Publikationsserver der RWTH Aachen University
Identifizierung regulatorischer Proteindomänen der zytolytischen Eigenschaften des humanen P2X7-Rezeptors
Abstract
dc:descriptionThe P2X7-receptor is a ligand-gated ion channel of the P2X-purigenic-receptor group. In different of the other receptors of the P2X-family the P2X7-receptor is able to build both an ion channel and a cytolytic-pore which leads to membrane-blebbing. The aim of this MD-thesis was to develop a method which provides evidence whether the activation of selected P2X7-receptor-mutants with agonists like ATP or the more potent BzATP mediates a cytolytic membrane pore. As cell-system we choose COS-7-cells because of the lack of an endogenous P2X7-Receptor. As the cytolytic pore is permeable for cations up to a molecular mass of 900 Da, its possible to detect the cytolytic pore through fluorescence-microscopy of diffused 320 Da Ethidium-bromid cations which intercalates with the cellular DNA. DIC-microscopy accorded as an easier way to detect membrane-blebbing with the same explanatory power. After addition of the agonists a Zeiss-cell-observer system acquires over a period of 30 minutes automatically a series of pictures. After 30 minutes of activation there were no further changes of the membrane-integrity detectable.The co-transfection of an EGFP-cDNA accorded to an advantageous way to differ between transfected from un-transfected cells. Furthermore with the co-transfection of the EGFP-cDNA it was possible to localize changings of plasmamembrane at an early state. To valdidate the developed method, I investigated COS7-cells expressing the wildtype-P2X7-cDNA and 264.7-RAW-macrophages which expresses an endogenous P2X7-receptor. Furthermore I investigated the previous published E496A-Polymorphismem. According to the literature the activation of transfected COS7-cells couldnt mediate membrane-blebbing. The expression of the investigated P2X7-receptor-mutants was verified by western blot. After developing a suitable assay to show the cytolytic pore-formation, some hP2X7-receptor mutants were cloned and investigated. An up to now unpublished glycin-arginin-polymorphism at position 441 leads to a extended activation period with BzATP. Remarkable is the great number of cystein-residues in the cytosolic localized c-terminal endodomain. The exchange of the 362 cystein-residue also leads to a extended activation period and the membrane-blebs are formed less than the wildtype. Its possible that the high amount of cystein-residues is responsible for the species-dependent difference of the cytolytic pore-formation kinetic. The truncation of the last 12 amino acids resulted to a receptor that looses pore forming abilities. If the tyrosine motif 400YSVF403 can also affect pore forming abilities is not investigated finally, because of weak expression of the 400YSVF403-P2X7-receptor in the COS7-cell line. Finally these experiments show that a lot of amino acids take influence of the hP2X7-receptor pore forming abilities.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2007
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Schmitz, Stephan
- Contributors dc:contributor
-
- Schmalzing, Günther
Subjects
dc:subject × 8Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- ger