RESUMEN
P2X4 is an ATP-gated nonselective cation channel highly permeable to calcium. There is increasing evidence that this homomeric purinoceptor, which is expressed in several neuronal and immune cell types, is involved in chronic pain and inflammation. The current paucity of unambiguous pharmacological tools available to interrogate or modulate P2X4 function led us to pursue the search for selective antagonists. In the high-throughput screen of a compound library, we identified the phenylurea BX430 (1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea, molecular weight = 413), with antagonist properties on human P2X4-mediated calcium uptake. Patch-clamp electrophysiology confirmed direct inhibition of P2X4 currents by extracellular BX430, with submicromolar potency (IC50 = 0.54 µM). BX430 is highly selective, having virtually no functional impact on all other P2X subtypes, namely, P2X1-P2X3, P2X5, and P2X7, at 10-100 times its IC50. Unexpected species differences were noticed, as BX430 is a potent antagonist of zebrafish P2X4 but has no effect on rat and mouse P2X4 orthologs. The concentration-response curve for ATP on human P2X4 in the presence of BX430 shows an insurmountable blockade, indicating a noncompetitive allosteric mechanism of action. Using a fluorescent dye uptake assay, we observed that BX430 also effectively suppresses ATP-evoked and ivermectin-potentiated membrane permeabilization induced by P2X4 pore dilation. Finally, in single-cell calcium imaging, we validated its selective inhibitory effects on native P2X4 channels at the surface of human THP-1 cells that were differentiated into macrophages. In summary, this ligand provides a novel molecular probe to assess the specific role of P2X4 in inflammatory and neuropathic conditions, where ATP signaling has been shown to be dysfunctional.
Asunto(s)
Aminopiridinas/farmacología , Compuestos de Fenilurea/farmacología , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X4/metabolismo , Regulación Alostérica , Aminopiridinas/química , Animales , Calcio/metabolismo , Bases de Datos de Compuestos Químicos , Células HEK293 , Humanos , Ratones , Técnicas de Placa-Clamp , Compuestos de Fenilurea/química , Antagonistas del Receptor Purinérgico P2X/química , Ratas , Receptores Purinérgicos P2X4/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad de la Especie , Pez CebraRESUMEN
We report a novel model in which remote activation of peripheral nociceptive pathways in transgenic mice is achieved optogenetically, without any external noxious stimulus or injury. Taking advantage of a binary genetic approach, we selectively targeted Nav1.8(+) sensory neurons for conditional expression of channelrhodopsin-2 (ChR2) channels. Acute blue light illumination of the skin produced robust nocifensive behaviors, evoked by the remote stimulation of both peptidergic and nonpeptidergic nociceptive fibers as indicated by c-Fos labeling in laminae I and II of the dorsal horn of the spinal cord. A non-nociceptive component also contributes to the observed behaviors, as shown by c-Fos expression in lamina III of the dorsal horn and the expression of ChR2-EYFP in a subpopulation of large-diameter Nav1.8(+) dorsal root ganglion neurons. Selective activation of Nav1.8(+) afferents in vivo induced central sensitization and conditioned place aversion, thus providing a novel paradigm to investigate plasticity in the pain circuitry. Long-term potentiation was similarly evoked by light activation of the same afferents in isolated spinal cord preparations. These findings demonstrate, for the first time, the optical control of nociception and central sensitization in behaving mammals and enables selective activation of the same class of afferents in both in vivo and ex vivo preparations. Our results provide a proof-of-concept demonstration that optical dissection of the contribution of specific classes of afferents to central sensitization is possible. The high spatiotemporal precision offered by this non-invasive model will facilitate drug development and target validation for pain therapeutics.
