The enpp4 ectonucleotidase regulates kidney patterning signalling networks in Xenopus embryos

Abstract The enpp ectonucleotidases regulate lipidic and purinergic signalling pathways by controlling the extracellular concentrations of purines and bioactive lipids. Although both pathways are key regulators of kidney physiology and linked to human renal pathologies, their roles during nephrogenesis remain poorly understood. We previously showed that the pronephros was a major site of enpp expression and now demonstrate an unsuspected role for the conserved vertebrate enpp4 protein during kidney formation in Xenopus . Enpp4 over-expression results in ectopic renal tissues and, on rare occasion, complete mini-duplication of the entire kidney. Enpp4 is required and sufficient for pronephric markers expression and regulates the expression of RA, Notch and Wnt pathway members. Enpp4 is a membrane protein that binds, without hydrolyzing, phosphatidylserine and its effects are mediated by the receptor s1pr5, although not via the generation of S1P. Finally, we propose a novel and non-catalytic mechanism by which lipidic signalling regulates nephrogenesis

Excitatory effect of ATP on rat area postrema neurons

ATP-induced inward currents and increases in the cytosolic Ca2+ concentration ([Ca]in) were investigated in neurons acutely dissociated from rat area postrema using whole-cell patch-clamp recordings and fura-2 microfluorometry, respectively. The ATP-induced current (IATP) and [Ca]in increases were mimicked by 2-methylthio-ATP and ATP-γS, and were inhibited by P2X receptor (P2XR) antagonists. The current–voltage relationship of the IATP exhibited a strong inward rectification, and the amplitude of the IATP was concentration-dependent. The IATP was markedly reduced in the absence of external Na+, and the addition of Ca2+ to Na+-free saline increased the IATP. ATP did not increase [Ca]in in the absence of external Ca2+, and Ca2+ channel antagonists partially inhibited the ATP-induced [Ca]in increase, indicating that ATP increases [Ca]in by Ca2+ influx through both P2XR channels and voltage-dependent Ca2+ channels. There was a negative interaction between P2XR- and nicotinic ACh receptor (nAChR)-channels, which depended on the amplitude and direction of current flow through either channel. Current occlusion was observed at Vhs between −70 and −10 mV when the IATP and ACh-induced current (IACh) were inward, but no occlusion was observed when these currents were outward at a Vh of +40 mV. The IATP was not inhibited by co-application of ACh when the IACh was markedly decreased either by removal of permeant cations, by setting Vh close to the equilibrium potential of IACh, or by the addition of d-tubocurarine or serotonin. These results suggest that the inhibitory interaction is attributable to inward current flow of cations through the activated P2XR- and nAChR-channels

ATP from synaptic terminals and astrocytes regulates NMDA receptors and synaptic plasticity through PSD-95 multi-protein complex

Recent studies highlighted the importance of astrocyte-secreted molecules, such as ATP, for the slow modulation of synaptic transmission in central neurones. Biophysical mechanisms underlying the impact of gliotransmitters on the strength of individual synapse remain, however, unclear. Here we show that purinergic P2X receptors can bring significant contribution to the signalling in the individual synaptic boutons. ATP released from astrocytes facilitates a recruitment of P2X receptors into excitatory synapses by Ca2+-dependent mechanism. P2X receptors, co-localized with NMDA receptors in the excitatory synapses, can be activated by ATP co-released with glutamate from pre-synaptic terminals and by glia-derived ATP. An activation of P2X receptors in turn leads to down-regulation of postsynaptic NMDA receptors via Ca2+-dependent de-phosphorylation and interaction with PSD-95 multi-protein complex. Genetic deletion of the PSD-95 or P2X4 receptors obliterated ATP-mediated down-regulation of NMDA receptors. Impairment of purinergic modulation of NMDA receptors in the PSD-95 mutants dramatically decreased the threshold of LTP induction and increased the net magnitude of LTP. Our findings show that synergistic action of glia- and neurone-derived ATP can pre-modulate efficacy of excitatory synapses and thereby can have an important role in the glia-neuron communications and brain meta-plasticity

Modulation des propriétés fonctionnelles des récepteurs P2X par des interactions avec les récepteurs-canaux du GABA et la protéine AP2

