title: Salt bridge switching from Arg290/Glu167 to Arg290/ATP promotes the closed-to-open transition of the P2X2 receptor.
abstract: P2X receptors are trimeric adenosine-5'-triphosphate (ATP)-gated cation channels involved in fast signal transduction in many cell types. In this study, we used homology modeling of the rat P2X2 receptor with the zebrafish P2X4 X-ray template to determine that the side chains of the Glu167 and Arg290 residues are in close spatial vicinity within the ATP-binding pocket when the rat P2X2 channel is closed. Through charge reversal mutation analysis and mutant cycle analysis, we obtained evidence that Glu167 and Arg290 form an electrostatic interaction. In addition, disulfide trapping indicated the close proximity of Glu167 and Arg290 when the channel is in the closed state, but not in the ATP-bound open state. Consistent with a gating-induced movement that disrupts the Glu167/Arg290 salt bridge, a comparison of the closed and open rat P2X2 receptor models revealed a significant rearrangement of the protein backbone and the side chains of the Glu167 and Arg290 residues during the closed-to-open transition. The associated release of the Glu167/Arg290 salt bridge during channel opening allows a strong ionic interaction between Arg290 and a γ-phosphate oxygen of ATP. We conclude from these results that the state-dependent salt bridge switching from Arg290/Glu167 to Arg290/ATP fulfills a dual role: to destabilize the closed state of the receptor and to promote the ionic coordination of ATP in the ATP-binding pocket.
authors.affiliation: Department of Molecular Pharmacology, RWTH Aachen University, Wendlingweg 2, D-52074 Aachen, Germany. rhausmann@ukaachen.de
authors.forename: Ralf
authors.initials: R
authors.lastname: Hausmann
authors.forename: Janka
authors.initials: J
authors.lastname: Günther
authors.forename: Achim
authors.initials: A
authors.lastname: Kless
authors.forename: Daniel
authors.initials: D
authors.lastname: Kuhlmann
authors.forename: Matthias U
authors.initials: MU
authors.lastname: Kassack
authors.forename: Gregor
authors.initials: G
authors.lastname: Bahrenberg
authors.forename: Fritz
authors.initials: F
authors.lastname: Markwardt
authors.forename: Günther
authors.initials: G
authors.lastname: Schmalzing
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000494:pharmacology; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D001665:Binding Sites; D005260:Female; D018698:Glutamic Acid / Q000737:chemistry*; D015640:Ion Channel Gating; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D058911:Purinergic P2X Receptor Agonists / Q000494:pharmacology; D058920:Purinergic P2X Receptor Antagonists / Q000494:pharmacology; D051381:Rats; D058476:Receptors, Purinergic P2X2 / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D058848:Receptors, Purinergic P2X4 / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D055672:Static Electricity; D014982:Xenopus laevis
chemical_list: D058911:Purinergic P2X Receptor Agonists; D058920:Purinergic P2X Receptor Antagonists; D058476:Receptors, Purinergic P2X2; D058848:Receptors, Purinergic P2X4; D011994:Recombinant Proteins; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D001120:Arginine