Asunto(s)
Vías Aferentes/metabolismo , Optogenética , Umbral del Dolor/fisiología , Dolor/patología , Vigilia/fisiología , Vías Aferentes/patología , Animales , Reacción de Prevención/efectos de los fármacos , Reacción de Prevención/fisiología , Células Cultivadas , Channelrhodopsins , Femenino , Ganglios Espinales/citología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hiperalgesia/genética , Hiperalgesia/fisiopatología , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Morfina/farmacología , Morfina/uso terapéutico , Canal de Sodio Activado por Voltaje NAV1.8/genética , Dolor/tratamiento farmacológico , Dolor/genética , Dolor/fisiopatología , Umbral del Dolor/efectos de los fármacos , Receptores Purinérgicos P2X3/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/fisiología , Valina/análogos & derivados , Valina/farmacología , Vigilia/genética , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
BACKGROUND: Prolonged intracellular calcium elevation contributes to sensitization of nociceptors and chronic pain in inflammatory conditions. The underlying molecular mechanisms remain unknown but store-operated calcium entry (SOCE) components participate in calcium homeostasis, potentially playing a significant role in chronic pain pathologies. Most G protein-coupled receptors activated by inflammatory mediators trigger calcium-dependent signaling pathways and stimulate SOCE in primary afferents. The aim of the present study was to investigate the role of TRPC3, a calcium-permeable non-selective cation channel coupled to phospholipase C and highly expressed in DRG, as a link between activation of pro-inflammatory metabotropic receptors and SOCE in nociceptive pathways. RESULTS: Using in situ hybridization, we determined that TRPC3 and TRPC1 constitute the major TRPC subunits expressed in adult rat DRG. TRPC3 was found localized exclusively in small and medium diameter sensory neurons. Heterologous overexpression of TRPC3 channel subunits in cultured primary DRG neurons evoked a significant increase of Gd3+-sensitive SOCE following thapsigargin-induced calcium store depletion. Conversely, using the same calcium add-back protocol, knockdown of endogenous TRPC3 with shRNA-mediated interference or pharmacological inhibition with the selective TRPC3 antagonist Pyr10 induced a substantial decrease of SOCE, indicating a significant role of TRPC3 in SOCE in DRG nociceptors. Activation of P2Y2 purinoceptors or PAR2 protease receptors triggered a strong increase in intracellular calcium in conditions of TRPC3 overexpression. Additionally, knockdown of native TRPC3 or its selective pharmacological blockade suppressed UTP- or PAR2 agonist-evoked calcium responses as well as sensitization of DRG neurons. These data show a robust link between activation of pro-inflammatory receptors and calcium homeostasis through TRPC3-containing channels operating both in receptor- and store-operated mode. CONCLUSIONS: Our findings highlight a major contribution of TRPC3 to neuronal calcium homeostasis in somatosensory pathways based on the unique ability of these cation channels to engage in both SOCE and receptor-operated calcium influx. This is the first evidence for TRPC3 as a SOCE component in DRG neurons. The flexible role of TRPC3 in calcium signaling as well as its functional coupling to pro-inflammatory metabotropic receptors involved in peripheral sensitization makes it a potential target for therapeutic strategies in chronic pain conditions.
Asunto(s)
Nociceptores/fisiología , Transducción de Señal/fisiología , Canales Catiónicos TRPC/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Ganglios Espinales/citología , Regulación de la Expresión Génica/efectos de los fármacos , Imidazoles/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2Y2/metabolismo , Transducción de Señal/efectos de los fármacos , Médula Espinal/citología , Canales Catiónicos TRPC/genética , Tapsigargina/farmacología , Factores de Tiempo , Fosfolipasas de Tipo C/metabolismoRESUMEN
The adenosine triphosphate (ATP)-gated channel P2X7 is encoded by a gene enriched for common nonsynonymous variants. Many of these variants have functional cellular effects, and some have been implicated in chronic pain. In this study, we first systematically characterized all 17 common nonsynonymous variants using whole-cell patch clamp electrophysiology. Then, we analyzed these variants for statistical association with chronic pain phenotypes using both individual P2RX7 variants as predictors and cumulative allele counts of same-direction cellular effect in univariate models. Association and validation analyses were conducted in the Orofacial Pain: Prospective Evaluation and Risk Assessment (OPPERA) cohort (N = 3260) and in the Complex Persistent Pain Conditions (CPPC) cohort (N = 900), respectively. Our results showed an association between allele A of rs7958311 and an increased risk of chronic pelvic pain, with convergent evidence for contribution to fibromyalgia and irritable bowel syndrome, confirmed in a meta-analysis. This allelic variant produced a unique cellular phenotype: a gain-of-function in channel opening, and a loss-of-function in pore opening. A computational study using a 12-state Markov model of ATP binding to the P2X7 receptor suggested that this cellular phenotype arises from an increased ATP binding affinity and an increased open channel conductance combined with a loss of sensitization. Cumulative allele count analysis did not provide additional insights. In conclusion, our results go beyond reproducing association for rs7958311 with chronic pain and suggest that its unique combination of gain-of-function in channel and loss-of-function in pore activity may explain why it is likely the only common P2RX7 variant with contribution to chronic pain. PERSPECTIVE: This study characterizes all common P2RX7 variants using cellular assays and statistical association analyses with chronic pain, with Markov state modeling of the most robustly associated variant.
