Molecular Dynamics Guided Analysis of Ligand Activation, Ion Permeation and Subtype-specific Ligand Interactions in P2X Receptors

• Applicant: Dr. Ralf Hausmann
• Subject Area: Pharmacology
• Term: from 2013 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 236175933

• Final Report Year: 2023

Final Report Abstract

The P2X3 receptor (P2X3R), an ATP-gated non-selective cation channel of the P2X receptor family, is involved in nociception and is therefore a promising target for new analgesics. Although new high-resolution structures of the human P2X3R (hP2X3R) in closed, open, and desensitized states were available at the start of the project, details of subtypeselective ligand binding, which are the basis for further development or even rational drug design, were poorly defined. Also, the biophysical mode of operation, or in detail the dynamic transitions between the different states as well as the determinants of cation selectivity were unclear. This knowledge, in addition to basic scientific interests in the functioning of these ion channels, is also valuable for rational drug design. Therefore, the main objectives of the present project were to identify and understand the molecular mechanisms of ligand activation, ion permeation, and subtype-specific ligand interactions of P2X receptors, especially the hP2X3R. Using all-atom molecular dynamics simulations and molecular docking as well as electrophysiological experiments, the conformational dynamics of the cytoplasmic cap during desensitization of the P2X3R was investigated very comprehensively and we were able to uncover an allosteric coupling mechanism between the cytoplasmic cap and the Ga-ting of the transmembrane ion pore in the P2X3R. The results show for the first time that only the distal C-terminus of the cytoplasmic cap unfolds during the transition from the ATP-bound open state to the desensitized state, whereas the N-terminus and proximal C-terminus maintain their folding. Furthermore, we demonstrated that the cation selectivity of hP2X3R (in contrast to a recent publication on P2X2R) is not only determined by residues in the N-terminus, but also significantly by transmembrane domain 2 (TM2) and residues in the external ion access portal. Our data complement the structural data from hP2X3 and provide, for the first time, a structural dynamic understanding of the functioning or dynamic transitions between conformations during desensitization of the channel. Furthermore, the molecular determinants of the interaction between individual negative allosteric as well as orthosteric competitive antagonists of the hP2X3R could be identified. Moreover, specific and rationally designed enhancement of the potency of both groups of antagonists could even be achieved by hP2X3R mutagenesis. These results impressively demonstrate that knowledge of the molecular determinants of ligand binding, together with a better structural-dynamical understanding of the mode of action, may allow rational enhancement of potency and possibly rational ligand design in the future.

Publications

• An ionic-lock mechanism stabilizes the closed-state conformation of the P2X2 receptor. Poster presentation at the 29th Ion Channel Meeting of the Association Canaux Ioniques of France in 2018, Sète, France
  Kuhlmann, D.; Kless, A.; Schmalzing, G. & Hausmann, R.
  
• The ASIC3/P2X3 cognate receptor is a pain-relevant and ligand-gated cationic channel. Nature Communications, 9(1).
  Stephan, Gabriele; Huang, Lumei; Tang, Yong; Vilotti, Sandra; Fabbretti, Elsa; Yu, Ye; Nörenberg, Wolfgang; Franke, Heike; Gölöncsér, Flóra; Sperlágh, Beáta; Dopychai, Anke; Hausmann, Ralf; Schmalzing, Günther; Rubini, Patrizia & Illes, Peter
  
• Bile acids are potent inhibitors of rat P2X2 receptors. Purinergic Signalling, 15(2), 213-221.
  Schmidt, Axel; Joussen, Sylvia; Hausmann, Ralf; Gründer, Stefan & Wiemuth, Dominik
  
• Identification of aurintricarboxylic acid as a potent allosteric antagonist of P2X1 and P2X3 receptors. Neuropharmacology, 158(2019, 11), 107749.
  Obrecht, Astrid S.; Urban, Nicole; Schaefer, Michael; Röse, Anni; Kless, Achim; Meents, Jannis E.; Lampert, Angelika; Abdelrahman, Aliaa; Müller, Christa E.; Schmalzing, Günther & Hausmann, Ralf
  
• Structural insights into ligand binding and dynamics of P2X receptors. Annals Royal Academy of Pharmacy of Spain, special issue of abstracts from 1st European Purine Meeting September 4-6, 2019; Symposium 11 (organized by Ralf Hausmann) at the 1st European Purine Meeting in Santiago di Compostela, Spain in 2019
  Hausmann, R. & Dal Ben, D.
  
• The molecular dynamics of ion conduction in P2X receptors. Annals Royal Academy of Pharmacy of Spain, special issue of abstracts from 1st European Purine Meeting September 4-6, 2019; Oral and poster presentation at the 1st European Purine Meeting in Santiago di Compostela, Spain in 2019
  Cönen, S.; Hausmann, R. & Machtens, J.P.
  
• Extracellular binding sites of positive and negative allosteric P2X4 receptor modulators. Life Sciences, 311(2022, 12), 121143.
  Weinhausen, Stephanie; Nagel, Jessica; Namasivayam, Vigneshwaran; Spanier, Claudia; Abdelrahman, Aliaa; Hanck, Theodor; Hausmann, Ralf & Müller, Christa E.
  
• Conformational dynamics of the cytoplasmic cap during desensitization of the hP2X3R. Biophysical Journal, 122(3), 247a-248a.
  Cheng, Linhan; Cönen, Saskia; Hausmann, Ralf & Machtens, J.-P.
  
• Diclofenac and other non-steroidal anti-inflammatory drugs (NSAIDs) are competitive antagonists of the human P2X3 receptor. Frontiers in Pharmacology, 14(2023, 3, 16).
  Grohs, Laura; Cheng, Linhan; Cönen, Saskia; Haddad, Bassam G.; Bülow, Astrid; Toklucu, Idil; Ernst, Lisa; Körner, Jannis; Schmalzing, Günther; Lampert, Angelika; Machtens, Jan-Philipp & Hausmann, Ralf