RESUMO
BACKGROUND: The purinergic ATP-gated P2X7 receptor (P2X7R) is increasingly recognized to contribute to pathological neuroinflammation and brain hyperexcitability. P2X7R expression has been shown to be increased in the brain, including both microglia and neurons, in experimental models of epilepsy and patients. To date, the cell type-specific downstream effects of P2X7Rs during seizures remain, however, incompletely understood. METHODS: Effects of P2X7R signaling on seizures and epilepsy were analyzed in induced seizure models using male mice including the kainic acid model of status epilepticus and pentylenetetrazole model and in male and female mice in a genetic model of Dravet syndrome. RNA sequencing was used to analyze P2X7R downstream signaling during seizures. To investigate the cell type-specific role of the P2X7R during seizures and epilepsy, we generated mice lacking exon 2 of the P2rx7 gene in either microglia (P2rx7:Cx3cr1-Cre) or neurons (P2rx7:Thy-1-Cre). To investigate the protective potential of overexpressing P2X7R in GABAergic interneurons, P2X7Rs were overexpressed using adeno-associated virus transduction under the mDlx promoter. RESULTS: RNA sequencing of hippocampal tissue from wild-type and P2X7R knock-out mice identified both glial and neuronal genes, in particular genes involved in GABAergic signaling, under the control of the P2X7R following seizures. Mice with deleted P2rx7 in microglia displayed less severe acute seizures and developed a milder form of epilepsy, and microglia displayed an anti-inflammatory molecular profile. In contrast, mice lacking P2rx7 in neurons showed a more severe seizure phenotype when compared to epileptic wild-type mice. Analysis of single-cell expression data revealed that human P2RX7 expression is elevated in the hippocampus of patients with temporal lobe epilepsy in excitatory and inhibitory neurons. Functional studies determined that GABAergic interneurons display increased responses to P2X7R activation in experimental epilepsy. Finally, we show that viral transduction of P2X7R in GABAergic interneurons protects against evoked and spontaneous seizures in experimental temporal lobe epilepsy and in mice lacking Scn1a, a model of Dravet syndrome. CONCLUSIONS: Our results suggest a dual and opposing action of P2X7R in epilepsy and suggest P2X7R overexpression in GABAergic interneurons as a novel therapeutic strategy for acquired and, possibly, genetic forms of epilepsy.
Assuntos
Modelos Animais de Doenças , Microglia , Neurônios , Receptores Purinérgicos P2X7 , Convulsões , Animais , Microglia/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2X7/genética , Masculino , Camundongos , Convulsões/metabolismo , Convulsões/genética , Neurônios/metabolismo , Feminino , Camundongos Endogâmicos C57BL , Ácido Caínico , Epilepsias Mioclônicas/metabolismo , Epilepsias Mioclônicas/genética , Hipocampo/metabolismo , Estado Epiléptico/metabolismo , Estado Epiléptico/genética , Camundongos Knockout , Pentilenotetrazol , Transdução de Sinais , Neurônios GABAérgicos/metabolismo , Epilepsia/metabolismo , Epilepsia/genética , Encéfalo/metabolismoRESUMO
P2X7 receptors are nonselective cation channels that are activated by extracellular ATP and play important roles in inflammation. They differ from other P2X family members by a large intracellular C-terminus that mediates diverse signaling processes that are little understood. A recent cryo-EM study revealed that the C-terminus of the P2X7 receptor forms a unique cytoplasmic ballast domain that possesses a GDP-binding site as well as a dinuclear Zn2+ site. However, the molecular basis for the regulatory function of the ballast domain as well as the interplay between the various ligands remain unclear. Here, we successfully expressed a soluble trimeric P2X7 ballast domain (P2X7BD) and characterized its ligand binding properties using a biophysical approach. We identified calmodulin (CaM)-binding regions within the ballast domain and found that binding of Ca2+-CaM and GDP to P2X7BD have opposite effects on its stability. Small-angle X-ray scattering experiments indicate that Ca2+-CaM binding disrupts the trimeric state of P2X7BD. Our results provide a possible framework for the intracellular regulation of the P2X7 receptor.
