Ethanol Promotes Clathrin Adaptor-Mediated Endocytosis via the Intracellular Domain of δ-Containing GABAAReceptors

Pharmacological and genetic evidence reveals that GABAAreceptor (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 toin vivoacute 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-R α4 and δ subunits at 5–15 min post-EtOH in hippocampus CA1 and 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 5 min post-EtOH, there is an initial 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 vitrostudies using glutathioneS-transferase fused to the δ subunit intracellular 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 μ2 binding, providing a possible mechanism that can explain the rapid downregulation of extrasynaptic α4βδ-GABAA-R followingin vivoEtOH administration, in which the δ-ICD increases in affinity for clathrin AP2-μ2 leading to endocytosis.