The P2X receptor binding domain: structure, conformational changes and coupling to the ion channel

P2X受体（P2XRs）是一种非选择性阳离子通道，在与细胞外ATP结合时被激活。它们具有不寻常的三聚体结构，这是迄今为止已知的离子通道家族中独一无二的。P2XRs与其他已知的离子通道或atp结合蛋白没有序列同源性。因此，关于它们的结构和ATP结合位点的位置以及导致通道打开的结构重排的信息仍然非常有限，主要来自诱变研究。在本研究中，我们建议使用详细的单通道分析和光亲和标记来确定打开通道所需的最小ATP分子数量，研究它们之间的结合协同性，并确定参与激动剂结合的蛋白质结构域。进一步，我们计划识别和分析导致通道打开和随后脱敏的结构重排。因此，我们将扩大突变受体的电生理分析，同时使用荧光技术，如时间分辨电压钳荧光法和荧光共振测量后，定点荧光标记或荧光配体的结合。

P2X receptors (P2XRs) are non-selective cation channels which are activated upon binding of extracellular ATP. They have an unusual trimeric structure which is unique among the ion channel families known so far. P2XRs show no sequence homology to any other known ion channel or ATP-binding protein. Thus, the information about their structure and location of the ATP binding site(s) as well as the structural rearrangements that lead to channel opening are still very limited and mainly derived from mutagenesis studies. In this study, we propose to use detailed single channel analysis and photoaffinity labeling to determine the minimum number of ATP molecules required to open the channel, investigate the cooperativity between their binding and identify protein domains which are involved in agonist binding. Further we plan to identify and analyze structural rearrangements leading to channel opening and subsequent desensitization. Therefore, we will expand the electrophysiological analysis of mutated receptors by the simultaneous use of fluorescence techniques such as time-resolved voltage-clamp fluorometry and fluorescence-resonance measurements after site directed fluorescence labeling or binding of fluorescent ligands.