Assuntos
Calmodulina , Receptores Purinérgicos P2X7 , Calmodulina/metabolismo , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Ligação Proteica , Sítios de Ligação , Domínios ProteicosRESUMO
OBJECTIVE: The P2X7 receptor (P2X7R) is an important contributor to neuroinflammation, responding to extracellularly released adenosine triphosphate. Expression of the P2X7R is increased in the brain in experimental and human epilepsy, and genetic or pharmacologic targeting of the receptor can reduce seizure frequency and severity in preclinical models. Experimentally induced seizures also increase levels of the P2X7R in blood. Here, we tested 18 F-JNJ-64413739, a positron emission tomography (PET) P2X7R antagonist, as a potential noninvasive biomarker of seizure-damage and epileptogenesis. METHODS: Status epilepticus was induced via an intra-amygdala microinjection of kainic acid. Static PET studies (30 min duration, initiated 30 min after tracer administration) were conducted 48 h after status epilepticus via an intravenous injection of 18 F-JNJ-64413739. PET images were coregistered with a brain magnetic resonance imaging atlas, tracer uptake was determined in the different brain regions and peripheral organs, and values were correlated to seizure severity during status epilepticus. 18 F-JNJ-64413739 was also applied to ex vivo human brain slices obtained following surgical resection for intractable temporal lobe epilepsy. RESULTS: P2X7R radiotracer uptake correlated strongly with seizure severity during status epilepticus in brain structures including the cerebellum and ipsi- and contralateral cortex, hippocampus, striatum, and thalamus. In addition, a correlation between radiotracer uptake and seizure severity was also evident in peripheral organs such as the heart and the liver. Finally, P2X7R radiotracer uptake was found elevated in brain sections from patients with temporal lobe epilepsy when compared to control. SIGNIFICANCE: Taken together, our data suggest that P2X7R-based PET imaging may help to identify seizure-induced neuropathology and temporal lobe epilepsy patients with increased P2X7R levels possibly benefitting from P2X7R-based treatments.
Assuntos
Epilepsia do Lobo Temporal , Estado Epiléptico , Camundongos , Humanos , Masculino , Animais , Epilepsia do Lobo Temporal/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2X7/uso terapêutico , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/diagnóstico por imagem , Estado Epiléptico/metabolismo , Convulsões/tratamento farmacológicoRESUMO
α-Conotoxins are well-known probes for the characterization of the various subtypes of nicotinic acetylcholine receptors (nAChRs). Identifying new α-conotoxins with different pharmacological profiles can provide further insights into the physiological or pathological roles of the numerous nAChR isoforms found at the neuromuscular junction, the central and peripheral nervous systems, and other cells such as immune cells. This study focuses on the synthesis and characterization of two novel α-conotoxins obtained from two species endemic to the Marquesas Islands, namely Conus gauguini and Conus adamsonii. Both species prey on fish, and their venom is considered a rich source of bioactive peptides that can target a wide range of pharmacological receptors in vertebrates. Here, we demonstrate the versatile use of a one-pot disulfide bond synthesis to achieve the α-conotoxin fold [Cys 1-3; 2-4] for GaIA and AdIA, using the 2-nitrobenzyl (NBzl) protecting group of cysteines for effective regioselective oxidation. The potency and selectivity of GaIA and AdIA against rat nicotinic acetylcholine receptors were investigated electrophysiologically and revealed potent inhibitory activities. GaIA was most active at the muscle nAChR (IC50 = 38 nM), whereas AdIA was most potent at the neuronal α6/3 ß2ß3 subtype (IC50 = 177 nM). Overall, this study contributes to a better understanding of the structure-activity relationships of α-conotoxins, which may help in the design of more selective tools.
Assuntos
Conotoxinas , Caramujo Conus , Receptores Nicotínicos , Animais , Ratos , Conotoxinas/farmacologia , Conotoxinas/química , Caramujo Conus/química , Caramujo Conus/fisiologia , Antagonistas Nicotínicos/farmacologia , Caramujos , PolinésiaRESUMO
The P2X7 receptor is a trimeric ligand-gated cation channel activated by extracellular adenosine 5'-triphosphate. The study of animals has greatly advanced the investigation of P2X7 and helped to establish the numerous physiological and pathophysiological roles of this receptor in human health and disease. Following a short overview of the P2X7 distribution, roles and functional properties, this article discusses how animal models have contributed to the generation of P2X7-specific antibodies and nanobodies (including biologics), recombinant receptors and radioligands to study P2X7 as well as to the pharmacokinetic testing of P2X7 antagonists. This article then outlines how mouse and rat models have been used to study P2X7. These sections include discussions on preclinical disease models, polymorphic P2X7 variants, P2X7 knockout mice (including bone marrow chimeras and conditional knockouts), P2X7 reporter mice, humanized P2X7 mice and P2X7 knockout rats. Finally, this article reviews the limited number of studies involving guinea pigs, rabbits, monkeys (rhesus macaques), dogs, cats, zebrafish, and other fish species (seabream, ayu sweetfish, rainbow trout and Japanese flounder) to study P2X7.
