Mechanistic insights from resolving ligand-dependent kinetics of conformational changes at ATP-gated P2X1R ion channels.
Structural studies of P2X receptors show a novel U shaped ATP orientation following binding. We used voltage clamp fluorometry (VCF) and molecular dynamics (MD) simulations to investigate agonist action. For VCF the P2X1 receptor (P2X1R) K190C mutant (adjacent to the agonist binding pocket) was labelled with the fluorophore MTS-TAMRA and changes in fluorescence on agonist treatment provided a real time measure of conformational changes. Studies with heteromeric channels incorporating a key lysine mutation (K68A) in the ATP binding site demonstrate that normally three molecules of ATP activate the receptor. The time-course of VCF responses to ATP, 2'-deoxy ATP, 3'-deoxy ATP, Ap5A and αβmeATP were agonist dependent. Comparing the properties of the deoxy forms of ATP demonstrated the importance of the 2' hydroxyl group on the ribose ring in determining agonist efficacy consistent with MD simulations showing that it forms a hydrogen bond with the γ-phosphate oxygen stabilizing the U-shaped conformation. Comparison of the recovery of fluorescence on agonist washout, with channel activation to a second agonist application for the partial agonists Ap5A and αβmeATP, showed a complex relationship between conformational change and desensitization. These results highlight that different agonists induce distinct conformational changes, kinetics and recovery from desensitization at P2X1Rs.
Department of Molecular and Cell Biology, University of Leicester, Leicester, LE1 7RH, United Kingdom.
Alistair G
AG
Fryatt
Department of Molecular and Cell Biology, University of Leicester, Leicester, LE1 7RH, United Kingdom.|Department of Chemistry, College of Science, University of Baghdad, Baghdad, Iraq.
Sudad
S
Dayl
Department of Chemistry, University of Leicester, Leicester, LE1 7RH, United Kingdom.
Paul M
PM
Cullis
Department of Molecular and Cell Biology, University of Leicester, Leicester, LE1 7RH, United Kingdom.|Leicester Institute of Structural and Chemical Biology, University of Leicester, Leicester, LE1 7RH, United Kingdom.
Ralf
R
Schmid
Department of Molecular and Cell Biology, University of Leicester, Leicester, LE1 7RH, United Kingdom.
Richard J
RJ
Evans
D000255:Adenosine Triphosphate / Q000819:agonists* / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D015226:Dinucleoside Phosphates / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009154:Mutation; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D011487:Protein Conformation; D058474:Receptors, Purinergic P2X1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis / Q000235:genetics*
D015226:Dinucleoside Phosphates; D008024:Ligands; D058474:Receptors, Purinergic P2X1; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; D000255:Adenosine Triphosphate