Regulation of the ion channel activities of the P2X7 receptor via its N and C terminal endodomains

P2X受体构成了一个配体门控离子通道家族，它们响应天然配体ATP，通过快速打开跨膜离子通道渗透到Na+， K+和Ca2+。在P2X家族中，P2X7受体（P2X7R）因其在炎性体激活、肿瘤生长和诱导细胞溶解大孔中的作用而引起了人们的特别关注。与长期持有的观点相反，大型有机阳离子（如NMDA+和TRIS+）的跨膜渗透不是由选择性过滤器的逐步扩张引起的，而是反映了开放P2X7R离子通道的稳定特性。在过去的报道中，我们已经通过半胱氨酸扫描诱变结合单通道记录和半胱氨酸反应性探针测试证明了这一点。在新项目中，我们将重点研究P2X7R的两个内结构域与跨膜离子通道的细胞质部分（即所谓的细胞质前庭）的相互作用在离子通道门控和细胞溶解大孔形成中的作用。为此，我们将在基于人类P2X3受体当前x射线结构的同源性模型的指导下进行蛋白质工程实验，其中包括（首次用于P2X受体）N端和c端内结构域的重要部分。对于C末端内结构域的远端部分，这是P2X受体家族中P2X7R所特有的，目前没有结构信息，我们将使用荧光（Förster）共振能量转移（FRET）方法结合蛋白质工程来探测N-和C端结构域与细胞质前庭之间的相互作用。我们期望从这个项目中对这种配体门控离子通道在分子水平上的不寻常性质有重要的新见解。

P2X receptors constitute a family of ligand-gated ion channels, which respond to their natural ligand ATP with the rapid opening of a transmembrane ion channel permeant to Na+, K+ and Ca2+. Within the P2X family, the P2X7 receptor (P2X7R) has attracted particular interest because of its role in inflammasome activation, tumor growth and induction of a cytolytic macropore. In contrast to a long hold view, the transmembrane permeation of large organic cations such as NMDA+ and TRIS+ does not result from a progressive dilatation of the selectivity filter, but reflects a stable peculiarity of the open P2X7R ion channel. This we have shown in the past reporting period by cysteine scanning mutagenesis combined with single channel recordings and testing with cysteine-reactive probes. In the new project, we will focus on the role of the interaction of the two endodomains of the P2X7R with the cytoplasmic part of the transmembrane ion channel, the so-called cytoplasmic vestibule, in ion channel gating and the formation of the cytolytic macropore. To this end, we will perform protein engineering experiments that are guided on a homology model based on the current X-ray structure of the human P2X3 receptor, which includes (for the first time for a P2X receptor) significant parts of the N- and C-terminal endodomains. For the more distal part of the C terminal endodomain, which is unique for the P2X7R within the P2X receptor family and for which currently no structure information is available, we will use fluorescence (Förster) resonance energy transfer (FRET) approaches combined with protein engineering to probe the interaction between N- and C-terminal domains and the cytoplasmic vestibule. We expect from this project significant new insights in the unusual properties of this ligand-gated ion channel at the molecular level.