The strength of synaptic inhibition can be controlled by the stability and endocytosis of surface and synaptic
GABA(A) receptors (
GABA(A)Rs), but the surface receptor dynamics that underpin
GABA(A)R recruitment to dendritic endocytic zones (EZs) have not been investigated. Stabilization of
GABA(A)Rs 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(A)R 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
GABA(A)R β3-subunit intracellular domain (ICD), responsible for the interaction with AP2 and
GABA(A)R 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
GABA(A)Rs exhibit highly confined behavior at EZs, which is dependent on AP2 interactions via this motif. Reduced stabilization of mutant
GABA(A)Rs 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(A)Rs in neurons, we also show that, under conditions of
oxygen-
glucose deprivation to mimic
cerebral ischemia,
GABA(A)Rs are depleted from synapses in dendrites, depending on the 405RRR407 motif. Thus, AP2 binding to an RRR motif in the
GABA(A)R β3-subunit ICD regulates
GABA(A)R residency time at EZs, steady-state
synaptic receptor levels, and pathological loss of
GABA(A)Rs from synapses during simulated
ischemia.