Bernier Louis-Philippe, Ase Ariel R, Tong Xinkang, Hamel Edith, Blais Dominique, Zhao Qi, Logothetis Diomedes E, Séguéla Philippe
Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.
Mol Pharmacol. 2008 Sep;74(3):785-92. doi: 10.1124/mol.108.047019. Epub 2008 Jun 3.
The P2X(1) 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 P2X(1) ATP receptors and their physiological consequences. In an isolated rat mesenteric artery preparation, we observed a strong inhibition of P2X(1)-mediated constrictive responses by depletion of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] with the phosphatidylinositol 4-kinase inhibitor wortmannin. Using the Xenopus laevis oocyte expression system, we provided electrophysiological evidence that lowering PI(4,5)P(2) levels with wortmannin significantly decreases P2X(1) current amplitude and recovery. Previously reported modulation of recovery of desensitized P2X(1) currents by phospholipase C-coupled 5-hydroxytryptamine(2A) metabotropic receptors was also found to be wortmannin-sensitive. Treatment with wortmannin alters the kinetics of P2X(1) activation and inactivation without changing its sensitivity to ATP. The functional impact of wortmannin on P2X(1) currents could be reversed by addition of intracellular PI(4,5)P(2), but not phosphatidylinositol 3,4,5-trisphosphate, and direct application of PI(4,5)P(2) to excised inside-out macropatches rescued P2X(1) currents from rundown. We showed that the proximal region of the intracellular C terminus of P2X(1) subunit directly binds to PI(4,5)P(2) and other anionic phospholipids, and we identified the basic residue Lys(364) as a critical determinant for phospholipid binding and sensitivity to wortmannin. Overall, these results indicate that PI(4,5)P(2) plays a key role in the expression of full native and heterologous P2X(1) function by regulating the amplitude, recovery, and kinetics of ionotropic ATP responses through direct receptor-lipid interactions.
细胞外ATP激活的P2X(1)受体通道，在啮齿动物和人类的血管床及泌尿生殖道平滑肌收缩的神经源性成分中发挥作用。在本研究中，我们研究了质膜磷酸肌醇与P2X(1) ATP受体的相互作用及其生理后果。在离体大鼠肠系膜动脉标本中，我们观察到用磷脂酰肌醇4-激酶抑制剂渥曼青霉素耗尽磷脂酰肌醇4,5-二磷酸[PI(4,5)P(2)]后，P2X(1)介导的收缩反应受到强烈抑制。利用非洲爪蟾卵母细胞表达系统，我们提供了电生理证据，表明渥曼青霉素降低PI(4,5)P(2)水平会显著降低P2X(1)电流幅度和恢复。先前报道的磷脂酶C偶联的5-羟色胺(2A)代谢型受体对脱敏P2X(1)电流恢复的调节也被发现对渥曼青霉素敏感。渥曼青霉素处理改变了P2X(1)激活和失活的动力学，而不改变其对ATP的敏感性。渥曼青霉素对P2X(1)电流的功能影响可通过添加细胞内PI(4,5)P(2)而不是磷脂酰肌醇3,4,5-三磷酸来逆转，并且将PI(4,5)P(2)直接应用于切除的内向外大膜片可挽救P2X(1)电流免于衰减。我们表明，P2X(1)亚基细胞内C末端的近端区域直接与PI(4,5)P(2)和其他阴离子磷脂结合，并且我们确定碱性残基Lys(364)是磷脂结合和对渥曼青霉素敏感性的关键决定因素。总体而言，这些结果表明，PI(4,5)P(2)通过直接的受体-脂质相互作用调节离子型ATP反应的幅度、恢复和动力学，在完整天然和异源P2X(1)功能的表达中起关键作用。
