Mechanistic insights from resolving ligand-dependent kinetics of conformational changes at ATP-gated P2X1R ion channels.

Alistair G Fryatt,Paul M Cullis,Sudad Dayl,Richard J Evans,Ralf Schmid

doi:10.1038/srep32918

Alistair G Fryatt, Paul M Cullis + Show 3 more

Open Access

https://doi.org/10.1038/srep32918

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Journal: Scientific Reports	Publication Date: Sep 12, 2016
Citations: 14	License type: CC BY 4.0

Affiliation: University of Leicester, University of Baghdad

Abstract

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.

Highlights

Extracellular ATP levels can be raised in a variety of ways including release from neurons, through pannexin hemichannels, and due to cell damage[1,2]
How many ATP molecules normally open the P2X1 receptor (P2X1R)? The crystal structures of ATP bound P2X receptor (P2XR) show the key role of a conserved lysine (K68, P2X1R numbering) that interacts with all three phosphate groups
We showed that the time-course of fluorescence change associated with channel activation and desensitization was indistinguishable from the channels with three normal binding sites and this is consistent with studies on P2X2R concatenated channels[14]

Summary

Introduction

Extracellular ATP levels can be raised in a variety of ways including release from neurons, through pannexin hemichannels, and due to cell damage[1,2]. ATP adopts an unusual U shaped conformation that involves rearrangement of the P2XR dorsal fin, left flipper and cysteine rich head region closing the agonist pocket. This is coupled through the lower body to the transmembrane fluke and opening the channel gate. In this study we have combined molecular modelling and VCF to address (i) how many molecules of ATP are normally required to open P2X1Rs, (ii) the importance of intramolecular hydrogen bonds for ATP agonist action and (iii) conformational changes associated with partial agonist action. Our work provides new insight into fundamental agonist dependent P2XR molecular transitions

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