RESUMEN
BACKGROUND AND PURPOSE: Many medications taste intensely bitter. The innate aversion to bitterness affects medical compliance, especially in children. There is a clear need to develop bitter blockers to suppress the bitterness of vital medications. Bitter taste is mediated by TAS2R receptors. Because different pharmaceutical compounds activate distinct sets of TAS2Rs, targeting specific receptors may only suppress bitterness for certain, but not all, bitter-tasting compounds. Alternative strategies are needed to identify universal bitter blockers that will improve the acceptance of every medication. Taste cells in the mouth transmit signals to afferent gustatory nerve fibres through the release of ATP, which activates the gustatory nerve-expressed purine receptors P2X2/P2X3. We hypothesized that blocking gustatory nerve transmission with P2X2/P2X3 inhibitors (e.g. 5-(5-iodo-4-methoxy-2-propan-2-ylphenoxy)pyrimidine-2,4-diamine [AF-353]) would reduce bitterness for all medications and bitter compounds. EXPERIMENTAL APPROACH: Human sensory taste testing and mouse behavioural analyses were performed to determine if oral application of AF-353 blocks perception of bitter taste and other taste qualities but not non-gustatory oral sensations (e.g. tingle). KEY RESULTS: Rinsing the mouth with AF-353 in humans or oral swabbing it in mice suppressed the bitter taste and avoidance behaviours of all compounds tested. We further showed that AF-353 suppressed other taste qualities (i.e. salt, sweet, sour and savoury) but had no effects on other oral or nasal sensations (e.g, astringency and oral tingle). CONCLUSION AND IMPLICATIONS: This is the first time a universal, reversible taste blocker in humans has been reported. Topical application of P2X2/P2X3 inhibitor to suppress bitterness may improve medical compliance.
Asunto(s)
Antagonistas del Receptor Purinérgico P2X , Receptores Purinérgicos P2X3 , Gusto , Humanos , Gusto/efectos de los fármacos , Animales , Receptores Purinérgicos P2X3/metabolismo , Masculino , Femenino , Antagonistas del Receptor Purinérgico P2X/farmacología , Antagonistas del Receptor Purinérgico P2X/administración & dosificación , Adulto , Ratones , Receptores Purinérgicos P2X2/metabolismo , Administración Tópica , Adulto Joven , Pirimidinas/farmacología , Pirimidinas/administración & dosificación , Ratones Endogámicos C57BLRESUMEN
P2X receptors are a family of ligand gated ion channels found in a range of eukaryotic species including humans but are not naturally present in the yeast Saccharomyces cerevisiae. We demonstrate the first recombinant expression and functional gating of the P2X2 receptor in baker's yeast. We leverage the yeast host for facile genetic screens of mutant P2X2 by performing site saturation mutagenesis at residues of interest, including SNPs implicated in deafness and at residues involved in native binding. Deep mutational analysis and rounds of genetic engineering yield mutant P2X2 F303Y A304W, which has altered ligand selectivity toward the ATP analog AMP-PNP. The F303Y A304W variant shows over 100-fold increased intracellular calcium amplitudes with AMP-PNP compared to the WT receptor and has a much lower desensitization rate. Since AMP-PNP does not naturally activate P2X receptors, the F303Y A304W P2X2 may be a starting point for downstream applications in chemogenetic cellular control. Interestingly, the A304W mutation selectively destabilizes the desensitized state, which may provide a mechanistic basis for receptor opening with suboptimal agonists. The yeast system represents an inexpensive, scalable platform for ion channel characterization and engineering by circumventing the more expensive and time-consuming methodologies involving mammalian hosts.