Les récepteurs P2X activés par l'ATP et la famille des récepteurs "Cys-loop" appartiennent à 2 familles de récepteurs-cabaux différents sur les plans structurels et fonctionnels. D'une part, nous avons montré que les récepteurs P2X2 et P2X3 excitateurs, et les récepteurs GABAA/C inhibiteurs, colocalisés dans de nombreuses structures du système nerveux, interagissent fonctionnellement et moléculairement. Le couplage fait intervenir la boucle intrtacellulaire des sous-unités GABAA/C et le domaine C-terminal des sous-unités P2X. Nous avons identifié un motif de 3 acides aminés (386QST388) de la sous-unité P2X3 comme étant indispensable au couplage des récepteurs exprimés dans des ovocytes de xénope et in situdans des neurones sensoriels en culture. D'autre part, nous avons mis en évidence la modulation des propriétés fonctionnelles des récepteurs P2X4 par leur interaction avec la protéine d'endocytose AP-2 Nos résultats suggèrent que la potentialisation des récepteurs P2X4, par l'ivermectine n'est pas un mécanisme allostérique, mais lié à une augmentation du nombre de récepteurs à la surface due à l'altération de leur zndocytose constitutive.P2X receptors, which are activated by ATP, and "Cys-loop" receptor family belong to two structurally- and functionally- distinct receptor-chanel families. In this study, we first characterized the functional and molecular interaction between P2X2 or P2X3 excitatory receptors and GABAA/C channels that are colocalized in many areas of the nervous system. This cross talk involved the intracellular loop of GABAA/C subunits and the C-terminal domain of P2X subunits. Furthermore, we identified a 3 amino-acid motif (QST) within the P2X3 subunit sequence which is necessary for the functional coupling between these receptors expressed in a heterologous system (Xenopus ovocytes) or in nociceptive sensory neurons in culture. We have also shown that the interaction between the AP2 endocytosis protein and P2X4 receptors caused a modulation in the latter's functional properties. Our results suggested that a potentiation of P2X4 receptors by ivermectin is not an allosteric mechanism as previously proposed, but is primarily caused by an increase in the number of cell surface receptors resulting from the impairment of the clathrin-mediated endocytosis mechanism.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Modulation des propriétés fonctionnelles des récepteurs P2X par des interactions avec les récepteurs-canaux du GABA et la protéine AP2

Les récepteurs P2X activés par l'ATP et la famille des récepteurs "Cys-loop" appartiennent à 2 familles de récepteurs-cabaux différents sur les plans structurels et fonctionnels. D'une part, nous avons montré que les récepteurs P2X2 et P2X3 excitateurs, et les récepteurs GABAA/C inhibiteurs, colocalisés dans de nombreuses structures du système nerveux, interagissent fonctionnellement et moléculairement. Le couplage fait intervenir la boucle intrtacellulaire des sous-unités GABAA/C et le domaine C-terminal des sous-unités P2X. Nous avons identifié un motif de 3 acides aminés (386QST388) de la sous-unité P2X3 comme étant indispensable au couplage des récepteurs exprimés dans des ovocytes de xénope et in situdans des neurones sensoriels en culture. D'autre part, nous avons mis en évidence la modulation des propriétés fonctionnelles des récepteurs P2X4 par leur interaction avec la protéine d'endocytose AP-2 Nos résultats suggèrent que la potentialisation des récepteurs P2X4, par l'ivermectine n'est pas un mécanisme allostérique, mais lié à une augmentation du nombre de récepteurs à la surface due à l'altération de leur zndocytose constitutive.P2X receptors, which are activated by ATP, and "Cys-loop" receptor family belong to two structurally- and functionally- distinct receptor-chanel families. In this study, we first characterized the functional and molecular interaction between P2X2 or P2X3 excitatory receptors and GABAA/C channels that are colocalized in many areas of the nervous system. This cross talk involved the intracellular loop of GABAA/C subunits and the C-terminal domain of P2X subunits. Furthermore, we identified a 3 amino-acid motif (QST) within the P2X3 subunit sequence which is necessary for the functional coupling between these receptors expressed in a heterologous system (Xenopus ovocytes) or in nociceptive sensory neurons in culture. We have also shown that the interaction between the AP2 endocytosis protein and P2X4 receptors caused a modulation in the latter's functional properties. Our results suggested that a potentiation of P2X4 receptors by ivermectin is not an allosteric mechanism as previously proposed, but is primarily caused by an increase in the number of cell surface receptors resulting from the impairment of the clathrin-mediated endocytosis mechanism.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF