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To celebrate the 50th anniversary of Cell Press and the Cell special issue focusing on structural biology, we want to highlight the rapid progress of cryo-EM related research in India in this collection of Voices. We have asked structural biologists to introduce their research and the national cryo-EM facilities throughout the country.
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Microscopía por Crioelectrón , IndiaRESUMEN
Precise synapse formation is essential for normal functioning of the nervous system. Retinal photoreceptors establish selective contacts with bipolar cells, aligning the neurotransmitter release apparatus with postsynaptic signaling cascades. This involves transsynaptic assembly between the dystroglycan-dystrophin complex on the photoreceptor and the orphan receptor GPR179 on the bipolar cell, which is mediated by the extracellular matrix protein pikachurin (also known as EGFLAM). This complex plays a critical role in the synaptic organization of photoreceptors and signal transmission, and mutations affecting its components cause blinding disorders in humans. Here, we investigated the structural organization and molecular mechanisms by which pikachurin orchestrates transsynaptic assembly and solved structures of the human pikachurin domains by x-ray crystallography and of the GPR179-pikachurin complex by single-particle, cryo-electron microscopy. The structures reveal molecular recognition principles of pikachurin by the Cache domains of GPR179 and show how the interaction is involved in the transsynaptic alignment of the signaling machinery. Together, these data provide a structural basis for understanding the synaptic organization of photoreceptors and ocular pathology.
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Proteínas de la Matriz Extracelular , Sinapsis , Humanos , Proteínas Portadoras/metabolismo , Microscopía por Crioelectrón , Proteínas de la Matriz Extracelular/metabolismo , Células Fotorreceptoras/metabolismo , Sinapsis/metabolismoRESUMEN
Continuous health monitoring through sensitive physiological signals (using a wearable device) is crucial for the early detection of heart diseases and breathing problems. Here, we have developed a flexible hBN/cotton hybrid device that can detect minor signals such as heartbeat and breathed-out air pressure. Systematic observation of the real-time motion sensing showed a peak-to-peak voltage output of â¼1.5 V for each heart rate pulse. The as-fabricated device showed a high voltage output of up to â¼10 V upon applying a pressure of â¼3 MPa. The FTIR results and DFT calculation suggested a chemical interaction between hBN and cellulose, giving rise to flat band characteristics and partially filled σ-bonding (sp2) hybridization. The atomic-scale chemical interface between atomically thin hBN and surface functional groups present on cotton resulted in charge localization and enhanced output voltage. An hBN/cotton hybrid device can bring new insights and opportunities to develop a self-charging and health-monitoring energy-harvesting cloth.
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GPR158 is an orphan G proteincoupled receptor (GPCR) highly expressed in the brain, where it controls synapse formation and function. GPR158 has also been implicated in depression, carcinogenesis, and cognition. However, the structural organization and signaling mechanisms of GPR158 are largely unknown. We used single-particle cryoelectron microscopy (cryo-EM) to determine the structures of human GPR158 alone and bound to an RGS signaling complex. The structures reveal a homodimeric organization stabilized by a pair of phospholipids and the presence of an extracellular Cache domain, an unusual ligand-binding domain in GPCRs. We further demonstrate the structural basis of GPR158 coupling to RGS7-Gß5. Together, these results provide insights into the unusual biology of orphan receptors and the formation of GPCR-RGS complexes.
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Subunidades beta de la Proteína de Unión al GTP/química , Proteínas RGS/química , Receptores Acoplados a Proteínas G/química , Sitios de Unión , Microscopía por Crioelectrón , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Humanos , Ligandos , Modelos Moleculares , Fosfolípidos/química , Unión Proteica , Conformación Proteica , Conformación Proteica en Hélice alfa , Dominios Proteicos , Multimerización de Proteína , Subunidades de Proteína/química , Proteínas RGS/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND AND PURPOSE: AMPA receptors, which shape excitatory postsynaptic currents and are directly involved in overactivation of synaptic function during seizures, represent a well-accepted target for anti-epileptic drugs. Trans-4-butylcyclohexane carboxylic acid (4-BCCA) has emerged as a new promising anti-epileptic drug in several in vitro and in vivo seizure models, but the mechanism of its action remained unknown. The purpose of this study is to characterize structure and dynamics of 4-BCCA interaction with AMPA receptors. EXPERIMENTAL APPROACH: We studied the molecular mechanism of AMPA receptor inhibition by 4-BCCA using a combination of X-ray crystallography, mutagenesis, electrophysiological assays, and molecular dynamics simulations. KEY RESULTS: We identified 4-BCCA binding sites in the transmembrane domain (TMD) of AMPA receptor, at the lateral portals formed by transmembrane segments M1-M4. At this binding site, 4-BCCA is very dynamic, assumes multiple poses, and can enter the ion channel pore. CONCLUSION AND IMPLICATIONS: 4-BCCA represents a low-affinity inhibitor of AMPA receptors that acts at the TMD sites distinct from non-competitive inhibitors, such as the anti-epileptic drug perampanel and the ion channel blockers. Further studies might examine the possibsility of synergistic use of these inhibitors in treatment of epilepsy and a wide range of neurological disorders and gliomas. LINKED ARTICLES: This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc.