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Receptores Purinérgicos P2X2 , Saccharomyces cerevisiae , Humanos , Sustitución de Aminoácidos , Ligandos , Ingeniería de Proteínas/métodos , Receptores Purinérgicos P2X2/metabolismo , Receptores Purinérgicos P2X2/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Modelos Moleculares , Estructura Terciaria de Proteína , Estructura Cuaternaria de Proteína , Homología Estructural de Proteína , MutaciónRESUMEN
BACKGROUND AND PURPOSE: The P2X3 receptor, a trimeric ionotropic purinergic receptor, has emerged as a potential therapeutic target for refractory chronic cough (RCC). Nevertheless, gefapixant/AF-219, the only marketed P2X3 receptor antagonist, might lead taste disorders by modulating the human P2X2/3 (hP2X2/3) heterotrimer. Hence, in RCC drug development, compounds exhibiting strong affinity for the hP2X3 homotrimer and a weak affinity for the hP2X2/3 heterotrimer hold promise. An example of such a molecule is sivopixant/S-600918, a clinical Phase II RCC candidate with a reduced incidence of taste disturbance compared to gefapixant. Sivopixant and its analogue, (3-(4-([3-chloro-4-isopropoxyphenyl]amino)-3-(4-methylbenzyl)-2,6-dioxo-3,6-dihydro-1,3,5-triazin-1(2H)-yl)propanoic acid (DDTPA), exhibit both high affinity and high selectivity for hP2X3 homotrimers, compared with hP2X2/3 heterotrimers. The mechanism underlying the druggable site and its high selectivity remains unclear. EXPERIMENTAL APPROACH: To analyse mechanisms that distinguish this drug candidate from other inhibitors of the P2X3 receptors we used a combination of chimera construction, site covalent occupation, metadynamics, mutagenesis and whole-cell recording. KEY RESULTS: The high affinity and selectivity of sivopixant/DDTPA for hP2X3 receptors was determined by the tri-symmetric site located close to the upper vestibule. Substitution of only four amino acids inside the upper body domain of hP2X2 with those of hP2X3, enabled the hP2X2/3 heterotrimer to exhibit a similar level of apparent affinity for sivopixant/DDTPA as the hP2X3 homotrimer. CONCLUSION AND IMPLICATIONS: From the receptor-ligand recognition perspective, we have elucidated the molecular basis of novel RCC clinical candidates' cough-suppressing properties and reduced side effects, offering a promising approach to the discovery of novel drugs that specifically target P2X3 receptors.
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Compuestos de Anilina , Bencenosulfonamidas , Carcinoma de Células Renales , Neoplasias Renales , Pirimidinas , Triazinas , Humanos , Carcinoma de Células Renales/inducido químicamente , Piridinas/uso terapéutico , Antagonistas del Receptor Purinérgico P2X/farmacología , Antagonistas del Receptor Purinérgico P2X/uso terapéutico , Tos/inducido químicamente , Receptores Purinérgicos P2X3 , Sulfonamidas , Neoplasias Renales/inducido químicamente , Receptores Purinérgicos P2X2RESUMEN
Heart failure is associated with multiple mechanisms, including sympatho-excitation, and is one of the leading causes of death worldwide. Enhanced carotid body chemoreflex function is strongly related to excessive sympathetic nerve activity and sleep-disordered breathing in heart failure. How to reduce the excitability of the carotid body is still scientifically challenging. Both clinical and experimental evidence have suggested that targeting purinergic receptors is of great potential to combat heart failure. In a recent study, Lataro et al. (Lataro et al. in Nat Commun 14:1725, 5) demonstrated that targeting purinergic P2X3 receptors in the carotid body attenuates the progression of heart failure. Using a series of molecular, biochemical, and functional assays, the authors observed that the carotid body generates spontaneous, episodic burst discharges coincident with the onset of disordered breathing in male rats with heart failure, which was generated by ligating the left anterior descending coronary artery. Moreover, P2X3 receptor expression was found to be upregulated in the petrosal ganglion chemoreceptive neurons of rats with heart failure. Of particular note, treatment with a P2X3 antagonist rescued pathological breathing disturbances, abolished episodic discharges, reinstated autonomic balance, attenuated cardiac dysfunction, and reduced the immune cell response and plasma cytokine levels in those rats.
