Functional properties of internalization-deficient P2X4 receptors reveal a novel mechanism of ligand-gated channel facilitation by ivermectin

Mol Pharmacol. 2006 Feb;69(2):576-87. doi: 10.1124/mol.105.018812. Epub 2005 Nov 10.


Although P2X receptors within the central nervous system mediate excitatory ATP synaptic transmission, the identity of central ATP-gated channels has not yet been elucidated. P2X(4), the most widely expressed subunit in the brain, was previously shown to undergo clathrin-dependent constitutive internalization by direct interaction between activator protein (AP)2 adaptors and a tyrosine-based sorting signal specifically present in the cytosolic C-terminal tail of mammalian P2X(4) sequences. In this study, we first used internalization-deficient P2X(4) receptor mutants to show that suppression of the endocytosis motif significantly increased the apparent sensitivity to ATP and the ionic permeability of P2X(4) channels. These unique properties, observed at low channel density, suggest that interactions with AP2 complexes may modulate the function of P2X(4) receptors. In addition, ivermectin, an allosteric modulator of several receptor channels, including mammalian P2X(4), did not potentiate the maximal current of internalization-deficient rat or human P2X(4) receptors. We demonstrated that binding of ivermectin onto wild-type P2X(4) channels increased the fraction of plasma membrane P2X(4) receptors, whereas surface expression of internalization-deficient P2X(4) receptors remained unchanged. Disruption of the clathrin-mediated endocytosis with the dominant-negative mutants Eps15 or AP-50 abolished the ivermectin potentiation of wild-type P2X(4) channel currents. Likewise, ivermectin increased the membrane fraction of nicotinic alpha7 acetylcholine (nalpha7ACh) receptors and the potentiation of acetylcholine current by ivermectin was suppressed when the same dominant-negative mutants were expressed. These data showed that potentiation by ivermectin of both P2X(4) and nalpha7ACh receptors was primarily caused by an increase in the number of cell surface receptors resulting from a mechanism dependent on clathrin/AP2-mediated endocytosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / pharmacology
  • Amino Acid Motifs
  • Animals
  • Antiparasitic Agents / pharmacology*
  • Cell Membrane / chemistry
  • Endocytosis / drug effects*
  • Endocytosis / genetics
  • Humans
  • Ion Channel Gating / drug effects*
  • Ion Channels / drug effects
  • Ivermectin / pharmacology*
  • Ligands
  • Mutation
  • Oocytes / chemistry
  • Oocytes / drug effects
  • Permeability / drug effects
  • Rats
  • Receptors, Nicotinic / analysis
  • Receptors, Nicotinic / metabolism
  • Receptors, Purinergic P2 / analysis
  • Receptors, Purinergic P2 / genetics
  • Receptors, Purinergic P2 / physiology*
  • Receptors, Purinergic P2X4
  • alpha7 Nicotinic Acetylcholine Receptor


  • Antiparasitic Agents
  • Chrna7 protein, human
  • Chrna7 protein, rat
  • Ion Channels
  • Ligands
  • P2RX4 protein, human
  • P2rx4 protein, rat
  • Receptors, Nicotinic
  • Receptors, Purinergic P2
  • Receptors, Purinergic P2X4
  • alpha7 Nicotinic Acetylcholine Receptor
  • Ivermectin
  • Adenosine Triphosphate