RESUMEN
Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labeling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labeling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-color receptor imaging; and pulse-chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions.
Asunto(s)
Neuronas , Proteínas , Ratones , Animales , Indicadores y Reactivos , Ligandos , EncéfaloRESUMEN
Rational synthetic expansion of photoresponsive ligands is important for photopharmacological studies. Adenosine A2A receptor (A2AR) is stimulated by adenosine and related in Parkinson's disease and other diseases. Here, we report the crystal structure of the A2AR in complex with the novel photoresponsive ligand photoNECA (blue) at 3.34 Å resolution. PhotoNECA (blue) was designed for this structural study and the cell-based assay showed a photoresponsive and receptor selective characteristics of photoNECA (blue) for A2AR. The crystal structure explains the binding mode, photoresponsive mechanism and receptor selectivity of photoNECA (blue). Our study would promote not only the rational design of photoresponsive ligands but also dynamic structural studies of A2AR.
Asunto(s)
Receptor de Adenosina A2A , Humanos , Adenosina/metabolismo , Ligandos , Enfermedad de Parkinson , Receptor de Adenosina A2A/química , Receptor de Adenosina A2A/metabolismo , Fotoquímica/métodos , Colorantes Fluorescentes/químicaRESUMEN
Proteins are the workhorse molecules performing various tasks to sustain life. To investigate the roles of a protein under physiological conditions, the rapid modulation of the protein with high specificity in a living system would be ideal, but achieving this is often challenging. To address this challenge, researchers have developed chemogenetic strategies for the rapid and selective modulation of protein function in live cells. Here, the target protein is modified genetically to become sensitive to a designer molecule that otherwise has no effect on other cellular biomolecules. One powerful chemogenetic strategy is to introduce a tethering point into the target protein, allowing covalent or non-covalent attachment of the designer molecule. In this tutorial review, we focus on tethering-based chemogenetic approaches for modulating protein function in live cells. We first describe genetic, optogenetic and chemical means to study protein function. These means lay the basis for the chemogenetic concept, which is explained in detail. The next section gives an overview, including advantages and limitations, of tethering tactics that have been employed for modulating cellular protein function. The third section provides examples of the modulation of cell-surface proteins using tethering-based chemogenetics through non-covalent tethering and covalent tethering for irreversible modulation or functional switching. The fourth section presents intracellular examples. The last section summarizes key considerations in implementing tethering-based chemogenetics and shows perspectives highlighting future directions and other applications of this burgeoning research field.
Asunto(s)
Proteínas/genética , Proteínas/metabolismo , Animales , Supervivencia Celular , Humanos , Optogenética , Proteínas/químicaRESUMEN
Zinc signaling and dynamics play significant roles in many physiological responses and diseases. To understand the physiological roles of zinc in detail, comprehensive identification of proteins under high concentration of mobile zinc ion is crucial. We developed a 'conditional proteomics' approach to identify proteins involved in zinc homeostasis based on a chemical proteomic strategy that utilizes designer zinc-responsive labeling reagents to tag such proteins and quantitative mass spectrometry for their identification. We used this method to elucidate zinc dyshomeostasis induced by nitric-oxide-triggered oxidative stress in glioma cells, and we unveiled dynamic changes of the zinc-related proteomes. Moreover, we characterized unknown zinc-rich vesicles generated by oxidative stress as endoplasmic-reticulum- and Golgi-related vesicles.
Asunto(s)
Proteínas Portadoras/metabolismo , Homeostasis/fisiología , Proteoma/metabolismo , Proteómica/métodos , Zinc/metabolismo , Sitios de Unión , Western Blotting , Proteínas Portadoras/genética , Línea Celular Tumoral , Electroforesis en Gel de Poliacrilamida , Glioma , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Microscopía Confocal , Óxido Nítrico/farmacología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Unión Proteica , Proteoma/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Coloración y EtiquetadoRESUMEN
Temperature influences the activities of living organisms at various levels. Cells not only detect environmental temperature changes through their unique temperature-sensitive molecular machineries but also muster an appropriate response to the temperature change to maintain their inherent functions. Despite the fundamental involvement of temperature in physiological phenomena, the mechanism by which cells produce and use heat is largely unknown. Recently, fluorescent thermosensors that function as thermometers in live cells have attracted much attention in biology. These new tools, made of various temperature-sensitive molecules, have allowed for intracellular thermometry at the single-cell level. Intriguing spatiotemporal temperature variations, including organelle-specific thermogenesis, have been revealed with these fluorescent thermosensors, which suggest an intrinsic connection between temperature and cell functions. Moreover, fluorescent thermosensors have shown that intracellular temperature changes at the microscopic level are largely different from those assumed for a water environment at the macroscopic level. Thus, the employment of fluorescent thermosensors will uncover novel mechanisms of intracellular temperature-assisted physiological functions.