Asunto(s)
Dolor Crónico , Receptores Purinérgicos P2X7 , Humanos , Adenosina Trifosfato , Enfermedad Crónica , Dolor Crónico/genética , Dimensión del Dolor , Receptores Purinérgicos P2X7/genéticaRESUMEN
ATP-gated P2X4 receptor channels expressed in spinal microglia actively participate in central sensitization, making their functional regulation a key process in chronic pain pathologies. P2Y6 metabotropic Gq -coupled receptors, also expressed in microglia, are involved in the initial response to nerve injury, triggering phagocytosis upon activation by UDP. It has been reported recently that expression of both P2X4 and P2Y6 is upregulated in activated microglia following nerve injury. We show here, in resting as well as LPS-activated primary microglia, that P2Y6 decreases P2X4-mediated calcium entry and inhibits the dilation of P2X4 channels into a large-conductance pore measured with a YO-PRO-1 uptake assay. Furthermore, P2Y6 activation modulates the ATP-dependent migration of microglia, a process likely involved in their shift from migratory to phagocytic phenotype. Reconstituting the P2X4-P2Y6 interaction in recombinant systems shows that P2Y6 activation decreases P2X4 current amplitude, activation and desensitization rates, and reduces P2X4 channel permeability to the large cation NMDG(+) . Phospholipase C-mediated hydrolysis of the phosphoinositide PI(4,5)P2 , a necessary cofactor for P2X4 channel function, underlies this inhibitory crosstalk. As extracellular levels of both ATP and UDP are increased in the spinal cord following nerve injury, the control of P2X4 activity by P2Y6 might play a critical role in regulating neuropathic pain-inducing microglial responses.
Asunto(s)
Calcio/metabolismo , Microglía/metabolismo , Receptores Purinérgicos P2X4/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfato/farmacología , Animales , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Lipopolisacáridos/farmacología , Ratones , Microglía/citología , Microglía/efectos de los fármacos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X4/genética , Regulación hacia Arriba , Uridina Difosfato/metabolismoRESUMEN
P2X4 ATP-gated cation channels have been shown to contribute to the microglial component of central sensitization, making their functional regulation a key element in chronic pain pathologies. Here we show that prolonged activation of native P2X4 receptor channels by ATP induces opening of a pore permeable to NMDG(+) and large fluorescent dyes in BV-2 microglial cells and primary murine microglia. This intrinsic pore formation mechanism is potentiated by LPS treatment, known to upregulate P2X4 expression in microglial cells and to mimic the microglial activation observed in neuropathic pain states. Sustained activation of the P2X7 channel subtype, also expressed in microglia, induces a pore formation that requires pannexin hemichannels and leads to plasma membrane blebbing and cytotoxicity. In contrast, P2X4 pore formation is unaffected by the pannexin blocker carbenoxolone, does not induce cytoskeletal rearrangements and does not lead to cell death. Furthermore, we show that P2X4 pore dilation is modulated by phosphoinositides (PIP(n) ) levels as it is inhibited by wortmannin, a blocker of PIP(n) synthesis, suggesting possible regulation by phospholipase C-coupled pathways. Nonlethal P2X4 pore dilation could play a role in neuropathic pain by allowing the flux of large organic molecules in microglia. Different outcomes of P2X4 and P2X7 membrane permeabilization point to subtype-specific microglial responses to ATP in normal and pathological neuro-immune crosstalks.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Microglía/metabolismo , Receptores Purinérgicos P2X4/metabolismo , Fase de Descanso del Ciclo Celular/fisiología , Animales , Animales Recién Nacidos , Pruebas Inmunológicas de Citotoxicidad , Femenino , Colorantes Fluorescentes/metabolismo , Glutamatos/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Microglía/citología , Receptores Purinérgicos P2X4/química , Xenopus laevisRESUMEN
Purinergic signaling is critical for neuron-glia communication. Glial cells participate in synaptic transmission and express metabotropic P2Y as well as ionotropic P2X ATP receptors. In astrocytes, endogenous ATP-evoked currents with kinetics and pharmacology characteristic of the heteromeric P2X1/5 receptor channel have recently been reported. We investigated the interaction of major phosphoinositides with heteromeric P2X1/5 channels. Using patch-clamp electrophysiology on enhanced green fluorescent protein-expressing astrocytes acutely isolated from cortical slices of transgenic mice, we report a strong modulation of P2X1/5-like currents by phosphoinositides. Wortmannin-induced depletion of phosphoinositides decreases the amplitude of both the fast and sustained component of the P2X1/5-like currents although recovery and kinetics remain intact. In transfected human embryonic kidney cells, we provide evidence that depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] levels significantly decreases P2X1/5 currents while intracellular application of PI(4,5)P(2) completely rescued P2X1/5 currents, ruling out the involvement of phosphatidylinositol 3,4,5-trisphosphate. In contrast to P2X1, homomeric P2X5 current responses were found insensitive to phosphoinositides, and the C-terminus of P2X5 subunit lacked binding to phospholipids in an overlay assay. Our results suggest that the contribution of calcium-permeable heteromeric P2X1/5 receptor channels to the excitability of astrocytes is modulated by PI(4,5)P(2) through the P2X1 lipid-binding domain.
