title: Stabilization of GABA(A) receptors at endocytic zones is mediated by an AP2 binding motif within the GABA(A) receptor 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 (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.
authors.affiliation: Department of Neuroscience, Physiology, and Pharmacology, University College London, London WC1E 6BT, UK.
authors.forename: Katharine R
authors.initials: KR
authors.lastname: Smith
authors.forename: James
authors.initials: J
authors.lastname: Muir
authors.forename: Yijian
authors.initials: Y
authors.lastname: Rao
authors.forename: Marietta
authors.initials: M
authors.lastname: Browarski
authors.forename: Marielle C
authors.initials: MC
authors.lastname: Gruenig
authors.forename: David F
authors.initials: DF
authors.lastname: Sheehan
authors.forename: Volker
authors.initials: V
authors.lastname: Haucke
authors.forename: Josef T
authors.initials: JT
authors.lastname: Kittler
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D004705:Endocytosis / Q000235:genetics*; D005260:Female; D008297:Male; D011485:Protein Binding / Q000235:genetics; D055550:Protein Stability; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: D033962:Adaptor Protein Complex 2; C546276:Gabrb3 protein, rat; D021122:Protein Subunits; D011963:Receptors, GABA-A