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Cuerpo Carotídeo , Insuficiencia Cardíaca , Ratas , Masculino , Animales , Cuerpo Carotídeo/metabolismo , Receptores Purinérgicos P2X/metabolismo , Insuficiencia Cardíaca/metabolismo , Neuronas/metabolismo , Sistema Nervioso Simpático , Receptores Purinérgicos P2X3/metabolismo , Receptores Purinérgicos P2X2/metabolismoRESUMEN
Major depressive disorder (MDD) is a devastating condition. Although progress has been made in the past seven decades, patients with MDD continue to receive an inadequate treatment, primarily due to the late onset of first-line antidepressant drugs and to their acute withdrawal symptoms. Resilience is the ability to rebound from adversity in a healthy manner and many people have psychological resilience. Revealing the mechanisms and identifying methods promoting resilience will hopefully lead to more effective prevention strategies and treatments for depression. In this study, we found that intermittent hypobaric hypoxia training (IHHT), a method for training pilots and mountaineers, enhanced psychological resilience in adult mice. IHHT produced a sustained antidepressant-like effect in mouse models of depression by inducing long-term (up to 3 months after this treatment) overexpression of hypoxia-inducible factor (HIF)-1α in the dorsal raphe nucleus (DRN) of adult mice. Moreover, DRN-infusion of cobalt chloride, which mimics hypoxia increasing HIF-1α expression, triggered a rapid and long-lasting antidepressant-like effect. Down-regulation of HIF-1α in the DRN serotonergic (DRN5-HT) neurons attenuated the effects of IHHT. HIF-1α translationally regulated the expression of P2X2, and conditionally knocking out P2rx2 (encodes P2X2 receptors) in DRN5-HT neurons, in turn, attenuated the sustained antidepressant-like effect of IHHT, but not its acute effect. In line with these results, a single sub-anesthetic dose of ketamine enhanced HIF-1α-P2X2 signaling, which is essential for its rapid and long-lasting antidepressant-like effect. Notably, we found that P2X2 protein levels were significantly lower in the DRN of patients with MDD than that of control subjects. Together, these findings elucidate the molecular mechanism underlying IHHT promoting psychological resilience and highlight enhancing HIF-1α-P2X2 signaling in DRN5-HT neurons as a potential avenue for screening novel therapeutic treatments for MDD.
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Trastorno Depresivo Mayor , Resiliencia Psicológica , Humanos , Ratones , Animales , Núcleo Dorsal del Rafe/metabolismo , Neuronas Serotoninérgicas/metabolismo , Serotonina/metabolismo , Serotonina/farmacología , Antidepresivos/farmacología , Hipoxia , Receptores Purinérgicos P2X2/metabolismoRESUMEN
The phenotypes of ATP-gated currents thought ionotropic P2X channels depend on the composition of the oligomeric receptor. We constructed chimeric P2X2/P2X7 receptors to study the effect of cytoplasmic domains on rectification of current flow through the open channel. We found that the identity of the N-terminus determines the pattern of rectification, with chimeric receptors containing the N-terminus of the P2X2 receptor displaying inward rectification, and chimeric receptors containing the N-terminus of the P2X7 receptor displaying slightly outward rectification. In contrast, rectification of current through chimeric receptors with swapped C-termini always mimicked the wild-type receptor. Thus, our findings suggest that the N-terminus of P2X receptors regulate ion flow through the channel pore and are responsible in part for determining current rectification.
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Adenosina Trifosfato , Receptores Purinérgicos P2X7 , Ratas , Animales , Receptores Purinérgicos P2X7/genética , Citoplasma , Citosol , Receptores Purinérgicos P2X2/genéticaRESUMEN
Neurosteroids are steroids synthesized de novo in the brain from cholesterol in an independent manner from peripheral steroid sources. The term "neuroactive steroid" includes all steroids independent of their origin, and newly synthesized analogs of neurosteroids that modify neuronal activities. In vivo application of neuroactive steroids induces potent anxiolytic, antidepressant, anticonvulsant, sedative, analgesic and amnesic effects, mainly through interaction with the γ-aminobutyric acid type-A receptor (GABAAR). However, neuroactive steroids also act as positive or negative allosteric regulators on several ligand-gated channels including N-methyl-d-aspartate receptors (NMDARs), nicotinic acetylcholine receptors (nAChRs) and ATP-gated purinergic P2X receptors. Seven different P2X subunits (P2X1-7) can assemble to form homotrimeric or heterotrimeric ion channels permeable for monovalent cations and calcium. Among them, P2X2, P2X4, and P2X7 are the most abundant within the brain and can be regulated by neurosteroids. Transmembrane domains are necessary for neurosteroid binding, however, no generic motif of amino acids can accurately predict the neurosteroid binding site for any of the ligand-gated ion channels including P2X. Here, we will review what is currently known about the modulation of rat and human P2X by neuroactive steroids and the possible structural determinants underlying neurosteroid-induced potentiation and inhibition of the P2X2 and P2X4 receptors. This article is part of the Special Issue on "Purinergic Signaling: 50 years".
