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.