P2X4 Activation Modulates Volume-sensitive Outwardly Rectifying Chloride Channels in Rat Hepatoma Cells

Abstract

Volume-sensitive outwardly rectifying (VSOR) Cl channels are critical for the regulatory volume decrease (RVD) response triggered upon cell swelling. Recent evidence indicates that H2O2 plays an essential role in the activation of these channels and that H2O2 per se activates the channels under isotonic isovolumic conditions. However, a significant difference in the time course for current onset betweenH2O2-induced and hypotonicity- mediated VSOR Cl activation is observed. In several cell types, cell swelling induced by hypotonic challenges triggers the release of ATP to the extracellular medium, which in turn, activates purinergic receptors and modulates cell volume regulation. In this study, we have addressed the effect of purinergic receptor activation on H2O2-induced and hypotonicity-mediated VSOR Cl current activation. Here we show that rat hepatoma cells (HTC) exposed to a 33% hypotonic solution responded by rapidly activating VSOR Cl current and releasing ATP to the extracellular medium. In contrast, cells exposed to 200 M H2O2 VSOR Cl current onset was significantly slower, and ATP release was not detected. In cells exposed to either 11% hypotonicity or 200 MH2O2, exogenous addition of ATP in the presence of extracellular Ca2 resulted in a decrease in the halftime for VSOR Cl current onset. Conversely, in cells that overexpress a dominant-negative mutant of the ionotropic receptor P2X4 challenged with a 33% hypotonic solution, the half-time for VSOR Cl current onset was significantly slowed down. Our results indicate that, at high hypotonic imbalances, swellinginduced ATP release activates the purinergic receptor P2X4, which in turn modulates the time course of VSOR Cl current onset in a extracellular Ca2 -dependent manner.