Constitutive GABAA Receptor Endocytosis Is Dynamin-mediated and Dependent on a Dileucine AP2 Adaptin-binding Motif within the β2 Subunit of the Receptor

Abstract

Receptor endocytosis is an important mechanism for regulating the synaptic efficacy of neurotransmitters. There is strong evidence that GABA A receptor endocytosis is clathrin-dependent; however, this process is not well understood. Here we demonstrate that in HEK 293 cells, endocytosis of GABA A receptors composed of either ␣ 1 ␤ 2 ␥ 2 L or ␣ 1 ␤ 2 subunits is blocked by the dominant negative dynamin construct K44A. Furthermore, we identify a dileucine AP2 adaptin-binding motif within the receptor ␤ 2 subunit that is critical for endocytosis. Internalization of GABA A receptors lacking this motif is dramatically inhibited, and the receptors appear to accumulate on the cell surface. Patch clamp analysis of receptors lacking the dileucine motif show that there is an increase in the peak amplitude of GABA-gated chloride currents compared with wild-type receptors. Additionally, GABA-gated chloride currents in HEK 293 cells expressing wild-type receptors are increased by introduction of a peptide corresponding to the dileucine motif region of the receptor ␤ 2 subunit but not by a control peptide containing alanine substitutions for the dileucine motif. In mouse brain cerebral cortical neurons, the dileucine motif peptide increases GABA-gated chloride currents of native GABA A receptors. This is the first report to our knowledge that an AP2 adaptin dileucine recognition motif is critical for the endocytosis of ligand-gated ion channels belonging to this superfamily.The GABA A receptor is a ligand-gated chloride channel that, upon activation by GABA 1 (␥-aminobutyric acid), mediates increases in chloride conductance resulting in membrane hyperpolarization and neuronal inhibition (1). The role of these receptors in hyperexcitability states, such as epilepsy and anxiety, is widely recognized. Importantly, GABA A receptors mediate the effects of benzodiazepines and barbiturates, two frequently prescribed classes of therapeutic agents. The GABA A receptor is a pentameric receptor composed of multiple subunits, each containing four membrane-spanning regions (M1-M4) with a large intracellular loop between M3 and M4. A number of subunits exist (␣ 1Ϫ6 , ␤ 1Ϫ3 , ␥ 1Ϫ3 , ␦, , ⑀, ), and receptors composed of ␣ 1 ␤ 2 ␥ 2 L subunits are believed to represent the predominant GABA A receptor subtype in the brain (1).Receptor endocytosis is known to regulate the cell surface expression of neurotransmitter receptors, and such regulation is an important mechanism for controlling the synaptic efficacy of neurotransmitters (2). Although GABA A receptors undergo endocytosis, the mechanism is not well understood. Several lines of evidence indicate that GABA A receptor endocytosis may be clathrin/dynamin-dependent. These include the presence of GABA A receptors in clathrin-coated vesicles isolated from brain (3), the colocalization of the receptor with transferrin receptors (4), and the colocalization and co-immunoprecipitation of hippocampal GABA A receptors with the clathrin adaptor complex AP2 adaptin (5). Additionally, peptides that dis...

β-Adrenergic Receptor Activation Induces Internalization of Cardiac Cav1.2 Channel Complexes through a β-Arrestin 1-mediated Pathway

“…The ␤-AR in the G i -coupled state can activate Src kinase, and subsequently the GTPase dynamin, which is known to play a role in the budding of vesicles from the plasma membrane during internalization (21,22). In addition, it has been shown that the ␤-AR can activate Src kinase in a G protein-independent manner (16,17).…”

Phospho-dependent binding of the clathrin AP2 adaptor complex to GABA A receptors regulates the efficacy of inhibitory synaptic transmission

“…2c) representing residues 401-412 to bind 2 (see below), we conclude that we have identified a previously uncharacterized atypical binding motif (between ␤-subunit residues 401 and 410) for the AP2 complex, which is conserved within the ICDs of all GABA A R ␤ subunits. Recently a dileucine motif (LL) in the intracellular domain of the ␤2 subunit (residues 344 and 345) has been suggested to be of significance in regulating GABA A R internalization via a clathrin-dependent mechanism, and this motif is also found in the ICDs of the ␤1 and ␤3 subunits (31). However, the direct binding of LL motifs to the 2 subunit of the AP2 complex is controversial (17)(18)(19).…”

Huntingtin-associated protein 1 regulates inhibitory synaptic transmission by modulating γ-aminobutyric acid type A receptor membrane trafficking

“…GABA A Rs undergo clathrin-mediated endocytosis and can be detected in endocytic structures (1,8,10,11,27). HAP1 has been localized to clathrin-coated vesicles and the endocytic pathway (23)(24)(25)(26) and has been implicated in endocytic protein trafficking (26), suggesting that HAP1 may be involved in the endocytic sorting of GABA A Rs.…”

“…Modifications of GABA A R cell surface number underlie changes in inhibitory postsynaptic current amplitude, providing an effective mechanism for regulating the efficacy of synaptic inhibition (3)(4)(5)(6)(7)(8)(9)(10). Under basal conditions, synaptic GABA A Rs are undergoing clathrin-dependent endocytosis (8,10,11). Given this constitutive endocytosis, the cellular fate of internalized GABA A receptors is critical as their recycling or degradation will affect the number of receptors on the cell surface, and hence the efficacy of synaptic inhibition.…”