Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
J Neurochem. 2012 Dec;123(5):725-35. doi: 10.1111/jnc.12012. Epub 2012 Oct 11.

Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes.

Author information

  • 1Department of Cell Physiology & Pharmacology, University of Leicester, Leicester, UK.

Abstract

Recombinant FlagHis(6) tagged Human P2X1 receptors expressed in HEK293 cells were purified, digested with trypsin and analysed by mass spectroscopy (96% coverage following de-glycosylation and reduction). The receptor was basally phosphorylated at residues S387, S388 and T389 in the carboxyl terminus, a triple alanine mutant of these residues had a modest ~ 25% increase in current amplitude and recovery from desensitization. Chemical modification showed that intracellular lysine residues close to the transmembrane domains and the membrane stabilization motif are accessible to the aqueous environment. The membrane-impermeant cross-linking reagent 3,3'-Dithiobis (sulfosuccinimidylpropionate) (DTSSP) reduced agonist binding and P2X1 receptor currents by > 90%, and modified lysine residues were identified by mass spectroscopy. Mutation to remove reactive lysine residues around the ATP-binding pocket had no effect on inhibtion of agonist evoked currents following DTSSP. However, agonist evoked currents were ~ 10-fold higher than for wild type following DTSSP treatment for mutants K199R, K221R and K199R-K221R. These mutations remove reactive residues distant from the agonist binding pocket that are close enough to cross-link adjacent subunits. These results suggest that conformational change in the P2X1 receptor is required for co-ordination of ATP action.

© 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.

PMID:
22971236
[PubMed - indexed for MEDLINE]
PMCID:
PMC3532615
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Icon for Blackwell Publishing Icon for PubMed Central
    Loading ...
    Write to the Help Desk