Louis-Philippe Bernier

Phosphoinositides Regulate P2X4 ATP-Gated Channels through Direct Interactions

P2X receptors are ATP-gated nonselective cation channels highly permeable to calcium that contribute to nociception and inflammatory responses. The P2X4 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 P2X4 function is crucial to the cellular mechanisms of neuropathic pain, however it remains poorly understood. Here, we show that the phosphoinositides PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3), products of phosphorylation by wortmannin-sensitive phosphatidylinositol 4-kinases and phosphatidylinositol 3-kinases, can modulate the function of native and recombinant P2X4 receptor channels. In BV-2 microglial cells, depleting the intracellular levels of PIP2 and PIP3 with wortmannin significantly decreased P2X4 current amplitude and P2X4-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 P2X4 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 PIP2 on excised inside-out macropatches rescued P2X4 currents from rundown. Moreover, we report the direct interaction of phospholipids with the proximal C-terminal domain of P2X4 subunit (Cys360–Val375) using an in vitro binding assay. These results demonstrate novel regulatory roles of the major signaling phosphoinositides PIP2 and PIP3 on P2X4 function through direct channel–lipid interactions.

Subtype-Specific Regulation of P2X3 and P2X2/3 Receptors by Phosphoinositides in Peripheral Nociceptors:

BackgroundP2X3 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.ResultsIn dissociated rat DRG neurons, incubation with the PI3K/PI4K inhibitor wortmannin at 35 μM induced a dramatic decrease in the amplitude of ATP- or α,β-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 μM 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 μM 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.ConclusionOur 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.

Ca2+ transients in astrocyte fine processes occur via Ca2+ influx in the adult mouse hippocampus

Astrocytes display complex morphologies with an array of fine extensions extending from the soma and the primary thick processes. Until the use of genetically encoded calcium indicators (GECIs) selectively expressed in astrocytes, Ca2+ signaling was only examined in soma and thick primary processes of astrocytes where Ca2+‐sensitive fluorescent dyes could be imaged. GECI imaging in astrocytes revealed a previously unsuspected pattern of spontaneous Ca2+ transients in fine processes that has not been observed without chronic expression of GECIs, raising potential concerns about the effects of GECI expression. Here, we perform two‐photon imaging of Ca2+ transients in adult CA1 hippocampal astrocytes using a new single‐cell patch‐loading strategy to image Ca2+‐sensitive fluorescent dyes in the cytoplasm of fine processes. We observed that astrocyte fine processes exhibited a high frequency of spontaneous Ca2+ transients whereas astrocyte soma rarely showed spontaneous Ca2+ oscillations similar to previous reports using GECIs. We exploited this new approach to show these signals were independent of neuronal spiking, metabotropic glutamate receptor (mGluR) activity, TRPA1 channels, and L‐ or T‐type voltage‐gated calcium channels. Removal of extracellular Ca2+ almost completely and reversibly abolished the spontaneous signals while IP3R2 KO mice also exhibited spontaneous and compartmentalized signals, suggesting they rely on influx of extracellular Ca2+. The Ca2+ influx dependency of the spontaneous signals in patch‐loaded astrocytes was also observed in astrocytes expressing GCaMP3, further highlighting the presence of Ca2+ influx pathways in astrocytes. The mechanisms underlying these localized Ca2+ signals are critical for understanding how astrocytes regulate important functions in the adult brain. GLIA 2016;64:2093–2103

Direct Modulation of P2X1 Receptor-Channels by the Lipid Phosphatidylinositol 4,5-Bisphosphate

The P2X1 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 P2X1 ATP receptors and their physiological consequences. In an isolated rat mesenteric artery preparation, we observed a strong inhibition of P2X1-mediated constrictive responses by depletion of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] with the phosphatidylinositol 4-kinase inhibitor wortmannin. Using the Xenopus laevis oocyte expression system, we provided electrophysiological evidence that lowering PI(4,5)P2 levels with wortmannin significantly decreases P2X1 current amplitude and recovery. Previously reported modulation of recovery of desensitized P2X1 currents by phospholipase C-coupled 5-hydroxytryptamine2A metabotropic receptors was also found to be wortmannin-sensitive. Treatment with wortmannin alters the kinetics of P2X1 activation and inactivation without changing its sensitivity to ATP. The functional impact of wortmannin on P2X1 currents could be reversed by addition of intracellular PI(4,5)P2, but not phosphatidylinositol 3,4,5-trisphosphate, and direct application of PI(4,5)P2 to excised inside-out macropatches rescued P2X1 currents from rundown. We showed that the proximal region of the intracellular C terminus of P2X1 subunit directly binds to PI(4,5)P2 and other anionic phospholipids, and we identified the basic residue Lys364 as a critical determinant for phospholipid binding and sensitivity to wortmannin. Overall, these results indicate that PI(4,5)P2 plays a key role in the expression of full native and heterologous P2X1 function by regulating the amplitude, recovery, and kinetics of ionotropic ATP responses through direct receptor-lipid interactions.

