Lê K T, Babinski K, Séguéla P
Cell Biology of Excitable Tissue Group, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A 2B4.
J Neurosci. 1998 Sep 15;18(18):7152-9. doi: 10.1523/JNEUROSCI.18-18-07152.1998.
Ionotropic ATP receptors are widely expressed in mammalian CNS. Despite extensive functional characterization of neuronal homomeric P2X receptors in heterologous expression systems, the subunit composition of native central P2X ATP-gated channels remains to be elucidated. P2X4 and P2X6 are major central subunits with highly overlapping mRNA distribution at both regional and cellular levels. When expressed alone in Xenopus oocytes, P2X6 subunits do not assemble into surface receptors responsive to ATP applications. On the other hand, P2X4 subunits assemble into bona fide ATP-gated channels, slowly desensitizing and weakly sensitive to the partial agonist alpha,beta-methylene ATP and to noncompetitive antagonists suramin and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid. We demonstrate here that the coexpression of P2X4 and P2X6 subunits in Xenopus oocytes leads to the generation of a novel pharmacological phenotype of ionotropic ATP receptors. Heteromeric P2X4+6 receptors are activated by low-micromolar alpha, beta-methylene ATP (EC50 = 12 microM) and are blocked by suramin and by Reactive Blue 2, which has the property, at low concentrations, to potentiate homomeric P2X4 receptors. The assembly of P2X4 with P2X6 subunits results from subunit-dependent interactions, as shown by their specific copurification from HEK-293 cells transiently transfected with various epitope-tagged P2X channel subunits. Our data strongly suggest that the numerous cases of neuronal colocalizations of P2X4 and P2X6 subunits observed in mammalian CNS reflect the native expression of heteromeric P2X4+6 channels with unique functional properties.
离子型ATP受体在哺乳动物中枢神经系统中广泛表达。尽管在异源表达系统中对神经元同聚体P2X受体进行了广泛的功能表征，但天然中枢P2X ATP门控通道的亚基组成仍有待阐明。P2X4和P2X6是主要的中枢亚基，在区域和细胞水平上mRNA分布高度重叠。当单独在非洲爪蟾卵母细胞中表达时，P2X6亚基不会组装成对ATP应用有反应的表面受体。另一方面，P2X4亚基组装成真正的ATP门控通道，对部分激动剂α,β-亚甲基ATP以及非竞争性拮抗剂苏拉明和5'-磷酸吡哆醛-6-偶氮苯-2',4'-二磺酸缓慢脱敏且敏感性较弱。我们在此证明，在非洲爪蟾卵母细胞中共表达P2X4和P2X6亚基会导致离子型ATP受体产生一种新的药理学表型。异聚体P2X4+6受体可被低微摩尔浓度的α,β-亚甲基ATP（EC50 = 12 μM）激活，并被苏拉明和活性蓝2阻断，活性蓝2在低浓度时具有增强同聚体P2X4受体的特性。P2X4与P2X6亚基的组装是由亚基依赖性相互作用导致的，这通过从瞬时转染了各种表位标签P2X通道亚基的HEK-293细胞中特异性共纯化得到证明。我们的数据强烈表明，在哺乳动物中枢神经系统中观察到的P2X4和P2X6亚基大量神经元共定位情况反映了具有独特功能特性的异聚体P2X4+6通道的天然表达。