Asunto(s)
Técnicas Biosensibles/métodos , Orgánulos/metabolismo , Termometría/métodos , Xantenos/química , Animales , Proteínas Fluorescentes Verdes/química , Humanos , Puntos Cuánticos/químicaRESUMEN
The fast inhibitory actions of γ-aminobutyric acid (GABA) are mainly mediated by GABAA receptors (GABAARs) in the brain. The existence of multiple ligand-binding sites and a lack of structural information have hampered the efficient screening of drugs capable of acting on GABAARs. We have developed semisynthetic fluorescent biosensors for orthosteric and allosteric GABAAR ligands on live cells via coupling of affinity-based chemical labeling reagents to a bimolecular fluorescence quenching and recovery system. These biosensors were amenable to the high-throughput screening of a chemical library, leading to the discovery of new small molecules capable of interacting with GABAARs. Electrophysiological measurements revealed that one hit, 4,4',4â³-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), was a novel negative allosteric modulator capable of strongly suppressing GABA-induced chloride currents. Thus, these semisynthetic biosensors represent versatile platforms for screening drugs to treat GABAAR-related neurological disorders, and this strategy can be extended to structurally complicated membrane proteins.
Asunto(s)
Fenoles/farmacología , Pirazoles/farmacología , Receptores de GABA-A/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Regulación Alostérica/efectos de los fármacos , Técnicas Biosensibles , Relación Dosis-Respuesta a Droga , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Estructura Molecular , Fenoles/química , Pirazoles/química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
In native systems, scaffolding proteins play important roles in assembling proteins into complexes to transduce signals. This concept is yet to be applied to the assembly of functional transmembrane protein complexes in artificial systems. To address this issue, DNA origami has the potential to serve as scaffolds that arrange proteins at specific positions in complexes. Herein, we report that Kir3 K+ channel proteins are assembled through zinc-finger protein (ZFP)-adaptors at specific locations on DNA origami scaffolds. Specific binding of the ZFP-fused Kir3 channels and ZFP-based adaptors on DNA origami were confirmed by atomic force microscopy and gel electrophoresis. Furthermore, the DNA origami with ZFP binding sites nearly tripled the K+ channel current activity elicited by heterotetrameric Kir3 channels in HEK293T cells. Thus, our method provides a useful template to control the oligomerization states of membrane protein complexes inâ vitro and in living cells.
RESUMEN
Catalyst-mediated protein modification is a powerful approach for the imaging and engineering of natural proteins. We have previously developed affinity-guided 4-dimethylaminopyridine (AGD) chemistry as an efficient protein modification method using a catalytic acyl transfer reaction. However, because of the high electrophilicity of the thioester acyl donor molecule, AGD chemistry suffers from nonspecific reactions to proteins other than the target protein in crude biological environments, such as cell lysates, live cells, and tissue samples. To overcome this shortcoming, we here report a new acyl donor/organocatalyst system that allows more specific and efficient protein modification. In this method, a highly nucleophilic pyridinium oxime (PyOx) catalyst is conjugated to a ligand specific to the target protein. The ligand-tethered PyOx selectively binds to the target protein and facilitates the acyl transfer reaction of a mild electrophilic N-acyl-N-alkylsulfonamide acyl donor on the protein surface. We demonstrated that the new catalytic system, called AGOX (affinity-guided oxime) chemistry, can modify target proteins, both in test tubes and cell lysates, more selectively and efficiently than AGD chemistry. Low-background fluorescence labeling of the endogenous cell-membrane proteins, carbonic anhydrase XII and the folate receptor, in live cells allowed for the precise quantification of diffusion coefficients in the protein's native environment. Furthermore, the excellent biocompatibility and bioorthogonality of AGOX chemistry were demonstrated by the selective labeling of an endogenous neurotransmitter receptor in mouse brain slices, which are highly complicated tissue samples.