P2X4 receptor channels form large noncytolytic pores in resting and activated microglia

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 (PIPn) levels as it is inhibited by wortmannin, a blocker of PIPn 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.

Modulation of heteromeric P2X1/5 receptors by phosphoinositides in astrocytes depends on the P2X1 subunit

J. Neurochem. (2010) 113, 1676–1684.

N-Cadherin Prodomain Processing Regulates Synaptogenesis

Classical cadherins, which are adhesion molecules functioning at the CNS synapse, are synthesized as adhesively inactive precursor proteins in the endoplasmic reticulum (ER). Signal sequence and prodomain cleavage in the ER and Golgi apparatus, respectively, activates their adhesive properties. Here, we provide the first evidence for sorting of nonadhesive precursor N-cadherin (ProN) to the neuronal surface, where it coexists with adhesively competent mature N-cadherin (N-cad), generating a spectrum of adhesive strengths. In cultured hippocampal neurons, a high ProN/N-cad ratio downregulates synapse formation. Neurons expressing genetically engineered uncleavable ProN make markedly fewer synapses. The synapse number can be rescued to normality by depleting surface ProN levels through prodomain cleavage by an exogenous protease. Finally, prodomain processing is developmentally regulated in the rat hippocampus. We conclude that it is the ProN/N-cad ratio and not mature N-cad alone that is critical for regulation of adhesion during synaptogenesis.

Post-translational regulation of P2X receptor channels: modulation by phospholipids

P2X receptor channels mediate fast excitatory signaling by ATP and play major roles in sensory transduction, neuro-immune communication and inflammatory response. P2X receptors constitute a gene family of calcium-permeable ATP-gated cation channels therefore the regulation of P2X signaling is critical for both membrane potential and intracellular calcium homeostasis. Phosphoinositides (PIPn) are anionic signaling phospholipids that act as functional regulators of many types of ion channels. Direct PIPn binding was demonstrated for several ligand- or voltage-gated ion channels, however no generic motif emerged to accurately predict lipid-protein binding sites. This review presents what is currently known about the modulation of the different P2X subtypes by phospholipids and about critical determinants underlying their sensitivity to PIPn levels in the plasma membrane. All functional mammalian P2X subtypes tested, with the notable exception of P2X5, have been shown to be positively modulated by PIPn, i.e., homomeric P2X1, P2X2, P2X3, P2X4, and P2X7, as well as heteromeric P2X1/5 and P2X2/3 receptors. Based on various results reported on the aforementioned subtypes including mutagenesis of the prototypical PIPn-sensitive P2X4 and PIPn-insensitive P2X5 receptor subtypes, an increasing amount of functional, biochemical and structural evidence converges on the modulatory role of a short polybasic domain located in the proximal C-terminus of P2X subunits. This linear motif, semi-conserved in the P2X family, seems necessary and sufficient for encoding direct modulation of ATP-gated channels by PIPn. Furthermore, the physiological impact of the regulation of ionotropic purinergic responses by phospholipids on pain pathways was recently revealed in the context of native crosstalks between phospholipase C (PLC)-linked metabotropic receptors and P2X receptor channels in dorsal root ganglion sensory neurons and microglia.

Purinergic regulation of inflammasome activation after central nervous system injury

The body’s response to immune challenges and tissue damage involves a complex inflammatory process. The innate immune system acts as the initial barrier that protects from infectious agents and also senses endogenous danger molecules released after traumatic insults to mediate the response to

Inhibition of P2X4 function by P2Y6 UDP receptors in microglia.

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. GLIA 2013;61:2038–2049