RESUMEN
Photoactivatable or "caged" pharmacological agents combine the high spatiotemporal specificity of light application with the molecular specificity of drugs. A key factor in all optopharmacology experiments is the mechanism of uncaging, which dictates the photochemical quantum yield and determines the byproducts produced by the light-driven chemical reaction. In previous work, we demonstrated that coumarin-based photolabile groups could be used to cage tertiary amine drugs as quaternary ammonium salts. Although stable, water-soluble, and useful for experiments in brain tissue, these first-generation compounds exhibit relatively low uncaging quantum yield (Φu < 1%) and release the toxic byproduct formaldehyde upon photolysis. Here, we elucidate the photochemical mechanisms of coumarin-caged tertiary amines and then optimize the major pathway using chemical modification. We discovered that the combination of 3,3-dicarboxyazetidine and bromine substituents shift the mechanism of release to heterolysis, eliminating the formaldehyde byproduct and giving photolabile tertiary amine drugs with Φu > 20%âa 35-fold increase in uncaging efficiency. This new "ABC" cage allows synthesis of improved photoactivatable derivatives of escitalopram and nicotine along with a novel caged agonist of the oxytocin receptor.
Asunto(s)
Aminas , Cumarinas , Procesos Fotoquímicos , Cumarinas/química , Aminas/química , Estructura Molecular , FotólisisRESUMEN
The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as viroporins. Here, we show that neither SARS-CoV-2 nor SARS-CoV-1 Orf3a form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a positively charged aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a's ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/metabolismo , Endosomas/metabolismo , Canales Iónicos/metabolismoRESUMEN
The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as a viroporin. Here we show that neither SARS-CoV-2 nor SARS-CoV-1 form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a basic aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.
RESUMEN
Chemical synapses between axons and dendrites mediate neuronal intercellular communication. Here, we describe a synapse between axons and primary cilia: the axo-ciliary synapse. Using enhanced focused ion beam-scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between brainstem serotonergic axons and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, the 5-hydroxytryptamine receptor 6 (5-HTR6). Using a cilia-targeted serotonin sensor, we show that opto- and chemogenetic stimulation of serotonergic axons releases serotonin onto cilia. Ciliary 5-HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway, which modulates nuclear actin and increases histone acetylation and chromatin accessibility. Ablation of this pathway reduces chromatin accessibility in CA1 pyramidal neurons. As a signaling apparatus with proximity to the nucleus, axo-ciliary synapses short circuit neurotransmission to alter the postsynaptic neuron's epigenetic state.
Asunto(s)
Axones/fisiología , Cromatina/química , Cilios , Sinapsis , Núcleo Celular/metabolismo , Cromatina/metabolismo , Cilios/metabolismo , Hipocampo/citología , Hipocampo/fisiología , Serotonina/metabolismo , Transducción de Señal , Sinapsis/fisiologíaRESUMEN
Chemical neurotransmission constitutes one of the fundamental modalities of communication between neurons. Monitoring release of these chemicals has traditionally been difficult to carry out at spatial and temporal scales relevant to neuron function. To understand chemical neurotransmission more fully, we need to improve the spatial and temporal resolutions of measurements for neurotransmitter release. To address this, we engineered a chemi-sensitive, two-dimensional composite nanofilm that facilitates visualization of the release and diffusion of the neurochemical dopamine with synaptic resolution, quantal sensitivity, and simultaneously from hundreds of release sites. Using this technology, we were able to monitor the spatiotemporal dynamics of dopamine release in dendritic processes, a poorly understood phenomenon. We found that dopamine release is broadcast from a subset of dendritic processes as hotspots that have a mean spatial spread of ≈ 3.2 µm (full width at half maximum [FWHM]) and are observed with a mean spatial frequency of one hotspot per ≈ 7.5 µm of dendritic length. Major dendrites of dopamine neurons and fine dendritic processes, as well as dendritic arbors and dendrites with no apparent varicose morphology participated in dopamine release. Remarkably, these release hotspots co-localized with Bassoon, suggesting that Bassoon may contribute to organizing active zones in dendrites, similar to its role in axon terminals.