Asunto(s)
Oximas/química , Proteínas/análisis , Coloración y Etiquetado/métodos , 4-Aminopiridina/análogos & derivados , 4-Aminopiridina/química , Células A549 , Acilación , Animales , Química Encefálica , Anhidrasas Carbónicas/análisis , Receptores de Folato Anclados a GPI/análisis , Células HEK293 , Células HeLa , Humanos , Proteínas de la Membrana/análisis , Ratones , Receptores de Neurotransmisores/análisisRESUMEN
Transient Receptor Potential (TRP) proteins form cation channels characterized by a wide variety of activation triggers. Here, we overview a group of TRP channels that respond to reactive redox species to transduce physiological signals, with a focus on TRPA1 and its role in oxygen physiology. Our systematic evaluation of oxidation sensitivity using cysteine-selective reactive disulphides with different redox potentials reveals that TRPA1 has the highest sensitivity to oxidants/electrophiles among the TRP channels, which enables it to sense O2. Proline hydroxylation by O2-dependent hydroxylases also regulates the O2-sensing function by inhibiting TRPA1 in normoxia; TRPA1 is activated by hypoxia through relief from the inhibition and by hyperoxia through cysteine oxidation that overrides the inhibition. TRPA1 enhances neuronal discharges induced by hyperoxia and hypoxia in the vagus to underlie respiratory adaptation to changes in O2 availability. This importance of TRPA1 in non-carotid body O2 sensors can be extended to the universal significance of redox-sensitive TRP channels in O2 adaptation.
Asunto(s)
Oxígeno/metabolismo , Canal Catiónico TRPA1/metabolismo , Secuencia de Aminoácidos , Animales , Evolución Molecular , Humanos , Hipoxia/metabolismo , Canal Catiónico TRPA1/químicaRESUMEN
Transient receptor potential canonical (TRPC) proteins form Ca(2+)-permeable cation channels activated upon stimulation of metabotropic receptors coupled to phospholipase C. Among the TRPC subfamily, TRPC3 and TRPC6 channels activated directly by diacylglycerol (DAG) play important roles in brain-derived neurotrophic factor (BDNF) signaling, promoting neuronal development and survival. In various disease models, BDNF restores neurologic deficits, but its therapeutic potential is limited by its poor pharmacokinetic profile. Elucidation of a framework for designing small molecules, which elicit BDNF-like activity via TRPC3 and TRPC6, establishes a solid basis to overcome this limitation. We discovered, through library screening, a group of piperazine-derived compounds that activate DAG-activated TRPC3/TRPC6/TRPC7 channels. The compounds [4-(5-chloro-2-methylphenyl)piperazin-1-yl](3-fluorophenyl)methanone (PPZ1) and 2-[4-(2,3-dimethylphenyl)piperazin-1-yl]-N-(2-ethoxyphenyl)acetamide (PPZ2) activated, in a dose-dependent manner, recombinant TRPC3/TRPC6/TRPC7 channels, but not other TRPCs, in human embryonic kidney cells. PPZ2 activated native TRPC6-like channels in smooth muscle cells isolated from rabbit portal vein. Also, PPZ2 evoked cation currents and Ca(2+) influx in rat cultured central neurons. Strikingly, both compounds induced BDNF-like neurite growth and neuroprotection, which were abolished by a knockdown or inhibition of TRPC3/TRPC6/TRPC7 in cultured neurons. Inhibitors of Ca(2+) signaling pathways, except calcineurin, impaired neurite outgrowth promotion induced by PPZ compounds. PPZ2 increased activation of the Ca(2+)-dependent transcription factor, cAMP response element-binding protein. These findings suggest that Ca(2+) signaling mediated by activation of DAG-activated TRPC channels underlies neurotrophic effects of PPZ compounds. Thus, piperazine-derived activators of DAG-activated TRPC channels provide important insights for future development of a new class of synthetic neurotrophic drugs.
Asunto(s)
Factores de Crecimiento Nervioso/metabolismo , Piperazinas/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Evaluación Preclínica de Medicamentos/métodos , Femenino , Células HEK293 , Humanos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/farmacología , Piperazinas/química , Piperazinas/farmacología , Conejos , Ratas , Ratas Wistar , Canales Catiónicos TRPC/agonistasRESUMEN
Protein functions are tightly regulated by their subcellular localization in live cells, and quantitative evaluation of dynamically altered proteomes in each organelle should provide valuable information. Here, we describe a novel method for organelle-focused chemical proteomics using spatially limited reactions. In this work, mitochondria-localizable reactive molecules (MRMs) were designed that penetrate biomembranes and spontaneously concentrate in mitochondria, where protein labeling is facilitated by the condensation effect. The combination of this selective labeling and liquid chromatography-mass spectrometry (LC-MS) based proteomics technology facilitated identification of mitochondrial proteomes and the profile of the intrinsic reactivity of amino acids tethered to proteins expressed in live cultured cells, primary neurons and brain slices. Furthermore, quantitative profiling of mitochondrial proteins whose expression levels change significantly during an oxidant-induced apoptotic process was performed by combination of this MRMs-based method with a standard quantitative MS technique (SILAC: stable isotope labeling by amino acids in cell culture). The use of a set of MRMs represents a powerful tool for chemical proteomics to elucidate mitochondria-associated biological events and diseases.