Assuntos
Receptores Purinérgicos P2X7 , Peixe-Zebra , Camundongos , Ratos , Humanos , Animais , Cães , Cobaias , Coelhos , Receptores Purinérgicos P2X7/genética , Macaca mulatta , Modelos Animais , Camundongos Knockout , Trifosfato de AdenosinaRESUMO
P2X7 receptors (P2X7Rs) are associated with numerous pathophysiological mechanisms, and this promotes them as therapeutic targets for certain neurodegenerative conditions. However, the identity of P2X7R-expressing cells in the nervous system remains contentious. Here, we examined P2X7R functionality in auditory nerve cells from rodents of either sex, and determined their functional and anatomic expression pattern. In whole-cell recordings from rat spiral ganglion cultures, the purinergic agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) activated desensitizing currents in spiral ganglion neurons (SGNs) but non-desensitizing currents in glia that were blocked by P2X7R-specific antagonists. In imaging experiments, BzATP gated sustained Ca2+ entry into glial cells. BzATP-gated uptake of the fluorescent dye YO-PRO-1 was reduced and slowed by P2X7R-specific antagonists. In rats, P2X7Rs were immuno-localized predominantly within satellite glial cells (SGCs) and Schwann cells (SCs). P2X7R expression was not detected in the portion of the auditory nerve within the central nervous system. Mouse models allowed further exploration of the distribution of cochlear P2X7Rs. In GENSAT reporter mice, EGFP expression driven via the P2rx7 promoter was evident in SGCs and SCs but was undetectable in SGNs. A second transgenic model showed a comparable cellular distribution of EGFP-tagged P2X7Rs. In wild-type mice the discrete glial expression was confirmed using a P2X7-specific nanobody construct. Our study shows that P2X7Rs are expressed by peripheral glial cells, rather than by afferent neurons. Description of functional signatures and cellular distributions of these enigmatic proteins in the peripheral nervous system (PNS) will help our understanding of ATP-dependent effects contributing to hearing loss and other sensory neuropathies.SIGNIFICANCE STATEMENT P2X7 receptors (P2X7Rs) have been the subject of much scrutiny in recent years. They have been promoted as therapeutic targets in a number of diseases of the nervous system, yet the specific cellular location of these receptors remains the subject of intense debate. In the auditory nerve, connecting the inner ear to the brainstem, we show these multimodal ATP-gated channels localize exclusively to peripheral glial cells rather than the sensory neurons, and are not evident in central glia. Physiologic responses in the peripheral glia display classical hallmarks of P2X7R activation, including the formation of ion-permeable and also macromolecule-permeable pores. These qualities suggest these proteins could contribute to glial-mediated inflammatory processes in the auditory periphery under pathologic disease states.
Assuntos
Cóclea/metabolismo , Nervo Coclear/metabolismo , Audição/fisiologia , Neuroglia/metabolismo , Receptores Purinérgicos P2X7/biossíntese , Animais , Cóclea/química , Cóclea/citologia , Nervo Coclear/química , Nervo Coclear/citologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuroglia/química , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X7/análise , RoedoresRESUMO
BACKGROUND: Previous studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple models of brain diseases. However, tools for the selective and efficient targeting of these receptors are lacking. The development of new P2X7-specific nanobodies (nbs) has enabled us to effectively block the P2X7 channel. METHODS: Temporary middle cerebral artery occlusion (tMCAO) in wild-type (wt) and P2X7 transgenic (tg) mice was used to model ischemic stroke. Adenosine triphosphate (ATP) release was assessed in transgenic ATP sensor mice. Stroke size was measured after P2X7-specific nbs were injected intravenously (iv) and intracerebroventricularly (icv) directly before tMCAO surgery. In vitro cultured microglia were used to investigate calcium influx, pore formation via 4,6-diamidino-2-phenylindole (DAPI) uptake, caspase 1 activation and interleukin (IL)-1ß release after incubation with the P2X7-specific nbs. RESULTS: Transgenic ATP sensor mice showed an increase in ATP release in the ischemic hemisphere compared to the contralateral hemisphere or the sham-treated mice up to 24 h after stroke. P2X7-overexpressing mice had a significantly greater stroke size 24 h after tMCAO surgery. In vitro experiments with primary microglial cells demonstrated that P2X7-specific nbs could inhibit ATP-triggered calcium influx and the formation of membrane pores, as measured by Fluo4 fluorescence or DAPI uptake. In microglia, we found lower caspase 1 activity and subsequently lower IL-1ß release after P2X7-specific nb treatment. The intravenous injection of P2X7-specific nbs compared to isotype controls before tMCAO surgery did not result in a smaller stroke size. As demonstrated by fluorescence-activated cell sorting (FACS), after stroke, iv injected nbs bound to brain-infiltrated macrophages but not to brain resident microglia, indicating insufficient crossing of the blood-brain barrier of the nbs. Therefore, we directly icv injected the P2X7-specific nbs or the isotype nbs. After icv injection of 30 µg of P2X7 specific nbs, P2X7 specific nbs bound sufficiently to microglia and reduced stroke size. CONCLUSION: Mechanistically, we can show that there is a substantial increase of ATP locally after stroke and that blockage of the ATP receptor P2X7 by icv injected P2X7-specific nbs can reduce ischemic tissue damage.
