral tension on the inner leaflet of the lipid bilayer caused by changes in turgor pressure. Computational dynamic analysis suggests how this domain couples to the TM portion of the molecule to open a transport pathway. Hydrogen/deuterium exchange mass spectrometry (HDXMS) experimentally confirms the conformational dynamics of these coupled domains. These studies provide a framework to understand the structural basis of proposed hyperosmolality sensing in a staple crop plant, extend our knowledge of the anoctamin superfamily important for plants and fungi, and provide a structural mechanism for potentially translating membrane stress to transport regulation.
Asunto(s)
Anoctaminas/ultraestructura , Proteínas de Arabidopsis/ultraestructura , Canales de Calcio/ultraestructura , Oryza/ultraestructura , Conformación Proteica , Secuencia de Aminoácidos/genética , Anoctaminas/química , Anoctaminas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Microscopía por Crioelectrón , Citoplasma/genética , Espectrometría de Masas , Potenciales de la Membrana/genética , Oryza/genética , Oryza/crecimiento & desarrollo , Presión Osmótica/fisiología , Agua/químicaRESUMEN
The TRPA1 ion channel (also known as the wasabi receptor) is a detector of noxious chemical agents encountered in our environment or produced endogenously during tissue injury or drug metabolism. These include a broad class of electrophiles that activate the channel through covalent protein modification. TRPA1 antagonists hold potential for treating neurogenic inflammatory conditions provoked or exacerbated by irritant exposure. Despite compelling reasons to understand TRPA1 function, structural mechanisms underlying channel regulation remain obscure. Here we use single-particle electron cryo- microscopy to determine the structure of full-length human TRPA1 to â¼4 Å resolution in the presence of pharmacophores, including a potent antagonist. Several unexpected features are revealed, including an extensive coiled-coil assembly domain stabilized by polyphosphate co-factors and a highly integrated nexus that converges on an unpredicted transient receptor potential (TRP)-like allosteric domain. These findings provide new insights into the mechanisms of TRPA1 regulation, and establish a blueprint for structure-based design of analgesic and anti-inflammatory agents.
Asunto(s)
Canales de Calcio/química , Canales de Calcio/ultraestructura , Microscopía por Crioelectrón , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/ultraestructura , Canales de Potencial de Receptor Transitorio/química , Canales de Potencial de Receptor Transitorio/ultraestructura , Regulación Alostérica , Analgésicos , Repetición de Anquirina , Antiinflamatorios , Sitios de Unión , Canales de Calcio/metabolismo , Citosol/metabolismo , Humanos , Modelos Moleculares , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Polifosfatos/metabolismo , Polifosfatos/farmacología , Estabilidad Proteica/efectos de los fármacos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Relación Estructura-Actividad , Canal Catiónico TRPA1 , Canales de Potencial de Receptor Transitorio/antagonistas & inhibidores , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
Target cell type-dependent differences in presynaptic release probability (Pr ) and short-term plasticity are intriguing features of cortical microcircuits that increase the computational power of neuronal networks. Here, we tested the hypothesis that different voltage-gated Ca2+ channel densities in presynaptic active zones (AZs) underlie different Pr values. Two-photon Ca2+ imaging, triple immunofluorescent labeling, and 3D electron microscopic (EM) reconstruction of rat CA3 pyramidal cell axon terminals revealed â¼1.7-1.9 times higher Ca2+ inflow per AZ area in high Pr boutons synapsing onto parvalbumin-positive interneurons (INs) than in low Pr boutons synapsing onto mGluR1α-positive INs. EM replica immunogold labeling, however, demonstrated only 1.15 times larger Cav2.1 and Cav2.2 subunit densities in high Pr AZs. Our results indicate target cell type-specific modulation of voltage-gated Ca2+ channel function or different subunit composition as possible mechanisms underlying the functional differences. In addition, high Pr synapses are also characterized by a higher density of docked vesicles, suggesting that a concerted action of these mechanisms underlies the functional differences.SIGNIFICANCE STATEMENT Target cell type-dependent variability in presynaptic properties is an intriguing feature of cortical synapses. When a single cortical pyramidal cell establishes a synapse onto a somatostatin-expressing interneuron (IN), the synapse releases glutamate with low probability, whereas the next bouton of the same axon has high release probability when its postsynaptic target is a parvalbumin-expressing IN. Here, we used combined molecular, imaging, and anatomical approaches to investigate the mechanisms underlying these differences. Our functional experiments implied an approximately twofold larger Ca2+ channel density in high release probability boutons, whereas freeze-fracture immunolocalization demonstrated only a 15% difference in Ca2+ channel subunit densities. Our results point toward a postsynaptic target cell type-dependent regulation of Ca2+ channel function or different subunit composition as the underlying mechanism.
