Stabilization of GABA A receptors at endocytic zones is mediated by an AP2 binding motif within the GABA Areceptor β3 subunit

Abstract

The strength of synaptic inhibition can be controlled by the stability and endocytosis of surface and synaptic GABA A receptors (GABA ARs), but the surface receptor dynamics that underpin GABA AR recruitment to dendritic endocytic zones (EZs) have not been investigated. Stabilization of GABA ARs at EZs is likely to be regulated by receptor interactions with the clathrin-adaptor AP2, but the molecular determinants of these associations remain poorly understood. Moreover, although surface GABA AR downmodulation plays akey role in pathological disinhibition in conditions such as ischemia and epilepsy, whether this occurs in an AP2-dependent manner also remains unclear. Here we report the characterization of a novel motif containing three arginine residues ( 405RRR 407) within the GABA AR β3-subunit intracellular domain (ICD), responsible for the interaction with AP2 and GABA AR internalization. When this motif is disrupted, binding to AP2 is abolished in vitro and in rat brain. Using single-particle tracking, we reveal that surfaceβ3-subunit-containing GABA ARs exhibit highly confined behavior at EZs, which is dependent on AP2 interactions via this motif. Reduced stabilization of mutant GABA ARs at EZs correlates with their reduced endocytosis and increased steady-state levels at synapses. By imaging wild-type or mutant super-ecliptic pHluorin-tagged GABA ARs in neurons, we also show that, under conditions of oxygen- glucose deprivation to mimic cerebral ischemia, GABA ARs are depleted from synapses in dendrites, depending on the 405RRR 407 motif. Thus, AP2 binding to an RRR motif in the GABA AR β3-subunit ICD regulates GABA AR residency time at EZs, steady-state synaptic receptor levels, and pathological loss of GABA ARs from synapses during simulated ischemia. © 2012 the authors.