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Ácidos Carboxílicos , Receptores AMPA , Ciclohexanos , Humanos , Receptores AMPA/metabolismo , ConvulsionesRESUMEN
Epithelial calcium channel TRPV6 plays vital roles in calcium homeostasis, and its dysregulation is implicated in multifactorial diseases, including cancers. Here, we study the molecular mechanism of selective nanomolar-affinity TRPV6 inhibition by (4-phenylcyclohexyl)piperazine derivatives (PCHPDs). We use x-ray crystallography and cryo-electron microscopy to solve the inhibitor-bound structures of TRPV6 and identify two types of inhibitor binding sites in the transmembrane region: (i) modulatory sites between the S1-S4 and pore domains normally occupied by lipids and (ii) the main site in the ion channel pore. Our structural data combined with mutagenesis, functional and computational approaches suggest that PCHPDs plug the open pore of TRPV6 and convert the channel into a nonconducting state, mimicking the action of calmodulin, which causes inactivation of TRPV6 channels under physiological conditions. This mechanism of inhibition explains the high selectivity and potency of PCHPDs and opens up unexplored avenues for the design of future-generation biomimetic drugs.
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Canales de Calcio , Canales Catiónicos TRPV , Calcio/metabolismo , Canales de Calcio/química , Calmodulina/metabolismo , Microscopía por Crioelectrón , Humanos , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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We present structures of mouse TRPV3 in temperature-dependent open, closed and intermediate states that suggest two-step activation of TRPV3 by heat. During the strongly temperature-dependent first step, sensitization, the channel pore remains closed while S6 helices undergo α-to-π transitions. During the weakly temperature-dependent second step, channel opening, tight association of the S1-S4 and pore domains is stabilized by changes in the carboxy-terminal and linker domains.
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Canales Catiónicos TRPV/química , Sensación Térmica , Animales , Microscopía por Crioelectrón , Calor , Ratones , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Canales Catiónicos TRPV/metabolismo , TemperaturaRESUMEN
Algae produce the largest amount of oxygen on earth and are invaluable for human nutrition and biomedicine, as well as for the chemical industry, energy production and agriculture. The mechanisms by which algae can detect and respond to changes in their environments can rely on membrane receptors, including TRP ion channels. Here we present a 3.5-Å resolution cryo-EM structure of the transient receptor potential (TRP) channel crTRP1 from the alga Chlamydomonas reinhardtii that opens in response to increased temperature and is positively regulated by the membrane lipid PIP2. The structure of crTRP1 significantly deviates from the structures of other TRP channels and has a unique 2-fold symmetrical rose-shape architecture with elbow domains and ankyrin repeat domains submerged and dipping into the membrane, respectively. Our study provides a structure of a TRP channel from a micro-organism and a structural framework for better understanding algae biology and TRP channel evolution.
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Chlamydomonas reinhardtii/metabolismo , Proteínas de Plantas/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Repetición de Anquirina/genética , Repetición de Anquirina/fisiología , Chlamydomonas reinhardtii/genética , Microscopía por Crioelectrón , Células HEK293 , Humanos , Proteínas de Plantas/genética , Estructura Secundaria de Proteína , Canales de Potencial de Receptor Transitorio/genéticaRESUMEN
Transient receptor potential (TRP) channels are polymodal sensory transducers that respond to chemicals, temperature, mechanical stress, and membrane voltage and are involved in vision, taste, olfaction, hearing, touch, thermal perception, and nociception. TRP channels are implicated in numerous devastating diseases, including various forms of cancer, and represent important drug targets. The large sizes, low expression levels, and conformational dynamics of TRP channels make them challenging targets for structural biology. Here, we present the methodology used in structural studies of TRPV6, a TRP channel that is highly selective for calcium and mediates Ca2+ uptake in epithelial tissues. We provide a protocol for the expression, purification, and crystallization of TRPV6. Similar approaches can be used to determine crystal structures of other membrane proteins, including different members of the TRP channel family.