Assuntos
Receptores Purinérgicos P2 , Anticorpos de Domínio Único , Acidente Vascular Cerebral , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Caspase 1/metabolismo , Infarto da Artéria Cerebral Média/patologia , Interleucina-1beta/metabolismo , Camundongos , Microglia/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Anticorpos de Domínio Único/metabolismo , Acidente Vascular Cerebral/metabolismoRESUMO
Several neonicotinoids have recently been shown to activate the nicotinic acetylcholine receptor (nAChR) on human neurons. Moreover, imidacloprid (IMI) and other members of this pesticide family form a set of diverse metabolites within crops. Among these, desnitro-imidacloprid (DN-IMI) is of special toxicological interest, as there is evidence (i) for human dietary exposure to this metabolite, (ii) and that DN-IMI is a strong trigger of mammalian nicotinic responses. We set out here to quantify responses of human nAChRs to DN-IMI and an alternative metabolite, IMI-olefin. To evaluate toxicological hazards, these data were then compared to those of IMI and nicotine. Ca2+-imaging experiments on human neurons showed that DN-IMI exhibits an agonistic effect on nAChRs at sub-micromolar concentrations (equipotent with nicotine) while IMI-olefin activated the receptors less potently (in a similar range as IMI). Direct experimental data on the interaction with defined receptor subtypes were obtained by heterologous expression of various human nAChR subtypes in Xenopus laevis oocytes and measurement of the transmembrane currents evoked by exposure to putative ligands. DN-IMI acted on the physiologically important human nAChR subtypes α7, α3ß4, and α4ß2 (high-sensitivity variant) with similar potency as nicotine. IMI and IMI-olefin were confirmed as nAChR agonists, although with 2-3 orders of magnitude lower potency. Molecular docking studies, using receptor models for the α7 and α4ß2 nAChR subtypes supported an activity of DN-IMI similar to that of nicotine. In summary, these data suggest that DN-IMI functionally affects human neurons similar to the well-established neurotoxicant nicotine by triggering α7 and several non-α7 nAChRs.
Assuntos
Imidazolinas/farmacologia , Neonicotinoides/farmacologia , Agonistas Nicotínicos/farmacologia , Nitrocompostos/farmacologia , Piridinas/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Alcenos/química , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Simulação de Acoplamento Molecular , Neonicotinoides/metabolismo , Neuroblastoma/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Nitrocompostos/metabolismo , Oócitos , Praguicidas/metabolismo , Praguicidas/farmacologia , Receptores Nicotínicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Xenopus laevisRESUMO
Cone snails are venomous marine predators that rely on fast-acting venom to subdue their prey and defend against aggressors. The conotoxins produced in the venom gland are small disulfide-rich peptides with high affinity and selectivity for their pharmacological targets. A dominant group comprises α-conotoxins, targeting nicotinic acetylcholine receptors. Here, we report on the synthesis, structure determination and biological activity of a novel α-conotoxin, CIC, found in the predatory venom of the piscivorous species Conus catus and its truncated mutant Δ-CIC. CIC is a 4/7 α-conotoxin with an unusual extended N-terminal tail. High-resolution NMR spectroscopy shows a major influence of the N-terminal tail on the apparent rigidity of the three-dimensional structure of CIC compared to the more flexible Δ-CIC. Surprisingly, this effect on the structure does not alter the biological activity, since both peptides selectively inhibit α3ß2 and α6/α3ß2ß3 nAChRs with almost identical sub- to low micromolar inhibition constants. Our results suggest that the N-terminal part of α-conotoxins can accommodate chemical modifications without affecting their pharmacology.
