The P2X receptor binding domain: structure, conformational changes and coupling to the ion channel
Zusammenfassung der Projektergebnisse
At the beginning of the project, structural information on the P2XR was very limited and mainly based on mutagenesis studies. Using a variety of biophysical approaches, we aimed to a) identify protein domains and amino acid residues in the P2XR that contribute to the Binding of ATP, b) determine the number of ATP-binding which have to be occupied to open or desensitize the receptor, c) analyze conformational changes that are associated with the activation and desensitization of the P2X1 receptor, and d) estimate distances between specific residues and the ATP binding site and relative movements during channel activation. After publication of a first P2X crystal structure in 2009, we moved the focus of the project on the investigation of basic principles of P2XR molecular function and dynamics (as addressed in b) and c)) that cannot be derived from a static crystal structure. To address the question of ATP binding and cooperativity during activation, desensitization, and recovery of the P2X receptor, we applied for the first time the photo-reactive agonist Bz-ATP and the fluorescent agonist Alexa-ATP for functional studies at the P2X receptor and developed and optimized novel methods to investigate and demonstrate the presence of allosteric interactions between P2X receptor subunits during both activation and desensitization. These studies allowed important insights in electrically silent processes that could not be accessed by electrophysiological methods alone and provided the basis for a simple reaction model for the P2X1 receptor. Using voltage clamp fluorometry, we further dissected molecular movements in P2X receptor activation and desensitization and, in combination with molecular modelling studies, confirmed the location and determined the orientation of ATP in its subunit interface binding site. In particular, we showed that the cys-rich head domain undergoes substantial movements during both receptor activation and desensitization and detected clear movements of the right flipper domain activation. Our study represents the first investigation of a P2X receptor by voltage clamp fluorometry and one of only very few studies investigating ligand-gated channels with this method. The project was interrupted several times due to several job changes.
Projektbezogene Publikationen (Auswahl)
- Agonist- and competitive antagonist-induced movement of loop 5 on the alpha subunit of the neuronal alpha4beta4 nicotinic acetylcholine receptor J Neurochem. 2008; 105: 413–24
Mourot A, Bamberg E, Rettinger J
(Siehe online unter https://doi.org/10.1111/j.1471-4159.2007.05151.x) - 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
(Siehe online unter https://doi.org/10.1074/jbc.M109.033134) - 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
(Siehe online unter https://doi.org/10.1007/s00249-008-0325-9) - Aminoglycoside block of P2X2 receptors heterologously expressed in Xenopus laevis oocytes. Purinergic Signal. 2010; 6: 393–403
Bongartz EV, Rettinger J, Hausmann R
(Siehe online unter https://doi.org/10.1007/s11302-010-9204-9) - Discovery of potent competitive antagonists and positive modulators of the P2X2 receptor. J Med Chem. 2011; 54: 817–30
Baqi Y, Hausmann R, Rosefort C, Rettinger J, Schmalzing G, Müller CE
(Siehe online unter https://doi.org/10.1007/s11302-010-9204-9) - Allosteric nature of P2X receptor activation probed by photoaffinity labelling. Br J Pharmacol. 2012; 167: 1301–10
Bhargava Y, Rettinger J, Mourot A
(Siehe online unter https://doi.org/10.1111/j.1476-5381.2012.02083.x) - Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor. Proc Natl Acad Sci U S A. 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
(Siehe online unter https://doi.org/10.1073/pnas.1118759109) - The phenothiazine-class antipsychotic drugs prochlorperazine and trifluoperazine are potent allosteric modulators of the human P2X7 receptor. Neuropharmacology. 2013; 75C: 365–379
Hempel C, Nörenberg W, Sobottka H, Urban N, Nicke A, Fischer W, Schaefer M
(Siehe online unter https://doi.org/10.1016/j.neuropharm.2013.07.027) - 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
(Siehe online unter https://doi.org/10.1016/j.bbrc.2013.07.058)