Asunto(s)
Encéfalo/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/química , Proteoma/metabolismo , Proteómica/métodos , Sitios de Unión , Técnicas de Cultivo de Célula , Cromatografía Líquida de Alta Presión , Células HeLa , Humanos , Marcaje Isotópico , Microscopía Confocal , Modelos Moleculares , Rodaminas/química , Espectrometría de Masas en TándemRESUMEN
Calcium current through voltage-gated calcium channels (VGCC) controls gene expression. Here, we describe a novel signalling pathway in which the VGCC Cacnb4 subunit directly couples neuronal excitability to transcription. Electrical activity induces Cacnb4 association to Ppp2r5d, a regulatory subunit of PP2A phosphatase, followed by (i) nuclear translocation of Cacnb4/Ppp2r5d/PP2A, (ii) association with the tyrosine hydroxylase (TH) gene promoter through the nuclear transcription factor thyroid hormone receptor alpha (TRα), and (iii) histone binding through association of Cacnb4 with HP1γ concomitantly with Ser(10) histone H3 dephosphorylation by PP2A. This signalling cascade leads to TH gene repression by Cacnb4 and is controlled by the state of interaction between the SH3 and guanylate kinase (GK) modules of Cacnb4. The human R482X CACNB4 mutation, responsible for a form of juvenile myoclonic epilepsy, prevents association with Ppp2r5 and nuclear targeting of the complex by altering Cacnb4 conformation. These findings demonstrate that an intact VGCC subunit acts as a repressor recruiting platform to control neuronal gene expression.
Asunto(s)
Canales de Calcio/biosíntesis , Canales de Calcio/genética , Epilepsias Mioclónicas/metabolismo , Regulación de la Expresión Génica , Transporte Activo de Núcleo Celular , Animales , Biofisica/métodos , Canales de Calcio/metabolismo , Electrofisiología/métodos , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Ratones , Mutación , Proteína Fosfatasa 2/metabolismo , Transducción de Señal , Receptores alfa de Hormona Tiroidea/metabolismo , Transcripción GenéticaRESUMEN
In mammals and birds, thermoregulation to conserve body temperature is vital to life. Multiple mechanisms of thermogeneration have been proposed, localized in different subcellular organelles. However, visualizing thermogenesis directly in intact organelles has been challenging. Here we have developed genetically encoded, GFP-based thermosensors (tsGFPs) that enable visualization of thermogenesis in discrete organelles in living cells. In tsGFPs, a tandem formation of coiled-coil structures of the Salmonella thermosensing protein TlpA transmits conformational changes to GFP to convert temperature changes into visible and quantifiable fluorescence changes. Specific targeting of tsGFPs enables visualization of thermogenesis in the mitochondria of brown adipocytes and the endoplasmic reticulum of myotubes. In HeLa cells, tsGFP targeted to mitochondria reveals heterogeneity in thermogenesis that correlates with the electrochemical gradient. Thus, tsGFPs are powerful tools to noninvasively assess thermogenesis in living cells.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Salmonella enterica/metabolismo , Temperatura , Adenoviridae/genética , Adipocitos Marrones/citología , Proteínas Bacterianas/química , ADN Complementario/metabolismo , Escherichia coli/metabolismo , Células HeLa , Calor , Humanos , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente/métodos , Mitocondrias/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Conformación ProteicaRESUMEN
Among amino acids, leucine is a potential signaling molecule to regulate cell growth and metabolism by activating mechanistic target of rapamycin complex 1 (mTORC1). To reveal the critical structures of leucine molecule to activate mTORC1, we examined the structure-activity relationships of leucine derivatives in HeLa S3 cells for cellular uptake and for the induction of phosphorylation of p70 ribosomal S6 kinase 1 (p70S6K), a downstream effector of mTORC1. The activation of mTORC1 by leucine and its derivatives was the consequence of two successive events: the cellular uptake by L-type amino acid transporter 1 (LAT1) responsible for leucine uptake in HeLa S3 cells and the activation of mTORC1 following the transport. The structural requirement for the recognition by LAT1 was to have carbonyl oxygen, alkoxy oxygen of carboxyl group, amino group and hydrophobic side chain. In contrast, the requirement for mTORC1 activation was more rigorous. It additionally required fixed distance between carbonyl oxygen and alkoxy oxygen of carboxyl group, and amino group positioned at α-carbon. L-Configuration in chirality and appropriate length of side chain with a terminal isopropyl group were also important. This confirmed that LAT1 itself is not a leucine sensor. Some specialized leucine sensing mechanism with rigorous requirement for agonistic structures should exist inside the cells because leucine derivatives not transported by LAT1 did not activate mTORC1. Because LAT1-mTOR axis is involved in the regulation of cell growth and cancer progression, the results from this study may provide a new insight into therapeutics targeting both LAT1 and leucine sensor.