To form the vast and complex network necessary for an organism to sense and react to the world, neurons must connect at highly specialized junctions. Individual cells communicate at these 'synapses' by releasing chemical signals (or neurotransmitters) such as dopamine, a molecule involved in learning and motivation. Despite the central role that synapses play in the brain, it remains challenging to measure exactly where neurotransmitters are released and how far they travel from their release site. Currently, most tools available to scientists only allow bulk measurements of neurotransmitter release. To tackle this limitation, Bulumulla et al. developed a new way to measure neurotransmitter release from neurons, harnessing a technique which uses fluorescent nanosensors that glow brighter when exposed to dopamine. These sensors form a very thin film upon which neurons can grow; when the cells release dopamine, the sensors 'light up' as they encounter the molecule. Dubbed DopaFilm, the technology reveals exactly where the neurotransmitter comes from and how it spreads between cells in real time. In particular, the approach showed that dopamine emerges from 'hot spots' at specific sites in cells; it also helped Bulumulla et al. study how dopamine is released from subcellular compartments that have previously not been well characterized. Improving the sensors so that the film could detect other neurotransmitters besides dopamine would broaden the use of this approach. In the future, combining this technology with other types of imaging should enable studies of individual synapses with intricate detail.
Asunto(s)
Dopamina , Transmisión Sináptica , Neuronas Dopaminérgicas , Terminales Presinápticos , Transmisión Sináptica/fisiologíaRESUMEN
Teeth are composed of many tissues, covered by an inflexible and obdurate enamel. Unlike most other tissues, teeth become extremely cold sensitive when inflamed. The mechanisms of this cold sensation are not understood. Here, we clarify the molecular and cellular components of the dental cold sensing system and show that sensory transduction of cold stimuli in teeth requires odontoblasts. TRPC5 is a cold sensor in healthy teeth and, with TRPA1, is sufficient for cold sensing. The odontoblast appears as the direct site of TRPC5 cold transduction and provides a mechanism for prolonged cold sensing via TRPC5's relative sensitivity to intracellular calcium and lack of desensitization. Our data provide concrete functional evidence that equipping odontoblasts with the cold-sensor TRPC5 expands traditional odontoblast functions and renders it a previously unknown integral cellular component of the dental cold sensing system.
RESUMEN
Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for electrophysiologists for many decades. The method known as "spermatozoan patch clamp" was introduced in 2006. It has enabled the direct recording of ion channel activity in whole-cell and cell-attached configurations and has been instrumental in describing sperm cell physiology and the molecular identity of various calcium, potassium, sodium, chloride, and proton ion channels. However, recording from single spermatozoa requires advanced skills and training in electrophysiology. This detailed protocol summarizes the step-by-step procedure and highlights several 'tricks-of-the-trade' in order to make it available to anyone who wishes to explore the fascinating physiology of the sperm cell. Specifically, the protocol describes recording from human and murine sperm cells but can be adapted to essentially any mammalian sperm cell of any species. The protocol covers important details of the application of this technique, such as isolation of sperm cells, selection of reagents and equipment, immobilization of the highly motile cells, formation of the tight (Gigaohm) seal between a recording electrode and the plasma membrane of the sperm cells, transition into the whole-spermatozoan mode (also known as break-in), and exemplary recordings of the sperm cell calcium ion channel, CatSper, from six mammalian species. The advantages and limitations of the sperm patch clamp method, as well as the most critical steps, are discussed.
Asunto(s)
Membrana Celular/fisiología , Fenómenos Electrofisiológicos , Espermatozoides/fisiología , Animales , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Tamaño de la Célula , Disección , Fenómenos Electrofisiológicos/efectos de los fármacos , Flagelos/efectos de los fármacos , Flagelos/fisiología , Humanos , Concentración de Iones de Hidrógeno , Transporte Iónico/efectos de los fármacos , Macaca mulatta , Masculino , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Perfusión , Progesterona/farmacología , Soluciones , Espermatozoides/citología , Espermatozoides/efectos de los fármacosRESUMEN
NaChBac, the first bacterial voltage-gated Na+ (Nav) channel to be characterized, has been the prokaryotic prototype for studying the structure-function relationship of Nav channels. Discovered nearly two decades ago, the structure of NaChBac has not been determined. Here we present the single particle electron cryomicroscopy (cryo-EM) analysis of NaChBac in both detergent micelles and nanodiscs. Under both conditions, the conformation of NaChBac is nearly identical to that of the potentially inactivated NavAb. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Nav channels in lipid bilayers. To study GMTs in mammalian Nav channels, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Nav1.7 replaced the corresponding sequence in NaChBac. Cryo-EM structures of the nanodisc-embedded chimera alone and in complex with HuwenToxin IV (HWTX-IV) were determined to 3.5 and 3.2 Å resolutions, respectively. Compared to the structure of HWTX-IV-bound human Nav1.7, which was obtained at an overall resolution of 3.2 Å, the local resolution of the toxin has been improved from â¼6 to â¼4 Å. This resolution enabled visualization of toxin docking. NaChBac can thus serve as a convenient surrogate for structural studies of the interactions between GMTs and Nav channels in a membrane environment.
