A dual polybasic motif determines phosphoinositide binding and regulation in the P2X channel family.
Phosphoinositides modulate the function of several ion channels, including most ATP-gated P2X receptor channels in neurons and glia, but little is known about the underlying molecular mechanism. We identified a phosphoinositide-binding motif formed of two clusters of positively charged amino acids located on the P2X cytosolic C-terminal domain, proximal to the second transmembrane domain. For all known P2X subtypes, the specific arrangement of basic residues in these semi-conserved clusters determines their sensitivity to membrane phospholipids. Neutralization of these positive charges disrupts the functional properties of the prototypical phosphoinositide-binding P2X4 subtype, mimicking wortmannin-induced phosphoinositide depletion, whereas adding basic residues at homologous positions to the natively insensitive P2X5 subtype establishes de novo phosphoinositide-mediated regulation. Moreover, biochemical evidence of in vitro P2X subunit-phospholipid interaction and functional intracellular phosphoinositide-binding assays demonstrate that the dual polybasic cluster is necessary and sufficient for regulation of P2X signaling by phospholipids.
Department of Neurology and Neurosurgery, Montreal Neurological Institute, Alan Edwards Centre for Research on Pain, McGill University, Montréal, Québec, Canada.
Louis-Philippe
LP
Bernier
Dominique
D
Blais
Éric
É
Boué-Grabot
Philippe
P
Séguéla
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D017124:Conserved Sequence; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010641:Phenotype; D010716:Phosphatidylinositols / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D058469:Receptors, Purinergic P2X / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D058474:Receptors, Purinergic P2X1 / Q000737:chemistry / Q000378:metabolism; D058485:Receptors, Purinergic P2X5 / Q000737:chemistry / Q000378:metabolism; D058486:Receptors, Purinergic P2X7 / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction
D010716:Phosphatidylinositols; D021122:Protein Subunits; D058469:Receptors, Purinergic P2X; D058474:Receptors, Purinergic P2X1; D058485:Receptors, Purinergic P2X5; D058486:Receptors, Purinergic P2X7