Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function

PLoS One. 2016 Mar 17;11(3):e0151862. doi: 10.1371/journal.pone.0151862. eCollection 2016.

Abstract

The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Calcium / metabolism
  • Calcium / physiology
  • Fluorescence Resonance Energy Transfer
  • HEK293 Cells
  • Humans
  • Immunoprecipitation
  • Patch-Clamp Techniques
  • Polymorphism, Single Nucleotide / genetics*
  • Polymorphism, Single Nucleotide / physiology
  • RNA, Small Interfering / metabolism
  • Real-Time Polymerase Chain Reaction
  • Receptors, Purinergic P2X7 / genetics
  • Receptors, Purinergic P2X7 / metabolism
  • Receptors, Purinergic P2X7 / physiology*
  • Signal Transduction / physiology

Substances

  • P2RX7 protein, human
  • RNA, Small Interfering
  • Receptors, Purinergic P2X7
  • Calcium

Grants and funding

This work was partially supported by grants from Affectis Pharmaceuticals AG (http://www.affectis.com/), the University of Buenos Aires (UBA) (http://www.uba.ar/), the CONICET (http://www.conicet.gov.ar/), FOCEMMercosur (COF 03/11) (http://www.mercosur.int/innovaportal/v/385/2/innova.front/fondo_para_la_convergencia_estructural_del_mercosur_focem) and Agencia Nacional de Promoción Científica y Tecnológica, Argentina (http://www.agencia.mincyt.gob.ar/) (E.A); by the German Federal Ministry of Education and Research (https://www.bmbf.de/en), within the framework of the e:Med research and funding concept (IntegraMent: Integrated Understanding of Causes and Mechanisms in Mental Disorders; FKZ 01ZX1314H), by the program for medical genome research within the framework of the NGFN-Plus (FKZ:01GS08151) (http://www.ngfn.de/en) (J.D.) and by the Volkswagen Stiftung (D.R.) (https://www.volkswagenstiftung.de). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.