Sites and Mechanisms of Ethanol Action in P2X receptors

描述（由申请人提供）：P2 X受体（P2 XR）构成最近克隆的配体门控离子通道超家族。P2 XR是通过突触释放的细胞外腺苷5 '-三磷酸（ATP）门控的快速作用的阳离子渗透性离子通道，并且广泛分布于CNS中。本实验室和其他实验室的研究表明：1）P2 XRs介导乙醇影响的行为; 2）P2 XRs调节被认为是介导乙醇诱导的行为的靶点的受体系统的活性; 3）乙醇抑制海马和延髓核神经元的ATP激活电流; 4）乙醇抑制在非洲爪蟾卵母细胞和HEK 293细胞中表达的野生型P2 XR中的ATP活化，以及5）P2 XR对乙醇的敏感性是亚基依赖性的。这些发现与乙醇诱导的P2 XR功能变化可能直接或间接在引起或调节某些乙醇行为效应中发挥作用的观点一致。证据必须等待选择性P2 XR拮抗剂的发展和足够的知识，在P2 XR的行动和分子靶乙醇。当前的提案通过开始回答三个关键问题来解决后者：1）哪些重组P2 XR亚型对乙醇敏感？2)哪些天然P2 XR亚型对乙醇敏感？这将需要比较非洲爪蟾卵母细胞和HEK 293细胞中表达的乙醇敏感性重组受体与海马和延髓核神经元中的天然P2 XR的药理学和电生理学特征，以及3）P2 XR中乙醇作用的分子位点/机制是什么？拟议的工作可能会贡献基础信息，这将导致乙醇在P2 XR中作用位点的初始分子模型，并将为未来的研究奠定基础，这些研究将利用这些知识开发敲入和无效突变（“敲除”）小鼠，这些小鼠可用于测试特定P2 XR在介导乙醇的生理和行为效应中发挥的作用。

DESCRIPTION (provided by applicant): P2X receptors (P2XRs) constitute the most recently cloned superfamily of ligand-gated ion channels LGICs). P2XRs are fast acting, cation-permeable ion channels that are gated by synaptically released extracellular adenosine 5'-triphosphate (ATP) and are widely distributed in the CNS. Studies to date by our lab and others show that: 1) P2XRs mediate behaviors that are affected by ethanol; 2) P2XRs modulate activity in receptors systems believed to be targets mediating ethanol-induced behaviors; 3) Ethanol inhibits ATP-activated current in hippocampal and nucleus accumbens neurons; 4) Ethanol inhibits ATP-activation in wildtype P2XRs expressed in Xenopus oocytes and HEK 293 cells and 5) Sensitivity to ethanol of P2XRs is subunit dependent. These findings are consistent with the notion that ethanol-induced changes in P2XR function may directly or indirectly play a role in causing or modulating some ethanol behavioral effects. Definitive evidence must await development of selective P2XR antagonists and sufficient knowledge regarding the actions and molecular targets of ethanol in P2XRs. The current proposal addresses the latter by beginning to answer three key questions: 1) What recombinant P2XR subtypes are sensitive to ethanol? 2) What native P2XR subtypes are sensitive to ethanol? This will require comparing the pharmacological and electrophysiological characteristics of ethanol sensitive recombinant receptors expressed in Xenopus oocytes and HEK293 cells and native P2XRs in hippocampal and nucleus accumbens neurons and 3) What are the molecular sites/mechanisms of ethanol action in P2XRs? The proposed work likely will contribute foundation information that will lead to initial molecular models of sites of action for ethanol in P2XRs and will set the stage for future investigations that will use this knowledge to develop knock in and null mutant ("knock out") mice that can be used to test the roles that specific P2XRs play in mediating physiological and behavioral effects of ethanol.