Asunto(s)
Proteínas Bacterianas/química , Microscopía por Crioelectrón/métodos , Nanoestructuras/química , Canales de Sodio Activados por Voltaje/química , Canales de Sodio Activados por Voltaje/metabolismo , Animales , Proteínas Bacterianas/genética , Humanos , Membrana Dobles de Lípidos/química , Modelos Moleculares , Conformación Proteica , Canales de Sodio , Venenos de Araña/química , Canales de Sodio Activados por Voltaje/genéticaRESUMEN
Targeting small-molecule fluorescent indicators using genetically encoded protein tags yields new hybrid sensors for biological imaging. Optimization of such systems requires redesign of the synthetic indicator to allow cell-specific targeting without compromising the photophysical properties or cellular performance of the small-molecule probe. We developed a bright and sensitive Ca2+ indicator by systematically exploring the relative configuration of dye and chelator, which can be targeted using the HaloTag self-labeling tag system. Our "isomeric tuning" approach is generalizable, yielding a far-red targetable indicator to visualize Ca2+ fluxes in the primary cilium.
RESUMEN
The transient receptor potential canonical subfamily member 5 (TRPC5), one of seven mammalian TRPC members, is a nonselective calcium-permeant cation channel. TRPC5 is of considerable interest as a drug target in the treatment of progressive kidney disease, depression, and anxiety. Here, we present the 2.8-Å resolution cryo-electron microscopy (cryo-EM) structure of the mouse TRPC5 (mTRPC5) homotetramer. Comparison of the TRPC5 structure to previously determined structures of other TRPC and TRP channels reveals differences in the extracellular pore domain and in the length of the S3 helix. The disulfide bond at the extracellular side of the pore and a preceding small loop are essential elements for its proper function. This high-resolution structure of mTRPC5, combined with electrophysiology and mutagenesis, provides insight into the lipid modulation and gating mechanisms of the TRPC family of ion channels.
Asunto(s)
Secuencia Conservada , Microscopía por Crioelectrón , Canales Catiónicos TRPC/metabolismo , Canales Catiónicos TRPC/ultraestructura , Animales , Sitios de Unión , Cationes , Gadolinio/farmacología , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Cinética , Lantano/farmacología , Lípidos/química , Ratones , Mutación/genética , Relación Estructura-Actividad , Canales Catiónicos TRPC/química , Canales Catiónicos TRPC/genéticaRESUMEN
Members of the transient receptor potential (TRP) ion channels conduct cations into cells. They mediate functions ranging from neuronally mediated hot and cold sensation to intracellular organellar and primary ciliary signaling. Here we report a cryo-electron microscopy (cryo-EM) structure of TRPC4 in its unliganded (apo) state to an overall resolution of 3.3 Å. The structure reveals a unique architecture with a long pore loop stabilized by a disulfide bond. Beyond the shared tetrameric six-transmembrane fold, the TRPC4 structure deviates from other TRP channels with a unique cytosolic domain. This unique cytosolic N-terminal domain forms extensive aromatic contacts with the TRP and the C-terminal domains. The comparison of our structure with other known TRP structures provides molecular insights into TRPC4 ion selectivity and extends our knowledge of the diversity and evolution of the TRP channels.