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Cristalografía por Rayos X/métodos , Canales Catiónicos TRPV/aislamiento & purificación , Animales , Baculoviridae , Calcio/metabolismo , Canales de Calcio/metabolismo , Células HEK293 , Humanos , Células Sf9 , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismoRESUMEN
Transient receptor potential vanilloid subfamily member 3 (TRPV3) channel plays a crucial role in skin physiology and pathophysiology. Mutations in TRPV3 are associated with various skin diseases, including Olmsted syndrome, atopic dermatitis, and rosacea. Here we present the cryo-electron microscopy structures of full-length mouse TRPV3 in the closed apo and agonist-bound open states. The agonist binds three allosteric sites distal to the pore. Channel opening is accompanied by conformational changes in both the outer pore and the intracellular gate. The gate is formed by the pore-lining S6 helices that undergo local α-to-π helical transitions, elongate, rotate, and splay apart in the open state. In the closed state, the shorter S6 segments are entirely α-helical, expose their nonpolar surfaces to the pore, and hydrophobically seal the ion permeation pathway. These findings further illuminate TRP channel activation and can aid in the design of drugs for the treatment of inflammatory skin conditions, itch, and pain.
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Activación del Canal Iónico , Canales Catiónicos TRPV/química , Canales Catiónicos TRPV/fisiología , Animales , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Conformación Proteica , Relación Estructura-Actividad , Canales Catiónicos TRPV/agonistasRESUMEN
Calcium (Ca2+) plays a major role in numerous physiological processes. Ca2+ homeostasis is tightly controlled by ion channels, the aberrant regulation of which results in various diseases including cancers. Calmodulin (CaM)-mediated Ca2+-induced inactivation is an ion channel regulatory mechanism that protects cells against the toxic effects of Ca2+ overload. We used cryo-electron microscopy to capture the epithelial calcium channel TRPV6 (transient receptor potential vanilloid subfamily member 6) inactivated by CaM. The TRPV6-CaM complex exhibits 1:1 stoichiometry; one TRPV6 tetramer binds both CaM lobes, which adopt a distinct head-to-tail arrangement. The CaM carboxyl-terminal lobe plugs the channel through a unique cation-π interaction by inserting the side chain of lysine K115 into a tetra-tryptophan cage at the pore's intracellular entrance. We propose a mechanism of CaM-mediated Ca2+-induced inactivation that can be explored for therapeutic design.
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Canales de Calcio/química , Calcio/metabolismo , Calmodulina/química , Canales Catiónicos TRPV/química , Animales , Sitios de Unión , Canales de Calcio/metabolismo , Calmodulina/metabolismo , Microscopía por Crioelectrón , Humanos , Unión Proteica , Conformación Proteica , Ratas , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/metabolismoRESUMEN
Transient receptor potential (TRP) channels are involved in various physiological processes, including sensory transduction. The TRP channel TRPV6 mediates calcium uptake in epithelia and its expression is dramatically increased in numerous types of cancer. TRPV6 inhibitors suppress tumor growth, but the molecular mechanism of inhibition remains unknown. Here, we present crystal and cryo-EM structures of human and rat TRPV6 bound to 2-aminoethoxydiphenyl borate (2-APB), a TRPV6 inhibitor and modulator of numerous TRP channels. 2-APB binds to TRPV6 in a pocket formed by the cytoplasmic half of the S1-S4 transmembrane helix bundle. Comparing human wild-type and high-affinity mutant Y467A structures, we show that 2-APB induces TRPV6 channel closure by modulating protein-lipid interactions. Mutagenesis and functional analyses suggest that the identified 2-APB binding site might be present in other members of vanilloid subfamily TRP channels. Our findings reveal a mechanism of ion channel allosteric modulation that can be exploited for therapeutic design.
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Compuestos de Boro/química , Canales de Calcio/metabolismo , Calcio/metabolismo , Neoplasias/patología , Canales Catiónicos TRPV/metabolismo , Animales , Sitios de Unión/genética , Cristalografía por Rayos X , Células HEK293 , Humanos , Metabolismo de los Lípidos/fisiología , Estructura Secundaria de Proteína , Ratas , Canales Catiónicos TRPV/antagonistas & inhibidoresRESUMEN
Transient receptor potential (TRP) ion channels are molecular sensors of a large variety of stimuli including temperature, mechanical stress, voltage, small molecules including capsaicin and menthol, and lipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). Since the same TRP channels may respond to different physical and chemical stimuli, they can serve as signal integrators. Many TRP channels are calcium permeable and contribute to Ca2+ homeostasis and signaling. Although the TRP channel family was discovered decades ago, only recently have the structures of many of these channels been solved, largely by cryo-electron microscopy (cryo-EM). Complimentary to cryo-EM, X-ray crystallography provides unique tools to unambiguously identify specific atoms and can be used to study ion binding in channel pores. In this review we describe crystallographic studies of the TRP channel TRPV6. The methodology used in these studies may serve as a template for future structural analyses of different types of TRP and other ion channels.
