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Kinetics of conformational changes revealed by voltage-clamp fluorometry give insight to desensitization at ATP-gated human P2X1 receptors.

journal contribution
posted on 18.02.2015, 11:36 by Alistair G. Fryatt, Richard J. Evans
ATP acts as an extracellular signaling molecule at cell-surface P2X receptors, mediating a variety of important physiologic and pathophysiologic roles. Homomeric P2X1 receptors open on binding ATP and then transition to an ATP-bound closed, desensitized state that requires an agonist-free washout period to recover. Voltage-clamp fluorometry was used to record ion channel activity and conformational changes simultaneously at defined positions in the extracellular loop of the human P2X1 receptor during not only agonist binding and desensitization but also during recovery. ATP evoked distinct conformational changes adjacent to the agonist binding pocket in response to channel activation and desensitization. The speed of recovery of the conformational change on agonist washout was state-dependent, with a faster time constant from the open (5 seconds) compared with the desensitized (75 seconds) form of the channel. The ability of ATP to evoke channel activity on washout after desensitization was not dependent on the degree of conformational rearrangement in the extracellular loop, and desensitization was faster from the partially recovered state. An intracellular mutation in the carboxyl terminus that slowed recovery of P2X1 receptor currents (7-fold less recovery at 30 seconds) had no effect on the time course of the extracellular conformational rearrangements. This study highlights that the intracellular portion of the receptor can regulate recovery and shows for the first time that this is by a mechanism independent of changes in the extracellular domain, suggesting the existence of a distinct desensitization gate in this novel class of ligand gated ion channels.

Funding

This work was supported by the British Heart Foundation [Grant PG/11/64/28772].

History

Citation

Mol Pharmacol, 2014, 86 (6), pp. 707-715

Author affiliation

/Organisation/COLLEGE OF MEDICINE, BIOLOGICAL SCIENCES AND PSYCHOLOGY/School of Biological Sciences/Department of Cell Physiology and Pharmacology

Version

AM (Accepted Manuscript)

Published in

Mol Pharmacol

Publisher

American Society for Pharmacology and Experimental Therapeutics (ASPET)

issn

0026-895X

eissn

1521-0111

Copyright date

2014

Publisher version

http://molpharm.aspetjournals.org/content/86/6/707

Notes

This article has supplemental material available at molpharm.aspetjournals.org.

Language

en

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