Assuntos
Conotoxinas/isolamento & purificação , Caramujo Conus/metabolismo , Venenos de Moluscos/química , Antagonistas Nicotínicos/isolamento & purificação , Animais , Conotoxinas/química , Conotoxinas/farmacologia , Espectroscopia de Ressonância Magnética , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismoRESUMO
Venom peptide toxins such as conotoxins play a critical role in the characterization of nicotinic acetylcholine receptor (nAChR) structure and function and have potential as nervous system therapeutics as well. However, the lack of solved structures of conotoxins bound to nAChRs and the large size of these peptides are barriers to their computational docking and design. We addressed these challenges in the context of the α4ß2 nAChR, a widespread ligand-gated ion channel in the brain and a target for nicotine addiction therapy, and the 19-residue conotoxin α-GID that antagonizes it. We developed a docking algorithm, ToxDock, which used ensemble-docking and extensive conformational sampling to dock α-GID and its analogs to an α4ß2 nAChR homology model. Experimental testing demonstrated that a virtual screen with ToxDock correctly identified three bioactive α-GID mutants (α-GID[A10V], α-GID[V13I], and α-GID[V13Y]) and one inactive variant (α-GID[A10Q]). Two mutants, α-GID[A10V] and α-GID[V13Y], had substantially reduced potency at the human α7 nAChR relative to α-GID, a desirable feature for α-GID analogs. The general usefulness of the docking algorithm was highlighted by redocking of peptide toxins to two ion channels and a binding protein in which the peptide toxins successfully reverted back to near-native crystallographic poses after being perturbed. Our results demonstrate that ToxDock can overcome two fundamental challenges of docking large toxin peptides to ion channel homology models, as exemplified by the α-GID:α4ß2 nAChR complex, and is extendable to other toxin peptides and ion channels. ToxDock is freely available at rosie.rosettacommons.org/tox_dock.
Assuntos
Algoritmos , Aplysia/química , Conotoxinas/química , Simulação de Acoplamento Molecular/métodos , Antagonistas Nicotínicos/química , Receptores Nicotínicos/química , Animais , HumanosRESUMO
P2X receptors (P2XRs) are ligand-gated ion channels activated by extracellular ATP. Although the crystal structure of the zebrafish P2X4R has been solved, the exact mode of ATP binding and the conformational changes governing channel opening and desensitization remain unknown. Here, we used voltage clamp fluorometry to investigate movements in the cysteine-rich head domain of the rat P2X1R (A118-I125) that projects over the proposed ATP binding site. On substitution with cysteine residues, six of these residues (N120-I125) were specifically labeled by tetramethyl-rhodamine-maleimide and showed significant changes in the emission of the fluorescence probe on application of the agonists ATP and benzoyl-benzoyl-ATP. Mutants N120C and G123C showed fast fluorescence decreases with similar kinetics as the current increases. In contrast, mutants P121C and I125C showed slow fluorescence increases that seemed to correlate with the current decline during desensitization. Mutant E122C showed a slow fluorescence increase and fast decrease with ATP and benzoyl-benzoyl-ATP, respectively. Application of the competitive antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) resulted in large fluorescence changes with the N120C, E122C, and G123C mutants and minor or no changes with the other mutants. Likewise, TNP-ATP-induced changes in control mutants distant from the proposed ATP binding site were comparably small or absent. Combined with molecular modeling studies, our data confirm the proposed ATP binding site and provide evidence that ATP orients in its binding site with the ribose moiety facing the solution. We also conclude that P2XR activation and desensitization involve movements of the cysteine-rich head domain.