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Canales Iónicos Activados por Ligandos , Neuroesteroides , Ratas , Humanos , Animales , Canales Iónicos Activados por Ligandos/metabolismo , Receptores Purinérgicos P2X/metabolismo , Encéfalo/metabolismo , Sitios de Unión , Adenosina Trifosfato/metabolismo , Receptores Purinérgicos P2X2/metabolismoRESUMEN
Extracellular adenosine 5'-triphosphate (ATP) acts as an autocrine and paracrine agent, the actions of which on affected cells are mediated by P2 receptors (P2R), which include trans cell-membrane cationic channels (P2XRs), and G protein coupled receptors (P2YRs). The mammalian P2X receptors form homotrimeric or heterotrimeric cationic channels, each of which contains three ATP-binding sites. There are seven homotrimeric P2X receptors (P2X1-7) and three heteromeric (P2X2/P2X3, P2X4/P2X6, P2X1/P2X5). In the lungs and airways, ATP activates P2X3 and P2X2/3 receptors (P2X3R, P2X2/3R, respectively) localized on vagal sensory nerve terminals resulting in bronchoconstriction, and cough, and probably also localized release of pro-inflammatory neuropeptides via the axon reflex. Currently, several P2X3R and P2X2/3R antagonists are being developed as drug-candidates for the treatment of chronic cough. This report presents the receptor affinity data of a novel water-soluble small molecule, DT-0111, that acts as a selective P2X3R antagonist.
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Tos , Receptores Purinérgicos P2X3 , Animales , Antagonistas del Receptor Purinérgico P2X/farmacología , Adenosina Trifosfato/metabolismo , Pulmón/metabolismo , Receptores Purinérgicos P2X2 , Mamíferos/metabolismoRESUMEN
P2X receptor channels are trimeric ATP-activated ion channels expressed in neuronal and non-neuronal cells that are attractive therapeutic targets for human disorders. Seven subtypes of P2X receptor channels have been identified in mammals that can form both homomeric and heteromeric channels. P2X1-4 and P2X7 receptor channels are cation-selective, whereas P2X5 has been reported to have both cation and anion permeability. P2X receptor channel structures reveal that each subunit is comprised of two transmembrane helices, with both N-and C-termini on the intracellular side of the membrane and a large extracellular domain that contains the ATP binding sites at subunit interfaces. Recent structures of ATP-bound P2X receptors with the activation gate open reveal the unanticipated presence of a cytoplasmic cap over the central ion permeation pathway, leaving lateral fenestrations that may be largely buried within the membrane as potential pathways for ions to permeate the intracellular end of the pore. In the present study, we identify a critical residue within the intracellular lateral fenestrations that is readily accessible to thiol-reactive compounds from both sides of the membrane and where substitutions influence the relative permeability of the channel to cations and anions. Taken together, our results demonstrate that ions can enter or exit the internal pore through lateral fenestrations that play a critical role in determining the ion selectivity of P2X receptor channels.