Asunto(s)
Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Leucina/farmacología , Complejos Multiproteicos/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Transporte Biológico , Expresión Génica , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Transportador de Aminoácidos Neutros Grandes 1/genética , Leucina/análogos & derivados , Leucina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/genética , Fosforilación/efectos de los fármacos , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Transducción de Señal , Relación Estructura-Actividad , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Cholinergically induced network activity is a useful analogue of theta rhythms involved in memory processing or epileptiform activity in the hippocampus, providing a powerful tool to elucidate the mechanisms of synchrony in neuronal networks. In absence epilepsy, although its association with cognitive impairments has been reported, the mechanisms underlying hippocampal synchrony remain poorly investigated. Here we simultaneously recorded electrical activities from 64 sites in hippocampal slices of CaV2.1 Ca(2+) channel mutant tottering (tg) mice, a well-established mouse model of spontaneous absence epilepsy, to analyze the spatiotemporal pattern of cholinergically induced hippocampal network activity. The cholinergic agonist carbachol induced oscillatory discharges originating from the CA3 region. In tg/tg mice, this hippocampal network activity was characterized by enhanced occupancy of discharges of relatively high frequency (6-10 Hz) compared to the wild type. Pharmacological analyses of slices, patch clamp electrophysiological characterization of isolated neurons, and altered patterns of hippocampal GABAA receptor subunit and Cl(-) transporter messenger RNA (mRNA) transcript levels revealed that this abnormality is attributable to a developmental retardation of GABAergic inhibition caused by immature intracellular Cl(-) regulation. These results suggest that the inherited CaV2.1 Ca(2+) channel mutation leads to developmental abnormalities in Cl(-) transporter expression and GABAA receptor compositions in hippocampal neurons and that compromised maturation of GABAergic inhibition contributes to the abnormal synchrony in the hippocampus of tg absence epileptic mice.
Asunto(s)
Región CA3 Hipocampal/metabolismo , Canales de Calcio Tipo N/metabolismo , Epilepsia/genética , Neuronas GABAérgicas/metabolismo , Inhibición Neural , Receptores de GABA-A/metabolismo , Potenciales de Acción , Animales , Región CA3 Hipocampal/citología , Región CA3 Hipocampal/crecimiento & desarrollo , Región CA3 Hipocampal/fisiopatología , Canales de Calcio Tipo N/genética , Células Cultivadas , Cloruros/metabolismo , Epilepsia/metabolismo , Epilepsia/fisiopatología , Neuronas GABAérgicas/fisiología , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de GABA-A/genética , Ácido gamma-Aminobutírico/metabolismoRESUMEN
S-Nitrosylation, the addition of a nitrosyl group to cysteine thiols, regulates various protein functions to mediate nitric oxide (NO) bioactivity. Recent studies have demonstrated that selectivity in protein S-nitrosylation signaling pathways is conferred through transnitrosylation, a transfer of the NO group, between proteins via interaction. We previously demonstrated that sensitivity to activation by synthetic NO-releasing agents via S-nitrosylation is a common feature of members of the transient receptor potential (TRP) family of Ca(2+)-permeable cation channels. However, strategies to confer subtype selectivity to nitrosylating agents targeted to TRP channels are yet to be developed. Here, we show selective activation of TRPA1 channels by novel NO donors derived from the ABBH (7-azabenzobicyclo[2.2.1]heptane) N-nitrosamines, which exhibit transnitrosylation reactivity to thiols without releasing NO. The NNO-ABBH1 (N-nitroso-2-exo,3-exo-ditrifluoromethyl-7-azabenzobicyclo[2.2.1]heptane) elicits S-nitrosylation of TRPA1 proteins, and dose-dependently induces robust Ca(2+) influx via both recombinant and native TRPA1 channels, but not via other NO-activated TRP channels. TRPA1 activation by NNO-ABBH1 is suppressed by specific cysteine mutations but not by NO scavenging, suggesting that cysteine transnitrosylation underlies the activation of TRPA1 by NNO-ABBH1. This is supported by the correlation of N-NO bond reactivity and TRPA1-activating potency in a congeneric series of ABBH N-nitrosamines. Interestingly, nonelectrophilic derivatives of ABBH also activate TRPA1 selectively, but less potently, compared with NNO-ABBH1. Thus, ABBH N-nitrosamines confer subtype selectivity on S-nitrosylation in TRP channels through synergetic effects of two chemical processes: cysteine transnitrosylation and molecular recognition of the nonelectrophilic moiety.
