Seminario

12938 • The Journal of Neuroscience, November 26, 2008 • 28(48):12938 –12945

Cellular/Molecular

Phosphoinositides Regulate P2X4 ATP-Gated Channels through Direct Interactions
´ Louis-Philippe Bernier,1 Ariel R. Ase,1 Stephanie Chevallier,1 Dominique Blais,1 Qi Zhao,2 Eric Boue-Grabot,3 ´ ´ 2 and Philippe Seguela1 Diomedes Logothetis, ´ ´
Montreal Neurological Institute, Department ofNeurology and Neurosurgery, McGill University, Montreal, Quebec, Canada H3A 2B4, 2Department of ´ ´ Structural and Chemical Biology, Mount Sinai School of Medicine, New York University, New York, New York 10029, and 3Universite de Bordeaux 2, Centre ´ National de la Recherche Scientifique, Unite Mixte de Recherche 5227, 33076 Bordeaux, France ´
1

P2X receptors are ATP-gated nonselective cationchannels 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 decreasedP2X4 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 ofphosphoinositides 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 signalingphosphoinositides PIP2 and PIP3 on P2X4 function through direct channel–lipid interactions. Key words: P2X; purinergic; PIP2; calcium; microglia; neuropathic pain

Introduction
Mammalian P2X receptors are ATP-gated nonselective cation channels, generated from the trimeric assembly of homologous subunits (P2X1–7). Each subunit is composed of two transmembrane domains connected by a large ectodomain, withboth N and C termini facing the intracellular side of the plasma membrane (North, 2002). In the nervous system, P2X receptor channels are expressed at the plasma membrane of neurons, astrocytes, and microglia, where they contribute to intercellular purinergic communication. The P2X4 subunit forms a functional homomeric channel with high permeability to calcium ions (Egan and Khakh, 2004). It can alsoheteromerize with P2X1 (Nicke et al., 2005), P2X6 (Le ˆ et al., 1998), or P2X7 subunits (Guo et al., 2007). It is expressed at high levels in central neurons and in microglial cells (Le et al., ˆ 1998; Rubio and Soto, 2001; Tsuda et al., 2003). In recent years,

Received July 1, 2008; revised Oct. 10, 2008; accepted Oct. 14, 2008. This work has been supported by operating grants from CanadianInstitutes of Health Research (CIHR) (P.S.), Agence Nationale pour la Recherche and Projet International de Cooperation Scientifique du Centre National de la ´ Recherche Scientifique (E.B.G.), and National Institutes of Health (D.L.). L.P.B. holds a studentship from the CIHR training grant “Pain: Molecules to Community.” Correspondence should be addressed to Dr. Philippe Seguela, Montreal...