Asunto(s)
Fosfatidilinositoles/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Androstadienos/farmacología , Animales , Animales Recién Nacidos , Astrocitos , Encéfalo/citología , Línea Celular Transformada , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica/métodos , Inhibidores Enzimáticos/farmacología , Proteína Ácida Fibrilar de la Glía/genética , Proteínas Fluorescentes Verdes/genética , Humanos , Lípidos de la Membrana/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Transgénicos , Técnicas de Placa-Clamp/métodos , Fosfatidilinositol 4,5-Difosfato/farmacología , Ratas , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X , Receptores Purinérgicos P2X5 , Transfección/métodos , WortmaninaRESUMEN
Microglia and hematogenous myeloid cells are prominent components of inflammatory central nervous system (CNS) lesions associated with tissue injury. To help define the basis for recruitment of such cells into lesions and their contribution to the disease process, we characterized the migratory and cytokine responses of human adult and fetal microglia in the presence of extracellular ATP comparing them to monocytes and macrophages. Adult microglia showed increased migration in response to low ATP concentrations (1-10 µM) whereas fetal microglia also migrated in response to higher ATP dosages (100-300 µM). The enhanced migration of microglia was reproduced with 2-MeSADP, a P2Y1/12/13 agonist. In contrast, the chemokine CCL2 did not promote migration of microglia, but promoted the migration of monocytes. Monocyte migration was also enhanced with low concentrations of ATP, whereas higher concentrations of ATP mediated an inhibitory effect. ATP had only an inhibitory effect on macrophages, which was not reproduced with hydrolysis products ADP or adenosine. ATP led to a decrease in LPS-induced pro-inflammatory cytokine release (TNFα, IL-6) in both microglia and macrophages without suppression of an anti-inflammatory response (IL-10). These in vitro based results suggest that ATP can selectively favor the recruitment of microglia rather than hematogenous myeloid cells while promoting an anti-inflammatory state in both hematogenous and resident myeloid cells of the CNS. Our results highlight the importance of environmental signals in shaping the properties of the innate immune response to injury in the CNS.
Asunto(s)
Adenosina Trifosfato/farmacología , Movimiento Celular/efectos de los fármacos , Citocinas/biosíntesis , Macrófagos/efectos de los fármacos , Microglía/efectos de los fármacos , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Encéfalo/citología , Quimiocina CCL2/farmacología , Femenino , Feto/citología , Citometría de Flujo , Humanos , Técnicas In Vitro , Indicadores y Reactivos , Interleucina-10/biosíntesis , Interleucina-6/biosíntesis , Lipopolisacáridos/farmacología , Embarazo , Agonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y1/efectos de los fármacos , Tionucleótidos/farmacología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Mutations in the parkin gene result in an autosomal recessive juvenile-onset form of Parkinson's disease. As an E3 ubiquitin-ligase, parkin promotes the attachment of ubiquitin onto specific substrate proteins. Defects in the ubiquitination of parkin substrates are therefore believed to lead to neurodegeneration in Parkinson's disease. Here, we identify the PSD-95/Discs-large/Zona Occludens-1 (PDZ) protein PICK1 as a novel parkin substrate. We find that parkin binds PICK1 via a PDZ-mediated interaction, which predominantly promotes PICK1 monoubiquitination rather than polyubiquitination. Consistent with monoubiquitination and recent work implicating parkin in proteasome-independent pathways, parkin does not promote PICK1 degradation. However, parkin regulates the effects of PICK1 on one of its other PDZ partners, the acid-sensing ion channel (ASIC). Overexpression of wild-type, but not PDZ binding- or E3 ubiquitin-ligase-defective parkin abolishes the previously described, protein kinase C-induced, PICK1-dependent potentiation of ASIC2a currents in non-neuronal cells. Conversely, the loss of parkin in hippocampal neurons from parkin knockout mice unmasks prominent potentiation of native ASIC currents, which is normally suppressed by endogenous parkin in wild-type neurons. Given that ASIC channels contribute to excitotoxicity, our work provides a mechanism explaining how defects in parkin-mediated PICK1 monoubiquitination could enhance ASIC activity and thereby promote neurodegeneration in Parkinson's disease.
Asunto(s)
Proteínas Portadoras/metabolismo , Activación del Canal Iónico , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Canales de Sodio/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Canales Iónicos Sensibles al Ácido , Aminoácidos , Animales , Células COS , Proteínas de Ciclo Celular , Chlorocebus aethiops , Células HeLa , Humanos , Ratones , Neuronas/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Ratas , Fracciones Subcelulares/metabolismo , Especificidad por SustratoRESUMEN
Itch and pain are distinct unpleasant sensations that can be triggered from the same receptive fields in the skin, raising the question of how pruriception and nociception are coded and discriminated. Here, we tested the multimodal capacity of peripheral first-order neurons, focusing on the genetically defined subpopulation of mouse C-fibers that express the chloroquine receptor MrgprA3. Using optogenetics, chemogenetics, and pharmacology, we assessed the behavioral effects of their selective stimulation in a wide variety of conditions. We show that metabotropic Gq-linked stimulation of these C-afferents, through activation of native MrgprA3 receptors or DREADDs, evokes stereotypical pruriceptive rather than nocifensive behaviors. In contrast, fast ionotropic stimulation of these same neurons through light-gated cation channels or native ATP-gated P2X3 channels predominantly evokes nocifensive rather than pruriceptive responses. We conclude that C-afferents display intrinsic multimodality, and we provide evidence that optogenetic and chemogenetic interventions on the same neuronal populations can drive distinct behavioral outputs.
