Stabilization of GABAA Receptors at Endocytic Zones Is Mediated by an AP2 Binding Motif within the GABAA Receptor β3 Subunit

Abstract

The strength of synaptic inhibition can be controlled by the stability and endocytosis of surface and synaptic GABAA receptors (GABAARs), but the surface receptor dynamics that underpin GABAAR recruitment to dendritic endocytic zones (EZs) have not been investigated. Stabilization of GABAARs 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 GABAAR downmodulation plays a key 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 (405RRR407) within the GABAAR β3-subunit intracellular domain (ICD), responsible for the interaction with AP2 and GABAAR internalization. When this motif is disrupted, binding to AP2 is abolished in vitro and in rat brain. Using single-particle tracking, we reveal that surface β3-subunit-containing GABAARs exhibit highly confined behavior at EZs, which is dependent on AP2 interactions via this motif. Reduced stabilization of mutant GABAARs at EZs correlates with their reduced endocytosis and increased steady-state levels at synapses. By imaging wild-type or mutant super-ecliptic pHluorin-tagged GABAARs in neurons, we also show that, under conditions of oxygen–glucose deprivation to mimic cerebral ischemia, GABAARs are depleted from synapses in dendrites, depending on the 405RRR407 motif. Thus, AP2 binding to an RRR motif in the GABAAR β3-subunit ICD regulates GABAAR residency time at EZs, steady- state synaptic receptor levels, and pathological loss of GABAARs from synapses during simulated ischemia.