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

点击翻译按钮获取中文摘要

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.