Asunto(s)
Compuestos Aza/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Canales de Calcio/metabolismo , Heptanos/farmacología , Proteínas del Tejido Nervioso/metabolismo , Donantes de Óxido Nítrico/farmacología , Nitrosaminas/farmacología , Canales de Potencial de Receptor Transitorio/metabolismo , Compuestos Aza/síntesis química , Compuestos Aza/química , Compuestos Bicíclicos Heterocíclicos con Puentes/síntesis química , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Células HEK293 , Heptanos/síntesis química , Heptanos/química , Humanos , Donantes de Óxido Nítrico/síntesis química , Donantes de Óxido Nítrico/química , Nitrosaminas/síntesis química , Nitrosaminas/química , Técnicas de Placa-Clamp , Canal Catiónico TRPA1RESUMEN
Synaptic AMPA receptors (AMPARs) on neuronal plasma membranes are correlated with learning and memory. Using a unique labeling and super-resolution imaging, we have visualized the nanoscale synaptic and extra-synaptic organization of native surface AMPARs for the first time in mouse brain slices as a function of brain region and tauopathy. We find that the fraction of surface AMPARs organized in synaptic clusters is two-times smaller in the hippocampus compared to the motor and somatosensory cortex. In 6 months old PS19 model of tauopathy, synaptic and extrasynaptic distributions are disrupted in the hippocampus but not in the cortex. Thus, this optimized super-resolution imaging tool allows us to observe synaptic deterioration at the onset of tauopathy before apparent neurodegeneration.
RESUMEN
The molecular mechanism underlying Ca(2+)/calmodulin (CaM)-dependent kinase II (CaMKII)-mediated regulation of the mouse transient receptor potential channel TRPC6 was explored by chimera, deletion and site-directed mutagenesis approaches. Induction of currents (ICCh) in TRPC6-expressing HEK293 cells by a muscarinic agonist carbachol (CCh; 100 µm) was strongly attenuated by a CaMKII-specific peptide, autocamtide-2-related inhibitory peptide (AIP; 10 µm). TRPC6/C7 chimera experiments showed that the TRPC6 C-terminal sequence is indispensable for ICCh to be sensitive to AIP-induced CaMKII inhibition. Further, deletion of a distal region (Gln(855)-Glu(877)) of the C-terminal CaM/inositol-1,4,5-trisphosphate receptor binding domain (CIRB) of TRPC6 was sufficient to abolish ICCh. Systematic alanine scanning for potential CaMKII phosphorylation sites revealed that Thr(487) was solely responsible for the activation of the TRPC6 channel by receptor stimulation. The abrogating effect of the alanine mutation of Thr(487) (T487A) was reproduced with other non-polar amino acids, namely glutamine or asparagine, while being partially rescued by phosphomimetic mutations with glutamate or aspartate. The cellular expression and distribution of TRPC6 channels did not significantly change with these mutations. Electrophysiological and immunocytochemical data with the Myc-tagged TRPC6 channel indicated that Thr(487) is most likely located at the intracellular side of the cell membrane. Overexpression of T487A caused significant reduction of endogenous TRPC6-like current induced by Arg(8)-vasopressin in A7r5 aortic myocytes. Based on these results, we propose that the optimal spatial arrangement of a C-terminal domain (presumably the distal CIRB region) around a single CaMKII phosphorylation site Thr(487) may be essential for CaMKII-mediated regulation of TRPC6 channels. This mechanism may be of physiological significance in a native environment such as in vascular smooth muscle cells.