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Adenosina Trifosfato , Canales Iónicos , Animales , Humanos , Canales Iónicos/metabolismo , Sitios de Unión , Estructura Secundaria de Proteína , Iones/metabolismo , Adenosina Trifosfato/metabolismo , Receptores Purinérgicos P2X2/metabolismo , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Mamíferos/metabolismoRESUMEN
Of the extended family of ATP-gated P2X ion-channels, the P2X5 receptor has received comparatively little attention since first cloned over 25 years ago. Disinterest in studying this P2X subtype stems from two commonly held beliefs: (i) canonical human P2X5 is non-functional because the P2X5 subunit is truncated (hP2X5A, 422 aa) and missing the critical peptide sequence (22 aa) encoded by exon 10; (ii) rat and mouse P2X5 subunits are fully formed (455 aa) but the receptor is only weakly functional, and successive ATP responses rapidly run down in amplitude. However, newer studies have re-evaluated these notions. First, a low proportion (around 10%) of humans possess full-length P2X5 subunits (444 aa) and can form competent P2X5 receptors. Full-length P2X5 has been identified only in black Americans, but may occur in a wider population as more ethnicities are screened. Second, replacement of one of three amino acids in rat P2X5 subunits with corresponding residues in human P2X5 subunits (V67I, S191F, or F195H) significantly improves the responsiveness of rat P2X5 to ATP. Replaced residues exert an allosteric action on the left flipper, allowing the docking jaw for ATP to flex the lower body of the subunit and fully open the ion pore. This proposed action may drive the search for naturally occurring modulators which act allosterically on wildtype rat P2X5. This review collates the available information on the structure and function of human and rat P2X5 receptors, with the view to rehabilitating the reputation of these ATP-gated ion channels and stimulating future lines of research.
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Receptores Purinérgicos P2 , Ratas , Humanos , Ratones , Animales , Receptores Purinérgicos P2/metabolismo , Secuencia de Aminoácidos , Adenosina Trifosfato/química , Receptores Purinérgicos P2X5/metabolismo , Receptores Purinérgicos P2X2/metabolismoRESUMEN
The adsorption of proteins onto surfaces significantly impacts biomaterials, medical devices, and biological processes. This study aims to provide insights into the irreversible adsorption process of multiprotein complexes, particularly focusing on the interaction between anti-His6 IgG antibodies and the His6-tagged P2X2 receptor. Traditional approaches to understanding protein adsorption have centered around kinetic and thermodynamic models, often examining individual proteins and surface coverage, typically through Molecular Dynamics (MD) simulations. In this research, we introduce a computational approach employing Autodesk Maya 3D software for the investigation of multiprotein complexes' adsorption behavior. Utilizing Atomic Force Microscopy (AFM) imaging and Maya 3D-based mechanical simulations, our study yields real-time structural and kinetic observations. Our combined experimental and computational findings reveal that the P2X2 receptor-IgG antibody complex likely undergoes absorption in an 'extended' configuration. Whereas the P2X2 receptor is less adsorbed once is complexed to the IgG antibody compared to its individual state, the opposite is observed for the antibody. This insight enhances our understanding of the role of protein-protein interactions in the process of protein adsorption.
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Inmunoglobulina G , Simulación de Dinámica Molecular , Adsorción , Receptores Purinérgicos P2X2 , Microscopía de Fuerza Atómica , Complejos MultiproteicosRESUMEN
Prostate cancer is one of the most common malignancies in men. Calcium signaling is implicated in the progression of prostate cancer and plays a critical role in immune cell function. However, whether specific calcium channel-related genes play a crucial role in the immune cell infiltration levels of prostate cancer requires further research. In this study, we performed an integrated analysis of transcriptional, clinical, and somatic mutation data from The Cancer Genome Atlas database and identified the hub calcium channel-related gene P2RX2 to be associated with the prognosis and immune infiltration of prostate cancer. P2RX2 expression was positively correlated with immune cell infiltration levels and the expression of immune checkpoint genes, and downregulation of P2RX2 led to poor survival in patients with prostate cancer. Furthermore, we validated the molecular and clinical characteristics of P2RX2 by using multiple databases and conducting in-vitro experiments. Additionally, drug sensitivity analysis revealed that patients with low P2RX2 expression were sensitive to docetaxel and Bicalutamide. In conclusion, we revealed an association between calcium channel-related genes and prostate cancer, and identified P2RX2 as a biomarker for early diagnosis, prognosis prediction, and aiding treatment decisions for patients with prostate cancer.
