Durner, Anna (2023): Improved voltage clamp fluorometry to investigate the molecular function of the P2X7 receptor C-terminus and characterization of novel P2X7 receptor ligands. Dissertation, LMU München: Faculty of Medicine |
Preview |
PDF
Durner_Anna.pdf 118MB |
Abstract
The ATP-gated, purinergic P2X7 receptor (P2X7R) is a trimeric, non-selective cation channel with key functions in inflammation and immunity. It mediates a multitude of cellular events that are normally not associated with ion channel functions (e.g. modification of the plasma membrane, cytokine release, and cell death). Compared to the other homologous P2X receptor subunits (P2X1-P2X6), the P2X7 subunit contains an unusually long intracellular C-terminus, which is thought to be essential for most of these cellular responses [4.1]. However, the underlying molecular mechanisms and signaling pathways are incompletely understood. The role of the P2X7R in immune signaling and especially its involvement in diverse pathophysiological processes such as inflammation, epilepsy, Alzheimer’s disease, and cancer makes it an interesting drug target. To validate the P2X7R as a drug target, detailed understanding of its molecular and physiological functions, cell-type specific expression, and selective ligands are required. A major aim of this work was the investigation of the molecular consequences of P2X7R activation and the identification of potent antagonists. In addition, I investigated the localization of the P2X7R and thereby contributed to the characterization of a novel P2X7 mouse model. To investigate conformational changes in the molecular structure of the P2X7R that are associated with its activation, I applied voltage clamp fluorometry (VCF) ([4.2]). To this end, I refined a method for the incorporation of the environment-sensitive fluorescent unnatural amino acid (fUAA) ANAP into Xenopus laevis oocyte-expressed receptors. Furthermore, I constructed a VCF setup optimized for the detection of ANAP-specific fluorescence changes. VCF measurements from ANAP-containing P2X7R-expressing oocytes provided evidence that the ATP-induced conformational changes in extracellular and transmembrane domains are not translated to the intra-cellular C-terminus and that the P2X7-characteristic current facilitation is an intrinsic receptor property and likely associated with change in its gating ([3.1]). To determine and compare the potency of novel small-molecule P2X7R antagonists, I performed two-electrode voltage clamp (TEVC) analysis on P2X7R-expressing X. laevis oocytes ([3.2], [4.3]). These data revealed a compound with nanomolar potency, for which I further analyzed association and dissociation kinetics and confirmed its binding to the P2X7R allosteric binding pocket by site-directed mutagenesis. Finally, I contributed to the validation of a novel BAC transgenic reporter mouse model and the determination of the controversially discussed P2X7 expression pattern ([4.4] and [3.3]) by performing immunohistochemical tissue stainings to compare endogenous and transgenic P2X7R expression patterns. Together, these studies advanced our knowledge about molecular principles underlying P2X7 signal transduction, identified a new potent drug lead, and helped to provide a novel mouse model to investigate P2X7R localization and physiological functions. In an unrelated publication ([3.4]), I helped to characterize nicotinic acetylcholine receptor ligands by TEVC.
Item Type: | Theses (Dissertation, LMU Munich) |
---|---|
Subjects: | 600 Technology, Medicine 600 Technology, Medicine > 610 Medical sciences and medicine |
Faculties: | Faculty of Medicine |
Language: | English |
Date of oral examination: | 30. August 2023 |
1. Referee: | Nicke, Annette |
MD5 Checksum of the PDF-file: | 770ff4ea4b9b3e29b5cd563ea0067768 |
Signature of the printed copy: | 0700/UMD 21388 |
ID Code: | 32536 |
Deposited On: | 06. Nov 2023 14:26 |
Last Modified: | 06. Nov 2023 15:14 |