Validation of Alexa-647-ATP as a powerful tool to study P2X receptor ligand binding and desensitization.
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.
['Bhargava|Yogesh|Y|', 'Nicke|Annette|A|', 'Rettinger|Jürgen|J|']
D000255:Adenosine Triphosphate D000818:Animals D002232:Carbocyanines D004791:Enzyme Inhibitors D005456:Fluorescent Dyes D009865:Oocytes D018408:Patch-Clamp Techniques D011485:Protein Binding D018075:RNA, Complementary D051381:Rats D058474:Receptors, Purinergic P2X1 D014982:Xenopus laevis
Alexa-647-ATP Binding Desensitization Ion channel P2X1 receptor Allosteric interaction MgORI ORI PAO VCF magnesium oocyte Ringer’s solution oocyte ringer’s solution phenyl arsine oxide voltage clamp fluorometry