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Canales de Calcio , Neoplasias de la Próstata , Canales de Calcio/genética , Docetaxel , Humanos , Masculino , Pronóstico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Receptores Purinérgicos P2X2RESUMEN
Rationale: Stress is a major risk factor for the development of depression. However, the underlying molecular mechanisms of stress vulnerability in depression are largely uncharacterized. Methods: P2X2 receptors (a major receptor for gliotransmitter-ATP) in the medial prefrontal cortex (mPFC) were identified by real-time qPCR, western blots and RNAscope in situ hybridization in chronic social defeat stress model (CSDS). We generated P2X2 conditional knockout mice and overexpressed AAV-P2X2 in CamkIIα-Cre mice. The depression-like behaviors were assessed via CSDS, subthreshold social defeat stress (SSDS), social interaction test (SI), forced interaction test (FIT), forced swimming test (FST), sucrose preference test (SPT), novel stressed feeding (NSF) and open field test (OFT). The neuronal activity and synapse function of P2X2 receptors in the mPFC were detected by in vivo fiber-photometry, patch-clamp techniques and neuronal morphometric analysis. Results: We identified that P2X2 receptors were increased in the mPFC of susceptible mice in CSDS. Conditional knockout of P2X2 receptors in pyramidal neurons promoted resilience of chronic stress-induced depressive-like behaviors, whereas pyramidal neurons - specific gain of P2X2 in the mPFC increased vulnerability to depressive-like behaviors. In vivo fiber-photometry, electrophysiology and neuronal morphometric analysis showed P2X2 receptors regulated neuronal activity and synapse function in the mPFC. Conclusions: Overall, our studies reveal a critical role of P2X2 in mediating vulnerability to chronic stress and identify P2X2 as a potential therapeutic target for treatment of stress-related mood disorders.
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Células Piramidales , Estrés Psicológico , Animales , Ratones , Ratones Endogámicos C57BL , Neuronas , Receptores Purinérgicos P2X2RESUMEN
A great number of pediatric patients undergoing varied procedures make neonatal surgery plus anesthesia become a matter of great concern owing to underlying neurotoxicity in developing brain. The authors set out to assess long-term effects of surgery plus anesthesia in mouse model. Six-day-old C57BL/6 mice were randomized to receive either anesthesia with 3% sevoflurane, abdominal surgery under the same anesthesia, or the control condition. These mice were examined of learning and memory at juvenile age in Morris water maze test. The brain tissues of mice were harvested for Western blot analysis, including purinergic receptors P2X family, CaMKII and NF-κB. Another battery of mice were administered with inhibitors of P2RX2/3 (e.g., A317491) into hippocampal dentate gyrus before behavioral testing. We found that neonatal surgery plus anesthesia, but not sevoflurane anesthesia alone, impaired the learning and memory of juvenile mice, as evidenced by delayed escape latency and reduced platform-crossing times. Immunoblotting analysis showed that behavioral abnormalities were associated with increased levels of P2RX2, phosphorylated-CaMKIIß and activated NF-κB in mouse hippocampus. Injection of A317491 ameliorated the impaired learning and memory of juvenile mice undergoing neonatal surgery plus anesthesia, and it also mitigated the neonatal surgery-induced signaling enhancement of P2RX2/CaMKII/NF-κB. Together, these results indicate that neonatal surgery plus anesthesia may cause long-term cognitive dysfunction, with potential mechanism of increasing P2RX2 and downstream signaling of phosphorylated-CaMKII and NF-κB. Our findings will promote more studies to assess detrimental effects of surgery and accompanying inflammation, diverse anesthetics and even sleeping deprivation on mouse neurodevelopment and neurobehavioral performance.
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Anestesia , Hipocampo , Aprendizaje por Laberinto , Trastornos de la Memoria , Anestesia/efectos adversos , Animales , Animales Recién Nacidos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Hipocampo/efectos de los fármacos , Hipocampo/fisiopatología , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Trastornos de la Memoria/epidemiología , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Receptores Purinérgicos P2X2 , Sevoflurano/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: P2X receptors are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding to their large extracellular domain. The seven known P2X subtypes can assemble as homotrimeric or heterotrimeric complexes and contribute to numerous physiological functions, including nociception, inflammation and hearing. The overall structure of P2X receptors is well established, but little is known about the range and prevalence of human genetic variations and the functional implications of specific domains. EXPERIMENTAL APPROACH: Here, we examine the impact of P2X2 receptor inter-subunit interface missense variants identified in the human population or by structural predictions. We test both single and double mutants through electrophysiological and biochemical approaches. KEY RESULTS: We demonstrate that predicted extracellular domain inter-subunit interfaces display a higher-than-expected density of missense variations and that the majority of mutations that disrupt putative inter-subunit interactions result in channels with higher apparent ATP affinity. Lastly, we show that double mutants at the subunit interface show significant energetic coupling, especially if located in close proximity. CONCLUSION AND IMPLICATIONS: We provide the first structural mapping of the mutational distribution across the human population in a ligand-gated ion channel and show that the density of missense mutations is constrained between protein domains, indicating evolutionary selection at the domain level. Our data may indicate that, unlike other ligand-gated ion channels, P2X2 receptors have evolved an intrinsically high threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation.
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Activación del Canal Iónico , Mutación Missense , Receptores Purinérgicos P2X2 , Adenosina Trifosfato/metabolismo , Humanos , Mutación , Receptores Purinérgicos P2X2/genéticaRESUMEN
P2X1 receptors are adenosine triphosphate (ATP)-gated cation channels that are functionally important for male fertility, bladder contraction, and platelet aggregation. The activity of P2X1 receptors is modulated by lipids and intracellular messengers such as cAMP, which can stimulate protein kinase A (PKA). Exchange protein activated by cAMP (EPAC) is another cAMP effector; however, its effect on P2X1 receptors has not yet been determined. Here, we demonstrate that P2X1 currents, recorded from human embryonic kidney (HEK) cells transiently transfected with P2X1 cDNA, were inhibited by the highly selective EPAC activator 007-AM. In contrast, EPAC activation enhanced P2X2 current amplitude. The PKA activator 6-MB-cAMP did not affect P2X1 currents, but inhibited P2X2 currents. The inhibitory effects of EPAC on P2X1 were prevented by triple mutation of residues 21 to 23 on the amino terminus of P2X1 subunits to the equivalent amino acids on P2X2 receptors. Double mutation of residues 21 and 22 and single mutation of residue 23 also protected P2X1 receptors from inhibition by EPAC activation. Finally, the inhibitory effects of EPAC on P2X1 were also prevented by NSC23766, an inhibitor of Rac1, a member of the Rho family of small GTPases. These data suggest that EPAC is an important regulator of P2X1 and P2X2 receptors.
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Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/farmacología , Riñón/metabolismo , Receptores Purinérgicos P2X1/metabolismo , Receptores Purinérgicos P2X2/metabolismo , Adenosina Trifosfato , Aminoquinolinas/farmacología , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Células HEK293 , Humanos , Riñón/efectos de los fármacos , Pirimidinas/farmacología , Receptores Purinérgicos P2X1/genética , Receptores Purinérgicos P2X2/genética , Proteína de Unión al GTP rac1/antagonistas & inhibidoresRESUMEN
Background: Hearing loss, a congenital genetic disorder in human, is difficult to diagnose. Whole exome sequencing is a powerful approach for ethiological disgnosis of such disorders. Methods: One Iranian family with two patients were attented in the study. Sequencing of known non-syndromic hearing loss genes was carried out to recognize the genetic causes of HL. Results: Molecular analyses identified a novel stop loss mutation, c.1048T>G (p.Term350Glu), whitin the P2RX2 gene, causing a termination-site modification.This event would lead to continued translation into the 3' UTR of the gene, which in turn may result in a longer protein product. The mutation was segregating with the disease phenotype and predicted to be pathogenic by bioinformatic tools. Conclusion: This study is the first Iranian case report of a diagnosis of autosomal dominant nonsyndromic hearing loss (ADNSHL) caused by P2RX2 mutation. The recognition of other causative mutations in P2RX2 gene more supports the probable function of this gene in causing ADNSHL.
