30 research outputs found
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-ÎłS, and were inhibited by P2X receptor (P2XR) antagonists. The currentâvoltage 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 â70 and â10 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 +40Â 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
Molecular and functional properties of P2X receptorsârecent progress and persisting challenges
Phosphoinositides Regulate P2X4 ATP-Gated Channels through Direct Interactions
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P2X Electrophysiology and Surface Trafficking in Xenopus Oocytes
International audienceXenopus oocytes serve as a standard heterologous expression system for the study of various ligand-gated ion channels including ATP P2X receptors. Here we describe the whole-cell two-electrode voltage clamp and biotinylation/Western blotting techniques to investigate the functional properties and surface trafficking from P2X-expressing oocytes
Protein interactions involving the Îł2 large cytoplasmic loop of GABAA receptors modulate conductance
Native GABAA channels display a single-channel conductance ranging between âŒ10 and 90 pS. Diazepam increases the conductance of some of these native channels but never those of recombinant receptors, unless they are coexpressed with GABARAP. This trafficking protein clusters recombinant receptors in the membrane, suggesting that high-conductance channels arise from receptors that are at locally high concentrations. The amphipathic (MA) helix that is present in the large cytoplasmic loop of every subunit of all ligand-gated ion channels mediates protein-protein interactions. Here we report that when applied to inside-out patches, a peptide mimicking the MA helix of the Îł2 subunit (Îł381-403) of the GABAA receptor abrogates the potentiating effect of diazepam on both endogenous receptors and recombinant GABAA receptors coexpressed with GABARAP, by substantially reducing their conductance. The protein interaction disrupted by the peptide did not involve GABARAP, because a shorter peptide (Îł386-403) known to compete with the Îł2-GABARAP interaction did not affect the conductance of recombinant αÎČÎł receptors coexpressed with GABARAP. The requirement for receptor clustering and the fact that the Îł2 MA helix is able to self-associate support a mechanism whereby adjacent GABAA receptors interact via their Îł2-subunit MA helices, altering ion permeation through each channel. Alteration of ion-channel function arising from dynamic interactions between ion channels of the same family has not been reported previously and highlights a novel way in which inhibitory neurotransmission in the brain may be differentially modulated.âEveritt, A. B., Seymour, V. A. L., Curmi, J., Laver, D. R., Gage, P. W., Tierney, M. L. Protein interactions involving the Îł2 large cytoplasmic loop of GABAA receptors modulate conductance