Asunto(s)
Canales Catiónicos TRPC/química , Animales , Microscopía por Crioelectrón , Disulfuros , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Ratones , Dominios Proteicos , Pliegue de ProteínaRESUMEN
The transient receptor potential ion channel subfamily M, member 7 (TRPM7), is a ubiquitously expressed protein that is required for mouse embryonic development. TRPM7 contains both an ion channel and an α-kinase. The channel domain comprises a nonselective cation channel with notable permeability to Mg2+ and Zn2+ Here, we report the closed state structures of the mouse TRPM7 channel domain in three different ionic conditions to overall resolutions of 3.3, 3.7, and 4.1 Å. The structures reveal key residues for an ion binding site in the selectivity filter, with proposed partially hydrated Mg2+ ions occupying the center of the conduction pore. In high [Mg2+], a prominent external disulfide bond is found in the pore helix, which is essential for ion channel function. Our results provide a structural framework for understanding the TRPM1/3/6/7 subfamily and extend the knowledge base upon which to study the diversity and evolution of TRP channels.
Asunto(s)
Embrión de Mamíferos , Desarrollo Embrionario , Evolución Molecular , Canales Catiónicos TRPM/química , Animales , Ratones , Dominios Proteicos , Canales Catiónicos TRPM/metabolismoRESUMEN
We report the near atomic resolution (3.3 Å) of the human polycystic kidney disease 2-like 1 (polycystin 2-l1) ion channel. Encoded by PKD2L1, polycystin 2-l1 is a calcium and monovalent cation-permeant ion channel in primary cilia and plasma membranes. The related primary cilium-specific polycystin-2 protein, encoded by PKD2, shares a high degree of sequence similarity, yet has distinct permeability characteristics. Here we show that these differences are reflected in the architecture of polycystin 2-l1.
Asunto(s)
Canales de Calcio/ultraestructura , Microscopía por Crioelectrón , Receptores de Superficie Celular/ultraestructura , Calcio , Canales de Calcio/química , Canales de Calcio/metabolismo , Cationes/metabolismo , Humanos , Modelos Moleculares , Conformación Proteica , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismoRESUMEN
Mutations in the polycystin genes, PKD1 or PKD2, results in Autosomal Dominant Polycystic Kidney Disease (ADPKD). Although a genetic basis of ADPKD is established, we lack a clear understanding of polycystin proteins' functions as ion channels. This question remains unsolved largely because polycystins localize to the primary cilium - a tiny, antenna-like organelle. Using a new ADPKD mouse model, we observe primary cilia that are abnormally long in cells associated with cysts after conditional ablation of Pkd1 or Pkd2. Using primary cultures of collecting duct cells, we show that polycystin-2, but not polycystin-1, is a required subunit for the ion channel in the primary cilium. The polycystin-2 channel preferentially conducts K+ and Na+; intraciliary Ca2+, enhances its open probability. We introduce a novel method for measuring heterologous polycystin-2 channels in cilia, which will have utility in characterizing PKD2 variants that cause ADPKD.
Asunto(s)
Cationes/metabolismo , Cilios/química , Células Epiteliales/química , Túbulos Renales/química , Potasio/metabolismo , Sodio/metabolismo , Canales Catiónicos TRPP/análisis , Animales , Humanos , Ratones , Ratones TransgénicosRESUMEN
Transient receptor potential melastatin subfamily member 4 (TRPM4) is a widely distributed, calcium-activated, monovalent-selective cation channel. Mutations in human TRPM4 (hTRPM4) result in progressive familial heart block. Here, we report the electron cryomicroscopy structure of hTRPM4 in a closed, Na+-bound, apo state at pH 7.5 to an overall resolution of 3.7 Å. Five partially hydrated sodium ions are proposed to occupy the center of the conduction pore and the entrance to the coiled-coil domain. We identify an upper gate in the selectivity filter and a lower gate at the entrance to the cytoplasmic coiled-coil domain. Intramolecular interactions exist between the TRP domain and the S4-S5 linker, N-terminal domain, and N and C termini. Finally, we identify aromatic interactions via π-π bonds and cation-π bonds, glycosylation at an N-linked extracellular site, a pore-loop disulfide bond, and 24 lipid binding sites. We compare and contrast this structure with other TRP channels and discuss potential mechanisms of regulation and gating of human full-length TRPM4.