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Sordera/genética , Mutación/genética , Receptores Purinérgicos P2X2/genética , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Familia , Femenino , Humanos , Irán , Masculino , Linaje , Receptores Purinérgicos P2X2/químicaRESUMEN
Amyloid beta peptide (Aß) is tightly associated with the physiopathology of Alzheimer's Disease (AD) as one of the most important factors in the evolution of the pathology. In this context, we previously reported that Aß increases the expression of ionotropic purinergic receptor 2 (P2×2R). However, its role on the cellular and molecular Aß toxicity is unknown, especially in human brain of AD patients. Using cellular and molecular approaches in hippocampal neurons, PC12 cells, and human brain samples of patients with AD, we evaluated the participation of P2×2R in the physiopathology of AD. Here, we reported that Aß oligomers (Aßo) increased P2×2 levels in mice hippocampal neurons, and that this receptor increases at late Braak stages of AD patients. Aßo also increases the colocalization of APP with Rab5, an early endosomes marker, and decreased the nuclear/cytoplasmic ratio of Fe65 and PGC-1α immunoreactivity. The overexpression in PC12 cells of P2×2a, but not P2×2b, replicated these changes in Fe65 and PGC-1α; however, both overexpressed isoforms increased levels of Aß. Taken together, these data suggest that P2×2 is upregulated in AD and it could be a key potentiator of the physiopathology of Aß. Our results point to a possible participation in a toxic cycle that increases Aß production, Ca2+ overload, and a decrease of PGC-1α. These novel findings put the P2×2R as a key novel pharmacological target to develop new therapeutic strategies to treat Alzheimer's Disease.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/metabolismo , Encéfalo/fisiopatología , Receptores Purinérgicos P2X2/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Femenino , Hipocampo/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Neuronas/metabolismo , Células PC12 , Ratas , Receptores Purinérgicos P2X2/genética , Regulación hacia ArribaRESUMEN
Gating of the ATP-activated channel P2X2 has been shown to be dependent not only on [ATP] but also on membrane voltage, despite the absence of a canonical voltage-sensor domain. We aimed to investigate the structural rearrangements of rat P2X2 during ATP- and voltage-dependent gating, using a voltage-clamp fluorometry technique. We observed fast and linearly voltage-dependent fluorescence intensity (F) changes at Ala337 and Ile341 in the TM2 domain, which could be due to the electrochromic effect, reflecting the presence of a converged electric field. We also observed slow and voltage-dependent F changes at Ala337, which reflect structural rearrangements. Furthermore, we determined that the interaction between Ala337 in TM2 and Phe44 in TM1, which are in close proximity in the ATP-bound open state, is critical for activation. Taking these results together, we propose that the voltage dependence of the interaction within the converged electric field underlies the voltage-dependent gating.
Asunto(s)
Adenosina Trifosfato/farmacología , Activación del Canal Iónico/efectos de los fármacos , Agonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X2/efectos de los fármacos , Animales , Fluorometría , Cinética , Potenciales de la Membrana , Microscopía Fluorescente , Mutación , Dominios Proteicos , Ratas , Receptores Purinérgicos P2X2/genética , Receptores Purinérgicos P2X2/metabolismo , Relación Estructura-Actividad , Xenopus laevisRESUMEN
Extracellular purines are important signaling molecules involved in numerous physiological and pathological processes via the activation of P2 receptors. Information about the spatial and temporal P2 receptor (P2R) expression and its regulation remains crucial for the understanding of the role of P2Rs in health and disease. To identify cells carrying P2X2Rs in situ, we have generated BAC transgenic mice that express the P2X2R subunits as fluorescent fusion protein (P2X2-TagRFP). In addition, we generated a BAC P2Y1R TagRFP reporter mouse expressing a TagRFP reporter for the P2RY1 gene expression. We demonstrate expression of the P2X2R in a subset of DRG neurons, the brain stem, the hippocampus, as well as on Purkinje neurons of the cerebellum. However, the weak fluorescence intensity in our P2X2R-TagRFP mouse precluded tracking of living cells. Our P2Y1R reporter mice confirmed the widespread expression of the P2RY1 gene in the CNS and indicate for the first time P2RY1 gene expression in mouse Purkinje cells, which so far has only been described in rats and humans. Our P2R transgenic models have advanced the understanding of purinergic transmission, but BAC transgenic models appeared not always to be straightforward and permanent reliable. We noticed a loss of fluorescence intensity, which depended on the number of progeny generations. These problems are discussed and may help to provide more successful animal models, even if in future more versatile and adaptable nuclease-mediated genome-editing techniques will be the methods of choice.