Assuntos
Cisteína/química , Receptores Purinérgicos P2X1/metabolismo , Animais , Cátions , Membrana Celular/metabolismo , Cristalografia por Raios X/métodos , DNA Complementar/metabolismo , Eletrofisiologia/métodos , Cinética , Maleimidas/química , Microscopia de Fluorescência/métodos , Mutação , Oócitos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Rodaminas/química , Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMO
Current treatments against organophosphate poisoning (OPP) do not directly address effects mediated by the overstimulation of nicotinic acetylcholine receptors (nAChR). Non-oxime bispyridinium compounds (BPC) promote acetylcholine esterase-independent recovery of organophosphate-induced paralysis. Here, we test the hypothesis that they act by positive modulatory action on nAChRs. Using two-electrode voltage clamp analysis in combination with mutagenesis and molecular docking analysis, the potency and molecular mode of action of a series of nine BPCs was investigated on human α7 and muscle-type nAChRs expressed in Xenopus laevis oocytes. The investigated BPCs inhibited α7 and/or muscle-type nAChRs with IC50 values in the high nanomolar to high micromolar range. Further analysis of the most potent analogues revealed a noncompetitive, voltage-dependent inhibition. Co-application with the α7-selective positive allosteric modulator PNU120596 and generation of α7/5HT3 receptor chimeras excluded direct interaction with the PNU120596 binding site and binding to the extracellular domain of the α7 nAChR, suggesting that they act as open channel blockers (OCBs). Molecular docking supported by mutagenesis localized the BPC binding area in the outer channel vestibule between the extracellular and transmembrane domains. Analysis of BPC action on other cation-selective channels suggests a rather nonspecific inhibition of pentameric cation channels. BPCs have been shown to ameliorate organophosphate-induced paralysis in vitro and in vivo. Our data support molecular action as OCBs at α7 and muscle-type nAChRs and suggest that their positive physiological effects are more complex than anticipated and require further investigation.
RESUMO
Introduction: P2X receptors are a family of homo- and heterotrimeric cation channels gated by extracellular ATP. The P2X4 and P2X7 subunits show overlapping expression patterns and have been involved in similar physiological processes, such as pain and inflammation as well as various immune cell functions. While formation of P2X2/P2X3 heterotrimers produces a distinct pharmacological phenotype and has been well established, functional identification of a P2X4/P2X7 heteromer has been difficult and evidence for and against a physical association has been found. Most of this evidence stems, however, from in vitro model systems. Methods: Here, we used a P2X7-EGFP BAC transgenic mouse model as well as P2X4 and P2X7 knock-out mice to re-investigate a P2X4-P2X7 interaction in mouse lung by biochemical and immunohistochemical experiments as well as quantitative expression analysis. Results: No detectable amounts of P2X4 could be co-purified from mouse lung via P2X7-EGFP. In agreement with these findings, immuno-histochemical analysis using a P2X7-specific nanobody revealed only limited overlap in the cellular and subcellular localizations of P2X4 and P2X7 in both the native lung tissue and primary cells. Comparison of P2X4 and P2X7 transcript and protein levels in the respective gene-deficient and wild type mice showed no mutual interrelation between their expression levels in whole lungs. However, a significantly reduced P2rx7 expression was found in alveolar macrophages of P2rx4 -/- mice. Discussion: In summary, our detailed analysis of the cellular and subcellular P2X4 and P2X7 localization and expression does not support a physiologically relevant direct association of P2X4 and P2X7 subunits or receptors in vivo.
Assuntos
Pulmão , Camundongos Knockout , Camundongos Transgênicos , Receptores Purinérgicos P2X4 , Receptores Purinérgicos P2X7 , Animais , Receptores Purinérgicos P2X4/metabolismo , Receptores Purinérgicos P2X4/genética , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Camundongos , Pulmão/metabolismo , Pulmão/imunologia , Camundongos Endogâmicos C57BL , Ligação ProteicaRESUMO
Ion channel opening and desensitization is a fundamental process in neurotransmission. The ATP-gated P2X1 receptor (P2X1R) shows rapid and long-lasting desensitization upon agonist binding. This makes the electrophysiological investigation of its desensitization process, agonist unbinding, and recovery from desensitization a challenging task. Here, we show that the fluorescent agonist Alexa-647-ATP is a potent agonist at the P2X1R and a versatile tool to directly visualize agonist binding and unbinding. We demonstrate that the long-lasting desensitization of the P2X1R is due to both slow unbinding of agonist from the desensitized receptor and agonist mediated receptor internalization. Furthermore, the unbinding of the agonist Alexa-647-ATP from the desensitized receptor is accelerated in the continuous presence of competitive ligand. Modeling of our data indicates that three agonist molecules are required to drive the receptor into desensitization. Direct visualization of ligand unbinding from the desensitized receptor demonstrates the cooperativity of this process.