Asunto(s)
Canales Catiónicos TRPM/química , Canales Catiónicos TRPM/metabolismo , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Sodio/química , Sodio/metabolismo , Canales Catiónicos TRPM/genéticaRESUMEN
TRPM6 and TRPM7 are members of the melastatin-related transient receptor potential (TRPM) subfamily of ion channels. Deletion of either gene in mice is embryonically lethal. TRPM6/7 are the only known examples of single polypeptides containing both an ion channel pore and a serine/threonine kinase (chanzyme). Here we show that the C-terminal kinase domain of TRPM6 is cleaved from the channel domain in a cell type-specific fashion and is active. Cleavage requires that the channel conductance is functional. The cleaved kinase translocates to the nucleus, where it is strictly localized and phosphorylates specific histone serine and threonine (S/T) residues. TRPM6-cleaved kinases (M6CKs) bind subunits of the protein arginine methyltransferase 5 (PRMT5) molecular complex that make important epigenetic modifications by methylating histone arginine residues. Histone phosphorylation by M6CK results in a dramatic decrease in methylation of arginines adjacent to M6CK-phosphorylated amino acids. Knockout of TRPM6 or inactivation of its kinase results in global changes in histone S/T phosphorylation and changes the transcription of hundreds of genes. We hypothesize that M6CK associates with the PRMT5 molecular complex in the nucleus, directing M6CK to a specific genomic location and providing site-specific histone phosphorylation. M6CK histone phosphorylation, in turn, regulates transcription by attenuating the effect of local arginine methylation.
Asunto(s)
Arginina/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , Canales Catiónicos TRPM/metabolismo , Arginina/química , Arginina/genética , Línea Celular , Histonas/química , Histonas/genética , Humanos , Metilación , Fosforilación , Dominios Proteicos , Serina/genética , Serina/metabolismo , Canales Catiónicos TRPM/química , Canales Catiónicos TRPM/genética , Treonina/genética , Treonina/metabolismoRESUMEN
TRPM7 (transient receptor potential cation channel subfamily M member 7) regulates gene expression and stress-induced cytotoxicity and is required in early embryogenesis through organ development. Here, we show that the majority of TRPM7 is localized in abundant intracellular vesicles. These vesicles (M7Vs) are distinct from endosomes, lysosomes, and other familiar vesicles or organelles. M7Vs accumulate Zn2+ in a glutathione-enriched, reduced lumen when cytosolic Zn2+ concentrations are elevated. Treatments that increase reactive oxygen species (ROS) trigger TRPM7-dependent Zn2+ release from the vesicles, whereas reduced glutathione prevents TRPM7-dependent cytosolic Zn2+ influx. These observations strongly support the notion that ROS-mediated TRPM7 activation releases Zn2+ from intracellular vesicles after Zn2+ overload. Like the endoplasmic reticulum, these vesicles are a distributed system for divalent cation uptake and release, but in this case the primary divalent ion is Zn2+ rather than Ca2.
Asunto(s)
Estrés Oxidativo , Proteínas Serina-Treonina Quinasas/metabolismo , Canales Catiónicos TRPM/metabolismo , Vesículas Transportadoras/metabolismo , Zinc/metabolismo , Desarrollo Embrionario , Glutatión/metabolismo , Células HEK293 , Humanos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
We report that the Gm7068 (CatSpere) and Tex40 (CatSperz) genes encode novel subunits of a 9-subunit CatSper ion channel complex. Targeted disruption of CatSperz reduces CatSper current and sperm rheotactic efficiency in mice, resulting in severe male subfertility. Normally distributed in linear quadrilateral nanodomains along the flagellum, the complex lacking CatSperζ is disrupted at ~0.8 µm intervals along the flagellum. This disruption renders the proximal flagellum inflexible and alters the 3D flagellar envelope, thus preventing sperm from reorienting against fluid flow in vitro and efficiently migrating in vivo. Ejaculated CatSperz-null sperm cells retrieved from the mated female uterus partially rescue in vitro fertilization (IVF) that failed with epididymal spermatozoa alone. Human CatSperε is quadrilaterally arranged along the flagella, similar to the CatSper complex in mouse sperm. We speculate that the newly identified CatSperζ subunit is a late evolutionary adaptation to maximize fertilization inside the mammalian female reproductive tract.