title: Validation of Alexa-647-ATP as a powerful tool to study P2X receptor ligand binding and desensitization.
abstract: Ion channel opening and desensitization is a fundamental process in neurotransmission. The ATP-gated P2X1 receptor (P2X1R) shows rapid and long-lasting desensitization upon agonist binding. This makes the electrophysiological investigation of its desensitization process, agonist unbinding, and recovery from desensitization a challenging task. Here, we show that the fluorescent agonist Alexa-647-ATP is a potent agonist at the P2X1R and a versatile tool to directly visualize agonist binding and unbinding. We demonstrate that the long-lasting desensitization of the P2X1R is due to both slow unbinding of agonist from the desensitized receptor and agonist mediated receptor internalization. Furthermore, the unbinding of the agonist Alexa-647-ATP from the desensitized receptor is accelerated in the continuous presence of competitive ligand. Modeling of our data indicates that three agonist molecules are required to drive the receptor into desensitization. Direct visualization of ligand unbinding from the desensitized receptor demonstrates the cooperativity of this process.
authors.affiliation: Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt am Main, Germany. yogesh.bhargava@gmail.com
authors.forename: Yogesh
authors.initials: Y
authors.lastname: Bhargava
authors.forename: Annette
authors.initials: A
authors.lastname: Nicke
authors.forename: Jürgen
authors.initials: J
authors.lastname: Rettinger
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002232:Carbocyanines / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology*; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D018075:RNA, Complementary / Q000378:metabolism; D051381:Rats; D058474:Receptors, Purinergic P2X1 / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: C569686:Alexa Fluor 647; D002232:Carbocyanines; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D018075:RNA, Complementary; D058474:Receptors, Purinergic P2X1; D000255:Adenosine Triphosphate
keywords: Alexa-647-ATP; Allosteric interaction; Binding; Desensitization; Ion channel; MgORI; ORI; P2X1 receptor; PAO; VCF; magnesium oocyte Ringer’s solution; oocyte ringer’s solution; phenyl arsine oxide; voltage clamp fluorometry