Asunto(s)
Canales de Calcio/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/citología , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Terminales Presinápticos/metabolismo , Probabilidad , Potenciales de Acción/fisiología , Animales , Animales Recién Nacidos , Axones/metabolismo , Calcio/metabolismo , Canales de Calcio/ultraestructura , Ácido Glutámico/clasificación , Técnicas In Vitro , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/ultraestructura , Parvalbúminas/metabolismo , Terminales Presinápticos/ultraestructura , Ratas , Ratas Wistar , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de Glutamato Metabotrópico/ultraestructura , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Cryo-electron microscopy (cryo-EM) is emerging as a real alternative for structural elucidation. In spite of this, very few cryo-EM structures have been described so far as successful platforms for in silico drug design. Gabapentin and pregabalin are some of the most successful drugs in the treatment of epilepsy and neuropathic pain. Although both are in clinical use and are known to exert their effects by binding to the regulatory α2δ subunit of voltage gated calcium channels, their binding modes have never been characterized. We describe here the successful use of an exhaustive protein-ligand sampling algorithm on the α2δ-1 subunit of the recently published cryo-EM structure, with the goal of characterizing the ligand entry path and binding mode for gabapentin, pregabalin, and several other amino acidic α2δ-1 ligands. Our studies indicate that (i) all simulated drugs explore the same path for accessing the occluded binding site on the interior of the α2δ-1 subunit; (ii) they all roughly occupy the same pocket; (iii) the plasticity of the binding site allows the accommodation of a variety of amino acidic modulators, driven by the flexible "capping loop" delineated by residues Tyr426-Val435 and the floppy nature of Arg217; (iv) the predicted binding modes are in line with previously available mutagenesis data, confirming Arg217 as key for binding, with Asp428 and Asp467 highlighted as additional anchoring points for all amino acidic drugs. The study is one of the first proofs that latest-generation cryo-EM structures combined with exhaustive computational methods can be exploited in early drug discovery.
Asunto(s)
Analgésicos/farmacología , Canales de Calcio/metabolismo , Gabapentina/farmacología , Pregabalina/farmacología , Algoritmos , Analgésicos/química , Sitios de Unión , Canales de Calcio/química , Canales de Calcio/ultraestructura , Microscopía por Crioelectrón , Gabapentina/química , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Pregabalina/química , Unión ProteicaAsunto(s)
Canales de Calcio/química , Canales de Calcio/ultraestructura , Microscopía por Crioelectrón , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/ultraestructura , Canales de Potencial de Receptor Transitorio/química , Canales de Potencial de Receptor Transitorio/ultraestructura , Humanos , Canal Catiónico TRPA1RESUMEN
Cholinergic inhibition of hair cells occurs by activation of calcium-dependent potassium channels. A near-membrane postsynaptic cistern has been proposed to serve as a store from which calcium is released to supplement influx through the ionotropic ACh receptor. However, the time and voltage dependence of acetylcholine (ACh)-evoked potassium currents reveal a more complex relationship between calcium entry and release from stores. The present work uses voltage steps to regulate calcium influx during the application of ACh to hair cells in the chicken basilar papilla. When calcium influx was terminated at positive membrane potential, the ACh-evoked potassium current decayed exponentially over â¼100 ms. However, at negative membrane potentials, this current exhibited a secondary rise in amplitude that could be eliminated by dihydropyridine block of the voltage-gated calcium channels of the hair cell. Calcium entering through voltage-gated channels may transit through the postsynaptic cistern, since ryanodine and sarcoendoplasmic reticulum calcium-ATPase blockers altered the time course and magnitude of this secondary, voltage-dependent contribution to ACh-evoked potassium current. Serial section electron microscopy showed that efferent and afferent synaptic structures are juxtaposed, supporting the possibility that voltage-gated influx at afferent ribbon synapses influences calcium homeostasis during long-lasting cholinergic inhibition. In contrast, spontaneous postsynaptic currents ("minis") resulting from stochastic efferent release of ACh were made briefer by ryanodine, supporting the hypothesis that the synaptic cistern serves primarily as a calcium barrier and sink during low-level synaptic activity. Hypolemmal cisterns such as that at the efferent synapse of the hair cell can play a dynamic role in segregating near-membrane calcium for short-term and long-term signaling.
Asunto(s)
Calcio/fisiología , Células Ciliadas Auditivas/fisiología , Órgano Espiral/fisiología , Sinapsis/fisiología , Animales , Canales de Calcio/fisiología , Canales de Calcio/ultraestructura , Embrión de Pollo , Femenino , Células Ciliadas Auditivas/ultraestructura , Masculino , Órgano Espiral/ultraestructura , Sinapsis/ultraestructuraRESUMEN
Although it has long been known that mitochondria take up Ca2+, the molecular identities of the channels and transporters involved in this process were revealed only recently. Here, we discuss the recent work that has led to the characterization of the mitochondrial calcium uniporter complex, which includes the channel-forming subunit MCU (mitochondrial calcium uniporter) and its regulators MICU1, MICU2, MCUb, EMRE, MCUR1 and miR-25. We review not only the biochemical identities and structures of the proteins required for mitochondrial Ca2+ uptake but also their implications in different physiopathological contexts.
Asunto(s)
Canales de Calcio/química , Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/fisiología , Enfermedades Mitocondriales/metabolismo , Animales , Canales de Calcio/ultraestructura , Humanos , Mitocondrias/química , Mitocondrias/ultraestructura , Enfermedades Mitocondriales/patología , Modelos Biológicos , Modelos Químicos , Relación Estructura-ActividadRESUMEN
AMPA-type glutamate receptors (AMPARs) mediate rapid signal transmission at excitatory synapses in the brain. Glutamate binding to the receptor's ligand-binding domains (LBDs) leads to ion channel activation and desensitization. Gating kinetics shape synaptic transmission and are strongly modulated by transmembrane AMPAR regulatory proteins (TARPs) through currently incompletely resolved mechanisms. Here, electron cryo-microscopy structures of the GluA1/2 TARP-γ8 complex, in both open and desensitized states (at 3.5 Å), reveal state-selective engagement of the LBDs by the large TARP-γ8 loop ('ß1'), elucidating how this TARP stabilizes specific gating states. We further show how TARPs alter channel rectification, by interacting with the pore helix of the selectivity filter. Lastly, we reveal that the Q/R-editing site couples the channel constriction at the filter entrance to the gate, and forms the major cation binding site in the conduction path. Our results provide a mechanistic framework of how TARPs modulate AMPAR gating and conductance.