Assuntos
Trifosfato de Adenosina/metabolismo , Carbocianinas/farmacologia , Corantes Fluorescentes/farmacologia , Receptores Purinérgicos P2X1/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Oócitos/citologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Ligação Proteica , RNA Complementar/metabolismo , Ratos , Xenopus laevisRESUMO
The large intracellular C-terminus of the pro-inflammatory P2X7 ion channel receptor (P2X7R) is associated with diverse P2X7R-specific functions. Cryo-EM structures of the closed and ATP-bound open full-length P2X7R recently identified a membrane-associated anchoring domain, an open-state stabilizing "cap" domain, and a globular "ballast domain" containing GTP/GDP and dinuclear Zn2+-binding sites with unknown functions. To investigate protein dynamics during channel activation, we improved incorporation of the environment-sensitive fluorescent unnatural amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP) into Xenopus laevis oocyte-expressed P2X7Rs and performed voltage clamp fluorometry. While we confirmed predicted conformational changes within the extracellular and the transmembrane domains, only 3 out of 41 mutants containing ANAP in the C-terminal domain resulted in ATP-induced fluorescence changes. We conclude that the ballast domain functions rather independently from the extracellular ATP binding domain and might require activation by additional ligands and/or protein interactions. Novel tools to study these are presented.
Assuntos
Trifosfato de Adenosina , Aminoácidos , Animais , Fluorometria/métodos , Domínios Proteicos , Xenopus laevis/metabolismo , Trifosfato de Adenosina/metabolismo , Receptores Purinérgicos P2X7/metabolismoRESUMO
BACKGROUND: Over recent years, increasing evidence suggests a causal relationship between neurofibrillary tangles (NFTs) formation, the main histopathological hallmark of tauopathies, including Alzheimer's disease (AD), and the ubiquitin-proteasome system (UPS) dysfunction detected in these patients. Nevertheless, the mechanisms underlying UPS failure and the factors involved remain poorly understood. Given that AD and tauopathies are associated with chronic neuroinflammation, here, we explore if ATP, one of the danger-associated molecules patterns (DAMPs) associated with neuroinflammation, impacts on AD-associated UPS dysfunction. METHODS: To evaluate if ATP may modulate the UPS via its selective P2X7 receptor, we combined in vitro and in vivo approaches using both pharmacological and genetic tools. We analyze postmortem samples from human AD patients and P301S mice, a mouse model that mimics pathology observed in AD patients, and those from the new transgenic mouse lines generated, such as P301S mice expressing the UPS reporter UbG76V-YFP or P301S deficient of P2X7R. RESULTS: We describe for the first time that extracellular ATP-induced activation of the purinergic P2X7 receptor (P2X7R) downregulates the transcription of ß5 and ß1 proteasomal catalytic subunits via the PI3K/Akt/GSK3/Nfr2 pathway, leading to their deficient assembly into the 20S core proteasomal complex, resulting in a reduced proteasomal chymotrypsin-like and postglutamyl-like activities. Using UPS-reported mice (UbGFP mice), we identified neurons and microglial cells as the most sensitive cell linages to a P2X7R-mediated UPS regulation. In vivo pharmacological or genetic P2X7R blockade reverted the proteasomal impairment developed by P301S mice, which mimics that were detected in AD patients. Finally, the generation of P301S;UbGFP mice allowed us to identify those hippocampal cells more sensitive to UPS impairment and demonstrate that the pharmacological or genetic blockade of P2X7R promotes their survival. CONCLUSIONS: Our work demonstrates the sustained and aberrant activation of P2X7R caused by Tau-induced neuroinflammation contributes to the UPS dysfunction and subsequent neuronal death associated with AD, especially in the hippocampus.
Assuntos
Doença de Alzheimer , Tauopatias , Camundongos , Humanos , Animais , Doença de Alzheimer/complicações , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Complexo de Endopeptidases do Proteassoma , Receptores Purinérgicos P2X7/genética , Ubiquitina , Doenças Neuroinflamatórias , Quinase 3 da Glicogênio Sintase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Camundongos Transgênicos , Trifosfato de Adenosina/metabolismoRESUMO
The blockade or deletion of the pro-inflammatory P2X7 receptor channel has been shown to reduce tissue damage and symptoms in models of inflammatory bowel disease, and P2X7 receptors on enteric neurons were suggested to mediate neuronal death and associated motility changes. Here, we used P2X7-specific antibodies and nanobodies, as well as a bacterial artificial chromosome transgenic P2X7-EGFP reporter mouse model and P2rx7-/- controls to perform a detailed analysis of cell type-specific P2X7 expression and possible overexpression effects in the enteric nervous system of the distal colon. In contrast to previous studies, we did not detect P2X7 in neurons but found dominant expression in glia and macrophages, which closely interact with the neurons. The overexpression of P2X7 per se did not induce significant pathological effects. Our data indicate that macrophages and/or glia account for P2X7-mediated neuronal damage in inflammatory bowel disease and provide a refined basis for the exploration of P2X7-based therapeutic strategies.
