RT Journal Article
SR Electronic
T1 Kinetics of Conformational Changes Revealed by Voltage Clamp Fluorometry Give Insight to Desensitization at ATP-gated Human P2X1 Receptors
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP mol.114.095307
DO 10.1124/mol.114.095307
A1 Alistair G Fryatt
A1 Richard J Evans
YR 2014
UL http://molpharm.aspetjournals.org/content/early/2014/10/08/mol.114.095307.abstract
AB ATP acts as an extracellular signalling molecule at cell surface P2X receptors mediating a variety of important physiological and pathophysiological 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 (VCF) was used to simultaneously record ion channel activity and conformational changes at defined positions in the extracellular loop of the human P2X1 receptor during not only agonist binding and desensitization, but also 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 s) compared to the desensitized (75 s) form of the channel. The ability of ATP to evoke channel activity on washout following desensitization was not dependent on the degree of conformational re-arrangement 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 30s) had no effect on the time-course of the extracellular conformational rearrangements. The study highlights that the intracellular portion of the receptor dominates 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 channel.