The P2X7 carboxyl tail is a regulatory module of P2X7 receptor channel activity

J Biol Chem. 2008 Sep 12;283(37):25725-25734. doi: 10.1074/jbc.M803855200. Epub 2008 Jul 10.

Abstract

P2X(7) receptors are ATP-gated cation channels composed of three identical subunits, each having intracellular amino and carboxyl termini and two transmembrane segments connected by a large ectodomain. Within the P2X family, P2X(7) subunits are unique in possessing an extended carboxyl tail. We expressed the human P2X(7) subunit as two complementary fragments, a carboxyl tail-truncated receptor channel core (residues 1-436 or 1-505) and a tail extension (residues 434-595) in Xenopus laevis oocytes. P2X(7) channel core subunits efficiently assembled as homotrimers that appeared abundantly at the oocyte surface, yet produced only approximately 5% of the full-length P2X(7) receptor current. Co-assembly of channel core subunits with full-length P2X(7) subunits inhibited channel current, indicating that the lack of a single carboxyl tail domain is dominant-negative for P2X(7) receptor activity. Co-expression of the tail extension as a discrete protein increased ATP-gated current amplitudes of P2X(7) channel cores 10-20-fold, fully reconstituting the wild type electrophysiological phenotype of the P2X(7) receptor. Chemical cross-linking revealed that the discrete tail extension bound with unity stoichiometry to the carboxyl tail of the P2X(7) channel core. We conclude that a non-covalent association of crucial functional importance exists between the carboxyl tail of the channel core and the tail extension. Using a slightly shorter P2X(7) subunit core and subfragments of the tail extension, this association could be narrowed down to include residues 409-436 and 434-494 of the split receptor. Together, these results identify the tail extension as a regulatory gating module, potentially making P2X(7) channel gating sensitive to intracellular regulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / chemistry
  • Animals
  • Cell Membrane / metabolism
  • Cross-Linking Reagents / pharmacology
  • DNA, Complementary / metabolism
  • Dose-Response Relationship, Drug
  • Humans
  • Hydrogen-Ion Concentration
  • Kinetics
  • Models, Biological
  • Oocytes / metabolism*
  • Patch-Clamp Techniques
  • Protein Structure, Tertiary
  • Receptors, Purinergic P2 / chemistry*
  • Receptors, Purinergic P2X7
  • Xenopus laevis / metabolism*

Substances

  • Cross-Linking Reagents
  • DNA, Complementary
  • P2RX7 protein, human
  • Receptors, Purinergic P2
  • Receptors, Purinergic P2X7
  • Adenosine Triphosphate