Asunto(s)
Channelrhodopsins/metabolismo , Fibras Nerviosas Amielínicas/metabolismo , Neuronas Aferentes/metabolismo , Nocicepción/fisiología , Dolor/metabolismo , Prurito/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Adenosina Trifosfato , Animales , Cloroquina , Ganglios Espinales/metabolismo , Péptido Liberador de Gastrina/metabolismo , Luz , Ratones , Neuronas Aferentes/fisiología , Optogenética , Receptores Opioides/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
P2X receptors are ATP-gated nonselective cation channels highly permeable to calcium that contribute to nociception and inflammatory responses. The P2X(4) subtype, upregulated in activated microglia, is thought to play a critical role in the development of tactile allodynia following peripheral nerve injury. Posttranslational regulation of P2X(4) function is crucial to the cellular mechanisms of neuropathic pain, however it remains poorly understood. Here, we show that the phosphoinositides PI(4,5)P(2) (PIP(2)) and PI(3,4,5)P(3) (PIP(3)), products of phosphorylation by wortmannin-sensitive phosphatidylinositol 4-kinases and phosphatidylinositol 3-kinases, can modulate the function of native and recombinant P2X(4) receptor channels. In BV-2 microglial cells, depleting the intracellular levels of PIP(2) and PIP(3) with wortmannin significantly decreased P2X(4) current amplitude and P2X(4)-mediated calcium entry measured in patch clamp recordings and ratiometric ion imaging, respectively. Wortmannin-induced depletion of phosphoinositides in Xenopus oocytes decreased the current amplitude of P2X(4) responses by converting ATP into a partial agonist. It also decreased their recovery from desensitization and affected their kinetics. Injection of phosphoinositides in wortmannin-treated oocytes reversed these effects and application of PIP(2) on excised inside-out macropatches rescued P2X(4) currents from rundown. Moreover, we report the direct interaction of phospholipids with the proximal C-terminal domain of P2X(4) subunit (Cys(360)-Val(375)) using an in vitro binding assay. These results demonstrate novel regulatory roles of the major signaling phosphoinositides PIP(2) and PIP(3) on P2X(4) function through direct channel-lipid interactions.
Asunto(s)
Adenosina Trifosfato/metabolismo , Inflamación/metabolismo , Activación del Canal Iónico/fisiología , Microglía/metabolismo , Fosfatidilinositoles/metabolismo , Receptores Purinérgicos P2/metabolismo , Androstadienos/farmacología , Animales , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Femenino , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatología , Inflamación/fisiopatología , Ratones , Oocitos , Técnicas de Placa-Clamp , Enfermedades del Sistema Nervioso Periférico/metabolismo , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/fisiología , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2X4 , Wortmanina , Xenopus laevisRESUMEN
BACKGROUND: P2X3 and P2X2/3 purinergic receptor-channels, expressed in primary sensory neurons that mediate nociception, have been implicated in neuropathic and inflammatory pain responses. The phospholipids phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) are involved in functional modulation of several types of ion channels. We report here evidence that these phospholipids are able to modulate the function of homomeric P2X3 and heteromeric P2X2/3 purinoceptors expressed in dorsal root ganglion (DRG) nociceptors and in heterologous expression systems. RESULTS: In dissociated rat DRG neurons, incubation with the PI3K/PI4K inhibitor wortmannin at 35 microM induced a dramatic decrease in the amplitude of ATP- or alpha,beta-meATP-evoked P2X3 currents, while incubation with 100 nM wortmannin (selective PI3K inhibition) produced no significant effect. Intracellular application of PIP2 was able to fully reverse the inhibition of P2X3 currents induced by wortmannin. In Xenopus oocytes and in HEK293 cells expressing recombinant P2X3, 35 microM wortmannin incubation induced a significant decrease in the rate of receptor recovery. Native and recombinant P2X2/3 receptor-mediated currents were inhibited by incubation with wortmannin both at 35 microM and 100 nM. The decrease of P2X2/3 current amplitude induced by wortmannin could be partially reversed by application of PIP2 or PIP3, indicating a sensitivity to both phosphoinositides in DRG neurons and Xenopus oocytes. Using a lipid binding assay, we demonstrate that the C-terminus of the P2X2 subunit binds directly to PIP2, PIP3 and other phosphoinositides. In contrast, no direct binding was detected between the C-terminus of P2X3 subunit and phosphoinositides. CONCLUSION: Our findings indicate a functional regulation of homomeric P2X3 and heteromeric P2X2/3 ATP receptors by phosphoinositides in the plasma membrane of DRG nociceptors, based on subtype-specific mechanisms of direct and indirect lipid sensing.
