title: Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes.
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.
authors.affiliation: Department of Cell Physiology & Pharmacology, University of Leicester, Leicester, UK.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Roberts
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Bottrill
authors.forename: Sharad
authors.initials: S
authors.lastname: Mistry
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Evans
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005071:Evoked Potentials / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D020836:Protein Structure, Quaternary; D058474:Receptors, Purinergic P2X1 / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D053719:Tandem Mass Spectrometry; D014982:Xenopus laevis
chemical_list: D058474:Receptors, Purinergic P2X1; D000255:Adenosine Triphosphate