Roles of ectodomain and transmembrane regions in ethanol and agonist action in purinergic P2X2 and P2X3 receptors
Roles of ectodomain and transmembrane regions in ethanol and agonist action in purinergic P2X2 and P2X3 receptors
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DOI：
10.1016/j.neuropharm.2008.06.044
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发表时间：
2008-10-01
影响因子：
4.7
通讯作者：
Davies, Daryl L.
中科院分区：
文献类型：
--
作者：
Asatryan, Liana;Popova, Maya;Davies, Daryl L.
本研究利用嵌合策略研究了乙醇在atp门控P2X受体（P2XRs）中的作用位点，利用了P2X2R（抑制）和P2X3R（增强）之间的乙醇反应差异。采用双电极电压钳（-70 mV）检测了乙醇（10-200 mM）对野生型P2X2、P2X3和嵌合型P2X2/P2X3Rs表达的ATP-和α、β -亚甲基ATP （α、β - meatp）诱导电流的影响。交换P2X2和P2X3Rs的外结构域可以逆转野生型对乙醇的反应。在含有P2X3R外域的嵌合体中，在TM界面上替换P2X2R外域的后部部分可以恢复野生型P2X2R样乙醇响应。用同源P2X2R残基取代P2X3Rs TM界面上的非保守外结构域残基的点突变鉴定出了逆转乙醇响应方向（304）或改变量级（53、55和313）的位置。P2X2Rs的同源取代没有显著改变野生型p2x2r样乙醇反应。这些发现表明，TM界面上的外结构域区段在决定P2X2和P2X3Rs对乙醇的定性和定量反应中起关键作用。用相应的P2X2R TM取代P2X3R TM区域的研究表明，决定乙醇反应大小的是TM1区域，而不是TM2区域。对ATP和α、β -肉ATP的研究支持了先前的证据，即TM区域在激动剂脱敏中起重要作用，并表明外畴和TM区域都在决定激动剂的效力和选择性中起作用。总的来说，这些发现首次确定了乙醇在P2X2和P2X3Rs中的潜在靶点，并为乙醇在其他P2XRs中的作用位点提供了见解。(C) 2008 Elsevier Ltd版权所有。
The present work investigated sites of ethanol action in ATP-gated P2X receptors (P2XRs) using chimeric strategies that exploited the differences in ethanol response between P2X2R (inhibition) and P2X3R (potentiation). We tested ethanol (10-200 mM) effects on ATP- and alpha,beta-methylene-ATP (alpha,beta-meATP)-induced currents in wildtype P2X2, P2X3 and chimeric P2X2/P2X3Rs expressed in Xenopus oocytes using two-electrode voltage-clamp (-70 mV). Exchanging ectodomain regions of P2X2 and P2X3Rs reversed wildtype ethanol responses. Substituting back portions of the P2X2R ectodomain at TM interfaces in chimeras that contained the P2X3R ectodomain restored wildtype P2X2R-like ethanol response. Point mutations that replaced non-conserved ectodomain residues at TM interfaces of P2X3Rs with homologous P2X2R residues identified positions that reversed the direction (304) or changed the magnitude (53, 55 and 313) of ethanol response. Homologous substitutions in P2X2Rs did not significantly alter wildtype P2X2R-like ethanol responses. These findings suggest that ectodomain segments at TM interfaces play key roles in determining qualitative and quantitative responses to ethanol of P2X2 and P2X3Rs. Studies that substituted TM regions of P2X3R with respective P2X2R TMs indicate that the TM1, but not the TM2, region plays a role in determining the magnitude of ethanol response. Studies with ATP and alpha,beta-meATP support prior indications that TM regions are important in agonist desensitization and suggest that both ectodomain and TM regions play roles in determining agonist potency and selectivity. Overall, these findings are the first to identify potential targets for ethanol in P2X2 and P2X3Rs and should provide insight into the sites of ethanol action in other P2XRs. (C) 2008 Elsevier Ltd. All rights reserved.