Asunto(s)
Fosfatidilinositol 4,5-Difosfato/fisiología , Fosfatos de Fosfatidilinositol/fisiología , Receptores Purinérgicos P2/metabolismo , Androstadienos/farmacología , Animales , Western Blotting , Línea Celular , Células Cultivadas , Ganglios Espinales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Técnicas de Placa-Clamp , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2X3 , Wortmanina , XenopusRESUMEN
P2X receptors constitute a gene family of cation channels gated by extracellular ATP. They mediate fast ionotropic purinergic signaling in neurons and non-excitable cell types in vertebrates. The highly calcium-permeable P2X4 subtype has been shown to play a significant role in cardiovascular physiology, inflammatory responses and neuro-immune communication. We previously reported the discovery of a P2X4-selective antagonist, the small organic compound BX430, with submicromolar potency for human P2X4 receptors and marked species-dependence (Ase et al., 2015). The present study investigates the molecular basis of P2X4 inhibition by the non-competitive blocker BX430 using a structural and functional approach relying on mutagenesis and electrophysiology. We provide evidence for the critical contribution of a single hydrophobic residue located in the ectodomain of P2X4 channel subunits, Ile312 in human P2X4, which determines blockade by BX430. We also show that the nature of this extracellular residue in various vertebrate P2X4 orthologs underlies their specific sensitivity or resistance to the inhibitory effects of BX430. Taking advantage of high-resolution crystallographic data available on zebrafish P2X4, we used molecular dynamics simulation to model the docking of BX430 on an allosteric binding site around Ile315 (zebrafish numbering) in the ectodomain of P2X4. We also observed that the only substitution I312D (human numbering) that renders P2X4 silent by itself has also a profound silencing effect on all other P2X subtypes tested when introduced at homologous positions. The generic impact of this aspartate mutation on P2X function indicates that the pre-TM2 subregion involved is conserved functionally and defines a novel allosteric inhibitory site present in all P2X receptor channels. This conserved structure-channel activity relationship might be exploited for the rational design of potent P2X subtype-selective antagonists of therapeutic value.
RESUMEN
The P2X(1) receptor-channels activated by extracellular ATP contribute to the neurogenic component of smooth muscle contraction in vascular beds and genitourinary tracts of rodents and humans. In the present study, we investigated the interactions of plasma membrane phosphoinositides with P2X(1) ATP receptors and their physiological consequences. In an isolated rat mesenteric artery preparation, we observed a strong inhibition of P2X(1)-mediated constrictive responses by depletion of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] with the phosphatidylinositol 4-kinase inhibitor wortmannin. Using the Xenopus laevis oocyte expression system, we provided electrophysiological evidence that lowering PI(4,5)P(2) levels with wortmannin significantly decreases P2X(1) current amplitude and recovery. Previously reported modulation of recovery of desensitized P2X(1) currents by phospholipase C-coupled 5-hydroxytryptamine(2A) metabotropic receptors was also found to be wortmannin-sensitive. Treatment with wortmannin alters the kinetics of P2X(1) activation and inactivation without changing its sensitivity to ATP. The functional impact of wortmannin on P2X(1) currents could be reversed by addition of intracellular PI(4,5)P(2), but not phosphatidylinositol 3,4,5-trisphosphate, and direct application of PI(4,5)P(2) to excised inside-out macropatches rescued P2X(1) currents from rundown. We showed that the proximal region of the intracellular C terminus of P2X(1) subunit directly binds to PI(4,5)P(2) and other anionic phospholipids, and we identified the basic residue Lys(364) as a critical determinant for phospholipid binding and sensitivity to wortmannin. Overall, these results indicate that PI(4,5)P(2) plays a key role in the expression of full native and heterologous P2X(1) function by regulating the amplitude, recovery, and kinetics of ionotropic ATP responses through direct receptor-lipid interactions.
