Functional Properties of Internalization-Deficient P2X4 Receptors Reveal a Novel Mechanism of Ligand-Gated Channel Facilitation by Ivermectin

Abstract

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. P2X₄, 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 P2X₄ sequences. In this study, we first used internalization-deficient P2X₄ receptor mutants to show that suppression of the endocytosis motif significantly increased the apparent sensitivity to ATP and the ionic permeability of P2X₄ channels. These unique properties, observed at low channel density, suggest that interactions with AP2 complexes may modulate the function of P2X₄ receptors. In addition, ivermectin, an allosteric modulator of several receptor channels, including mammalian P2X₄, did not potentiate the maximal current of internalization-deficient rat or human P2X₄ receptors. We demonstrated that binding of ivermectin onto wild-type P2X₄ channels increased the fraction of plasma membrane P2X₄ receptors, whereas surface expression of internalization-deficient P2X₄ 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 P2X₄ channel currents. Likewise, ivermectin increased the membrane fraction of nicotinic α7 acetylcholine (nα7ACh) 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 P2X₄ and nα7ACh receptors was primarily caused by an increase in the number of cell surface receptors resulting from a mechanism dependent on clathrin/AP2-mediated endocytosis.