Ethanol promotes clathrin adaptor-mediated endocytosis via the intracellular domain of δ-containing GABAA receptors

Abstract

Pharmacological and genetic evidence reveals that GABAA receptor (GABAA-R) expression and localization are modulated in response to acute and chronic ethanol (EtOH) exposure. To determine molecular mechanisms of GABAA-R plasticity in response to in vivo acute EtOH, we measured early time changes in GABAA-R subunit localization. Single doses of EtOH (3 g/kg via i.p. injection in rats) produced decreases in surface levels of GABAA-Rα4 and δ subunits at 5-15 min post-EtOH in hippocampusCA1and dentate gyrus, verifying our earlier report (Liang et al., 2007). Here we also examined the β3 subunit and its phosphorylation state during internalization. β3 also was internalized during 5-15 min after EtOH exposure, while phosphorylation of β3 was increased, then decreased at later times, ruling out β3 dephosphorylation-dependent endocytosis. As early as 5minpost-EtOH, there isaninitial increase in association between the δ subunits with clathrin adaptor proteins AP2-μ2 revealed by coimmunoprecipitation, followed by a decrease in association 15 min post-EtOH. In vitro studies using glutathione S-transferase fused to the δ subunit intra cellular domain (ICD) show that two regions, one containing a classical YxxΦ motif and the other an atypical R/K-rich motif, directly and differentially bind to AP2-μ2, with the former YRSV exhibiting higher affinity. Mutating both regions in the δ-ICD abolishes μ2binding, providing a possible mechanism that can explain the rapid down regulation of extra synaptic α4βδ-GABAA-Rfollowing in vivo EtOH administration, in which the δ-ICD increases in affinity for clathrin AP2-μ2 leading to endocytosis. © 2012 the authors.