Asunto(s)
Activación del Canal Iónico/efectos de los fármacos , Fosfatidilinositol 4,5-Difosfato/farmacología , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Androstadienos/farmacología , Animales , Electrofisiología , Técnicas In Vitro , Cinética , Lisina/metabolismo , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiología , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Ratas , Receptor de Serotonina 5-HT2A/metabolismo , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2X , Proteínas Recombinantes/metabolismo , Vasoconstricción/efectos de los fármacos , Wortmanina , XenopusRESUMEN
Chronic pain is a highly prevalent debilitating condition for which treatment options remain limited for many patients. Ionotropic ATP signalling through excitatory and calcium-permeable P2X receptor channels is now rightfully considered as a critical player in pathological pain generation and maintenance; therefore, their selective targeting represents a therapeutic opportunity with promising yet untapped potential. Recent advances in the structural, functional and pharmacological characterization of rodent and human ATP-gated P2X receptor channels have shed brighter light on the role of specific subtypes in the pathophysiology of chronic inflammatory, neuropathic or cancer pain. Here, we will review the contribution of P2X3, P2X4 and P2X7 receptors to chronic pain and discuss the opportunities and challenges associated with the pharmacological manipulation of their function. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.
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Dolor Crónico/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Animales , HumanosRESUMEN
The sensory neuron of Aplysia californica participates in several forms of presynaptic plasticity including homosynaptic depression, heterosynaptic depression, facilitation and the reversal of depression. The calcium channel triggering neurotransmitter release at most synapses is CaV2, consisting of the pore forming α1 subunit (CaV2α1), and auxiliary CaVß, and CaVα2δ subunits. To determine the role of the CaV2 channel in presynaptic plasticity in Aplysia, we cloned Aplysia CaV2α1, CaVß, and CaVα2δ and over-expressed the proteins in Aplysia sensory neurons (SN). We show expression of exogenous CaV2α1 in the neurites of cultured Aplysia SN. One proposed mechanism for heterosynaptic depression in Aplysia is through inhibition of CaV2. Here, we demonstrate that heterosynaptic depression of the CaV2 calcium current is inhibited when a channel with a Y-F mutation at the conserved Src phosphorylation site is expressed, showing the strong conservation of this mechanism over evolution. We also show that the Y-F mutation reduces heterosynaptic inhibition of neurotransmitter release, highlighting the physiological importance of this mechanism for the regulation of synaptic efficacy. These results also demonstrate our ability to replace endogenous CaV2 channels with recombinant channels allowing future examination of the structure function relationship of CaV2 in the regulation of transmitter release in this system.
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Aplysia , Canales de Calcio/metabolismo , Plasticidad Neuronal , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Células Receptoras Sensoriales/fisiología , Sustitución de Aminoácidos , Animales , Canales de Calcio/genética , Células Cultivadas , Clonación Molecular , Análisis Mutacional de ADN , Motivos EF Hand , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Células Receptoras Sensoriales/enzimología , Tirosina/genéticaRESUMEN
Activation of microglia has been implicated in many neurological conditions including Alzheimer's disease and neuropathic pain. Recent studies provide evidence that P2X ATP receptors on the surface of microglia play a crucial role in initiation of inflammatory cascades. We investigated changes in surface P2X receptors in BV-2 murine microglial cells following their activation by pro-inflammatory bacterial lipopolysaccharides (LPS). mRNA analysis using RT-PCR confirmed the presence of P2X4 and P2X7 as the main P2X subunits. Application of ATP at low (< or =100 microM) and high (> or =1 mM) concentrations, as well as BzATP, activated inward currents in BV-2 cells. Current responses of P2X4 and P2X7 subtypes could be distinguished based on their respective sensitivity to the positive modulator ivermectin and to the antagonist Brilliant Blue G. Treatment of BV-2 cells with LPS leads to a transient increase in ivermectin-sensitive P2X4 currents, while dominant P2X7 currents remain largely unaffected. This increase in P2X4 function was concomitant with higher receptor protein expression, itself related to an upregulation of P2X4 mRNA levels that peaked at 48 h post-LPS treatment. Our data demonstrate that although LPS activation has a minor impact on P2X7 receptors that remain the major ionotropic ATP receptors in microglia, it specifically enhances responses to low ATP concentrations mediated by P2X4 receptors, highlighting the significant contribution of both subtypes to neuroinflammatory mechanisms and pathologies.
