P2X-Rezeptoren - von der molekularen Struktur zur physiologischen Funktion
Final Report Abstract
In the course of the Heisenberg-Stipendium, I have continued my long-standing research on the molecular structure function analysis of P2X receptors and, beyond that, significantly extended my expertise by the generation of BAC transgenic mouse models that allow the investigation of the localisation, protein-protein interactions, and physiological functions of the P2X7 receptor. Starting from the original determination of the trimeric assembly and the localization of the ATP- binding site at the subunit interfaces of homo-and hetero-trimeric P2X receptors, we next aimed to identify the exact binding mode and number of ATP molecules necessary to open the channel as well as the associated conformational changes. We validated Alexa-ATP as a potent P2X1 agonist and used it in a fluorometric approach to demonstrate the cooperativity between P2X subunits, and to determine a reaction scheme in which three ATP molecules have to bind to desensitize the channel. After publication of a first P2X crystal structure by the group of E. Gouaux in 2009, the focus of this project was shifted towards the investigation of conformational changes using voltage clamp fluorometry (vcf). We successfully installed and optimized a new vcf set-up and, in combination with molecular modelling studies, determined the exact localization and orientation of ATP in its binding site and dissected molecular movements associated with the opening and desensitization of the receptor. The generation of BAC transgenic P2X4, P2X6, and P2X7 reporter mouse models for conditional expression of the respective tagged P2X subunits was held up by inefficient recombination of the rather complex BAC constructs. Therefore a simpler conventional strategy was finally chosen and the project was focused on the generation of BAC transgenic mice that overexpress EGFP-tagged P2X7 polymorphic variants. Mouse lines with different overexpression levels were obtained and their identical expression patterns confirmed. Initial imunohistochemical and biochemical analysis shows that the EGFP-tagged P2X7 receptors are properly folded and assembled and targeted to specific localizations in the plasma membrane. They can be directly visualized by fluorescence and 2-photon microscopy and were efficiently purified via the introduced EGFP and hexahistidyl-tags. An obvious motor phenotype is observed in the mouse line with the highest P2X7 overexpression level. Thus, our mice provide valuable models to clarify the major unresolved questions in the field, such as the specific localization of the P2X7 receptor as well as its protein interactions and involvement in pathophysiological processes. A first publication of these mice is in preparation. In the course of this project, we also clarified the homomeric assembly of the P2X7 receptor in native tissues, identified and characterized a functional P2X7 splice variant that escapes the gene deletion strategy in a published P2X7 KO mouse model and contributed to the identification of novel P2X7 ligands. Furthermore, several side projects in the field of structure activity-analysis of α-conotoxins at nAChRs were successfully completed. Despite the initial delay in the two main projects and the fact that we had to face a variety of novel methodological challenges, the most important aims were successfully achieved. Both projects are currently continued.
Publications
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Homotrimeric complexes are the dominant assembly state of P2X7 subunits in native tissues. Biochem. Biophys. Res. Commun. 2008, 377:803-8
Nicke, A
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A functional P2X7 splice variant with an alternative transmembrane domain 1 escapes gene inactivation in P2X7 KO mice. J. Biol. Chem. 2009; 284: 25813-22
Nicke A, Kuan Y-H, Masin M, Rettinger J, Marquez-Klaka B, Bender O, Gorecki D, Murrell-Lagnado R, Soto F
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Inter-subunit disulfide cross-linking in homomeric and heteromeric P2X receptors. Eur. Biophys. J. 2009; 38: 329-38
Marquez-Klaka B, Rettinger J, Nicke A
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Alternative Splicing of the N-Terminal Cytosolic and Transmembrane Domains of P2X7 Controls Gating of the Ion Channel by ADP-Ribosylation. PLoS One 2012; 7:e41269
Schwarz N, Drouot L, Nicke A, Fliegert R, Boyer O, Guse AH, Haag F, Adriouch S, Koch-Nolte
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Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor. Proc Natl Acad Sci USA 2012; 109: 11396-401
Lörinczi E, Bhargava Y, Marino SF, Taly A, Kaczmarek-Hájek K, Barrantes-Freer A, Dutertre S, Grutter T, Rettinger J, Nicke A
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Molecular and functional properties of P2X receptors – recent progress and persisting challenges. Purinergic Signal. 2012; 8: 375-417
Kaczmarek-Hájek K, Lörinczi E, Hausmann R, Nicke A
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Heteromeric assembly of P2X subunits. Front. Cell. Neurosci. 2013; 7: 250-250
Saul A, Hausmann R, Kless A, Nicke A
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The phenothiazine-class antipsychotic drugs prochlorperazine and trifluoperazine are potent allosteric modulators of the human P2X7 receptor. 2013; Neuropharmacology 75C: 365-379
Hempel, C, Nörenberg W, Sobottka H, Urban N, Nicke A, Fischer W, Schäfer M
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Validation of Alexa-647-ATP as a powerful tool to study P2X receptor ligand binding and desensitization. Biochem Biophys Res Commun. 2013; 438: 295-300
Bhargava Y, Nicke A, Rettinger J
