Selective knock-down of P2X7 ATP receptor function by dominant-negative subunits

Mol Pharmacol. 2004 Mar;65(3):646-54. doi: 10.1124/mol.65.3.646.

Abstract

Among the family of P2X ATP-gated cation channels, the P2X7 receptor is a homomeric subtype highly expressed in immune cells of the monocyte-macrophage lineage. We report here that the WC167-168AA mutation in the ectodomain of P2X7 produced nonfunctional subunits with strong dominant-negative effect on wild-type P2X7 receptors (77% inhibition with cotransfection of wild-type and mutant DNA at a ratio of 3:1). The C168A single mutant was also very effective in suppressing P2X7 receptor function (72% reduction at a DNA ratio of 3:1), indicating the major role played by the C168A mutation in this inhibition. The dominant-negative effect is selective; the mutant subunit did not suppress the function of other receptor-channel subtypes. The reduced current responses in cells coexpressing wild-type and dominant-negative subunits display wild-type characteristics in both agonist affinity and ionic selectivity, strongly suggesting that the heteromeric channels are functionally impaired. The mutant subunits also suppressed the P2X7-dependent pore formation as assessed by uptake of the propidium dye YO-PRO-1 (Molecular Probes, Eugene, OR) in response to 2',3'-O-(4-benzoyl)-benzoyl-ATP (BzATP) in transfected human embryonic kidney 293 cells. Native responses to BzATP as well as ATP-induced ethidium dye uptake were significantly knocked down (31 +/- 9% and 25 +/- 7% of control, respectively) in mouse macrophage cell line RAW264.7 transfected with the mutant subunits. Therefore, these dominant-negative subunits provide selective genetic tools to investigate the functional roles of native P2X7 receptors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Electrophysiology
  • Humans
  • Mice
  • Mutagenesis
  • Protein Subunits / genetics
  • Protein Subunits / metabolism*
  • Rats
  • Receptors, Purinergic P2 / genetics
  • Receptors, Purinergic P2 / metabolism*
  • Receptors, Purinergic P2X7
  • Subcellular Fractions
  • Transfection

Substances

  • P2RX7 protein, human
  • P2rx7 protein, mouse
  • Protein Subunits
  • Receptors, Purinergic P2
  • Receptors, Purinergic P2X7