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Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Microglía/fisiología , Receptores Purinérgicos P2/genética , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Línea Celular , Potenciales Evocados/efectos de los fármacos , Humanos , Inmunohistoquímica , Riñón , Ratones , Microglía/efectos de los fármacos , Técnicas de Placa-Clamp , Receptores Purinérgicos P2X4 , Receptores Purinérgicos P2X7 , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Primary C-fiber nociceptors are broadly divided into peptidergic and nonpeptidergic afferents. TRPV1 is a thermosensitive cation channel mainly localized in peptidergic nociceptors, whereas MrgD is a sensory G protein-coupled receptor expressed in most nonpeptidergic nociceptive afferents. TRPV1 and MrgD fibers have been reported to be primarily involved in thermal and mechanical nociception, respectively. Yet, their functional assessment in somatosensory transmission relied on ablation strategies that do not account for compensatory mechanisms. To achieve selective activation of these 2 major subsets of C-fibers in vivo in adult mice, we used optogenetics to specifically deliver the excitatory opsin channelrhodopsin-2 (ChR2) to TRPV1 or MrgD primary sensory neurons, as confirmed by histology and electrophysiology. This approach allowed, for the first time, the characterization of behavioral responses triggered by direct noninvasive activation of peptidergic TRPV1 or nonpeptidergic MrgD fibers in freely moving mice. Transdermal blue light stimulation of the hind paws of transgenic mice expressing ChR2 in TRPV1 neurons generated nocifensive behaviors consisting mainly of paw withdrawal and paw licking, whereas paw lifting occurrence was limited. Conversely, optical activation of cutaneous MrgD afferents produced mostly withdrawal and lifting. Of interest, in a conditioned place avoidance assay, blue light induced aversion in TRPV1-ChR2 mice, but not in MrgD-ChR2 mice. In short, we present novel somatosensory transgenic models in which control of specific subsets of peripheral unmyelinated nociceptors with distinct functions can be achieved with high spatiotemporal precision. These new tools will be instrumental in further clarifying the contribution of genetically identified C-fiber subtypes to chronic pain.
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Conducta Animal/fisiología , Fibras Nerviosas Amielínicas/metabolismo , Optogenética , Dolor/fisiopatología , Animales , Ganglios Espinales/fisiopatología , Ratones Transgénicos , Nociceptores/fisiología , Células Receptoras Sensoriales/fisiología , Piel/fisiopatología , Canales Catiónicos TRPV/genéticaRESUMEN
We report a novel transgenic mouse model in which the terminals of peripheral nociceptors can be silenced optogenetically with high spatiotemporal precision, leading to the alleviation of inflammatory and neuropathic pain. Inhibitory archaerhodopsin-3 (Arch) proton pumps were delivered to Nav1.8(+) primary afferents using the Nav1.8-Cre driver line. Arch expression covered both peptidergic and nonpeptidergic nociceptors and yellow light stimulation reliably blocked electrically induced action potentials in DRG neurons. Acute transdermal illumination of the hindpaws of Nav1.8-Arch(+) mice significantly reduced mechanical allodynia under inflammatory conditions, while basal mechanical sensitivity was not affected by the optical stimulation. Arch-driven hyperpolarization of nociceptive terminals was sufficient to prevent channelrhodopsin-2 (ChR2)-mediated mechanical and thermal hypersensitivity in double-transgenic Nav1.8-ChR2(+)-Arch(+) mice. Furthermore, prolonged optical silencing of peripheral afferents in anesthetized Nav1.8-Arch(+) mice led to poststimulation analgesia with a significant decrease in mechanical and thermal hypersensitivity under inflammatory and neuropathic conditions. These findings highlight the role of peripheral neuronal inputs in the onset and maintenance of pain hypersensitivity, demonstrate the plasticity of pain pathways even after sensitization has occurred, and support the involvement of Nav1.8(+) afferents in both inflammatory and neuropathic pain. Together, we present a selective analgesic approach in which genetically identified subsets of peripheral sensory fibers can be remotely and optically inhibited with high temporal resolution, overcoming the compensatory limitations of genetic ablations.
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Analgesia/métodos , Modelos Animales de Enfermedad , Inflamación/complicaciones , Canal de Sodio Activado por Voltaje NAV1.8/fisiología , Neuralgia/prevención & control , Neuralgia/fisiopatología , Nociceptores/fisiología , Optogenética/métodos , Potenciales de Acción , Animales , Proteínas Arqueales/genética , Femenino , Ganglios Espinales/fisiopatología , Miembro Posterior/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Vías Nerviosas/fisiopatología , Neuronas/fisiología , Nociceptores/metabolismo , Dolor/fisiopatología , Dolor/prevención & control , Umbral del DolorRESUMEN
OBJECTIVE: To investigate and measure the functional significance of altered P2Y12 expression in the context of human microglia activation. METHODS: We performed in vitro and in situ experiments to measure how P2Y12 expression can influence disease-relevant functional properties of classically activated (M1) and alternatively activated (M2) human microglia in the inflamed brain. RESULTS: We demonstrated that compared to resting and classically activated (M1) human microglia, P2Y12 expression is increased under alternatively activated (M2) conditions. In response to ADP, the endogenous ligand of P2Y12, M2 microglia have increased ligand-mediated calcium responses, which are blocked by selective P2Y12 antagonism. P2Y12 antagonism was also shown to decrease migratory and inflammatory responses in human microglia upon exposure to nucleotides that are released during CNS injury; no effects were observed in human monocytes or macrophages. In situ experiments confirm that P2Y12 is selectively expressed on human microglia and elevated under neuropathologic conditions that promote Th2 responses, such as parasitic CNS infection. CONCLUSION: These findings provide insight into the roles of M2 microglia in the context of neuroinflammation and suggest a mechanism to selectively target a functionally unique population of myeloid cells in the CNS.