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Canales de Potencial de Receptor Transitorio/química , Animales , Cristalografía por Rayos X , Humanos , Modelos MolecularesRESUMEN
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.
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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
Tetrameric ion channels have either swapped or non-swapped arrangements of the S1-S4 and pore domains. Here we show that mutations in the transmembrane domain of TRPV6 can result in conversion from a domain-swapped to non-swapped fold. These results reveal structural determinants of domain swapping and raise the possibility that a single ion channel subtype can fold into either arrangement in vivo, affecting its function in normal or disease states.
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Canales de Calcio/química , Canales de Calcio/genética , Dominios y Motivos de Interacción de Proteínas , Canales Catiónicos TRPV/química , Canales Catiónicos TRPV/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Calcio/metabolismo , Canales de Calcio/metabolismo , Genes Reporteros , Células HEK293 , Humanos , Ligandos , Modelos Moleculares , Conformación Molecular , Mutación , Relación Estructura-Actividad Cuantitativa , Canales Catiónicos TRPV/metabolismoRESUMEN
Excitatory neurotransmission plays a key role in epileptogenesis. Correspondingly, AMPA-subtype ionotropic glutamate receptors, which mediate the majority of excitatory neurotransmission and contribute to seizure generation and spread, have emerged as promising targets for epilepsy therapy. The most potent and well-tolerated AMPA receptor inhibitors act via a noncompetitive mechanism, but many of them produce adverse side effects. The design of better drugs is hampered by the lack of a structural understanding of noncompetitive inhibition. Here, we report crystal structures of the rat AMPA-subtype GluA2 receptor in complex with three noncompetitive inhibitors. The inhibitors bind to a novel binding site, completely conserved between rat and human, at the interface between the ion channel and linkers connecting it to the ligand-binding domains. We propose that the inhibitors stabilize the AMPA receptor closed state by acting as wedges between the transmembrane segments, thereby preventing gating rearrangements that are necessary for ion channel opening.
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Anticonvulsivantes/farmacología , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/química , Animales , Anticonvulsivantes/química , Benzodiazepinas/química , Benzodiazepinas/farmacología , Sitios de Unión/efectos de los fármacos , Cristalización , Simulación del Acoplamiento Molecular , Nitrilos , Piridonas/química , Piridonas/farmacología , Quinazolinas/química , Quinazolinas/farmacología , Ratas , Receptores AMPA/metabolismoRESUMEN
Precise regulation of calcium homeostasis is essential for many physiological functions. The Ca(2+)-selective transient receptor potential (TRP) channels TRPV5 and TRPV6 play vital roles in calcium homeostasis as Ca(2+) uptake channels in epithelial tissues. Detailed structural bases for their assembly and Ca(2+) permeation remain obscure. Here we report the crystal structure of rat TRPV6 at 3.25 Å resolution. The overall architecture of TRPV6 reveals shared and unique features compared with other TRP channels. Intracellular domains engage in extensive interactions to form an intracellular 'skirt' involved in allosteric modulation. In the K(+) channel-like transmembrane domain, Ca(2+) selectivity is determined by direct coordination of Ca(2+) by a ring of aspartate side chains in the selectivity filter. On the basis of crystallographically identified cation-binding sites at the pore axis and extracellular vestibule, we propose a Ca(2+) permeation mechanism. Our results provide a structural foundation for understanding the regulation of epithelial Ca(2+) uptake and its role in pathophysiology.
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Epitelio , Canales Catiónicos TRPV/química , Regulación Alostérica , Animales , Sitios de Unión , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Ratas , Relación Estructura-Actividad , Canales Catiónicos TRPV/metabolismoRESUMEN
AMPA subtype ionotropic glutamate receptors (iGluRs) mediate the majority of fast neurotransmission across excitatory synapses in the central nervous system. Each AMPA receptor is composed of four multi-domain subunits that are organized into layers of two amino-terminal domain (ATD) dimers, two ligand-binding domain (LBD) dimers, transmembrane domains and carboxy-terminal domains. We introduced cysteine substitutions at the intersubunit interfaces of AMPA receptor subunit GluA2 and confirmed substituted cysteine crosslink formation by SDS-PAGE. The functional consequence of intersubunit crosslinks was assessed by recording GluA2-mediated currents in reducing and non-reducing conditions. Strong redox-dependent changes in GluA2-mediated currents were observed for cysteine substitutions at the LBD dimer-dimer interface but not at the ATD dimer-dimer interface. We conclude that during gating, LBD dimers undergo significant relative displacement, while ATD dimers either maintain their relative positioning, or their relative displacement has no appreciable effect on AMPA receptor function.