Assuntos
Colite , Doenças Inflamatórias Intestinais , Camundongos , Animais , Colite/metabolismo , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios , Doenças Inflamatórias Intestinais/metabolismo , Camundongos Transgênicos , Macrófagos/metabolismo , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismoRESUMO
BACKGROUND AND PURPOSE: Neonatal seizures represent a clinical emergency. However, current anti-seizure medications fail to resolve seizures in ~50% of infants. The P2X7 receptor (P2X7R) is an important driver of inflammation, and evidence suggests that P2X7R contributes to seizures and epilepsy in adults. However, no genetic proof has yet been provided to determine what contribution P2X7R makes to neonatal seizures, its effects on inflammatory signalling during neonatal seizures, and the therapeutic potential of P2X7R-based treatments on long-lasting brain excitability. EXPERIMENTAL APPROACH: Neonatal seizures were induced by global hypoxia in 7-day-old mouse pups (P7). The role of P2X7Rs during seizures was analysed in P2X7R-overexpressing and knockout mice. Treatment of wild-type mice after hypoxia with the P2X7R antagonist JNJ-47965567 was used to determine the effects of the P2X7R on long-lasting brain hyperexcitability. Cell type-specific P2X7R expression was analysed in P2X7R-EGFP reporter mice. RNA sequencing was used to monitor P2X7R-dependent hippocampal downstream signalling. KEY RESULTS: P2X7R deletion reduced seizure severity, whereas P2X7R overexpression exacerbated seizure severity and reduced responsiveness to anti-seizure medication. P2X7R deficiency led to an anti-inflammatory phenotype in microglia, and treatment of mice with a P2X7R antagonist reduced long-lasting brain hyperexcitability. RNA sequencing identified several pathways altered in P2X7R knockout mice after neonatal hypoxia, including a down-regulation of genes implicated in inflammation and glutamatergic signalling. CONCLUSION AND IMPLICATIONS: Treatments based on targeting the P2X7R may represent a novel therapeutic strategy for neonatal seizures with P2X7Rs contributing to the generation of neonatal seizures, driving inflammatory processes and long-term hyperexcitability states.
Assuntos
Receptores Purinérgicos P2X7 , Convulsões , Animais , Camundongos , Animais Recém-Nascidos , Encéfalo/metabolismo , Epilepsia/induzido quimicamente , Epilepsia/metabolismo , Hipóxia/complicações , Inflamação/tratamento farmacológico , Camundongos Knockout , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Convulsões/metabolismoRESUMO
Understanding the determinants of α-conotoxin (α-CTX) selectivity for different nicotinic acetylcholine receptor (nAChR) subtypes is a prerequisite for the design of tool compounds to study nAChRs. However, selectivity optimization of these small, disulfide-rich peptides is difficult not only because of an absence of α-CTX/nAChR co-structures but also because it is challenging to predict how a mutation to an α-CTX will alter its potency and selectivity. As a prototypical system to investigate selectivity, we employed the α-CTX LvIA that is 25-fold selective for the α3ß2 nAChR over the related α3ß4 nAChR subtype, which is a target for nicotine addiction. Using two-electrode voltage clamp electrophysiology, we identified LvIA[D11R] that is 2-fold selective for the α3ß4 nAChR, reversing the subtype preference. This effect is specifically due to the change in charge and not shape of LvIA[D11R], as substitution of D11 with citrulline retains selectivity for the α3ß2 nAChR. Furthermore, LvIA[D11K] shows a stronger reversal, with 4-fold selectivity for the α3ß4 nAChR. Motivated by these findings, using site-directed mutagenesis, we found that ß2[K79A] (I79 on ß4), but not ß2[K78A] (N78 on ß4), largely restores the potency of basic mutants at position 11. Finally, to understand the structural basis of this effect, we used AlphaFold2 to generate models of LvIA in complex with both nAChR subtypes. Both models confirm the plausibility of an electrostatic mechanism to explain the data and also reproduce a broad range of potency and selectivity structure-activity relationships for LvIA mutants, as measured using free energy perturbation simulations. Our work highlights how electrostatic interactions can drive α-CTX selectivity and may serve as a strategy for optimizing the selectivity of LvIA and other α-CTXs.
Assuntos
Conotoxinas , Receptores Nicotínicos , Conotoxinas/genética , Conotoxinas/farmacologia , Eletricidade Estática , Receptores Nicotínicos/genética , Mutação/genética , Peptídeos , Antagonistas Nicotínicos/farmacologiaRESUMO
The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and over 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16178. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.