Stabilization of GABA_AReceptors 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 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 ( 405 RRR 407 ) within the GABA A R ␤3-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 ␤3-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 405 RRR 407 motif. Thus, AP2 binding to an RRR motif in the GABA A R ␤3-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.

Proteomic Characterization of Inhibitory Synapses Using a Novel pHluorin-tagged γ-Aminobutyric Acid Receptor, Type A (GABAA), α2 Subunit Knock-in Mouse

“…7D; ␤3 p Ͻ 0.001, ␥2 p Ͻ 0.0001). The GTPase Rab5 is found at endosomes, phagosomes, caveosome, and the plasma membrane (36) and has been shown to colocalize with the GABA A R ␤3 subunit (37). Consistent with these results, Rab5 bound GST-␤3 and ␥2 ( Fig.…”

Gephyrin Cleavage in In Vitro Brain Ischemia Decreases GABAA Receptor Clustering and Contributes to Neuronal Death

“…First, the synaptic population of GABA A R is characterized by a lower affinity for the neurotransmitter GABA when compared with the extrasynaptic receptors, due to their distinct molecular composition [36,47] and, therefore, the neurotransmitter concentration in the extrasynaptic regions may not be high enough to allow the activation of synaptic receptors displaced to extrasynaptic sites. Furthermore, the latter population of receptors may be internalized, by a mechanism dependent on the dephosphorylation of β3 subunits, as shown for synaptic GABA A R in hippocampal neurons subjected to OGD [5,9]. In contrast with the OGD-induced loss of synaptic clustering of GABA A R, which is accompanied by receptor internalization, the decrease in synaptic clustering of GABA A R following sustained network activity is not accompanied by an upregulation in receptor internalization [48].…”

“…Recent studies showed a decrease in the interaction of GABA A receptors (GABA A R) with the scaffold protein gephyrin in cultured hippocampal neurons subjected to oxygen-glucose deprivation (OGD), an in vitro model of brain ischemia, by a mechanism dependent on protein phosphatase activity [5]. The reduced interaction of GABA A R with the submembrane gephyrin lattice after OGD [6][7][8], together with the dephosphorylation of the receptors, facilitates their internalization by a clathrin-dependent mechanism [5,9].…”

Compromising the phosphodependent regulation of the GABA A R β3 subunit reproduces the core phenotypes of autism spectrum disorders

“…Central to these regulatory processes is the phosphorylation of S408/9 in the β3 subunit, which reduces the affinity of GABA A Rs for the clathrin adaptor protein AP2, as measured by using synthetic peptides corresponding to the β3 subunit and purified AP2 (9,13). The significance of these findings has recently been questioned by studies that suggest that the arginine residues flanking S408/9 are the principal determinants of AP2 binding in the β3 subunit (14). To examine the significance of S408/9 further, we expressed the intracellular domain of the β3 subunit as a GST fusion protein (GSTβ3) or a fusion protein in which the respective residues were mutated to alanines (GSTβ3S408/9A) in Escherichia coli.…”