GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition

Abstract

GABA (gamma-aminobutyric acid) type A receptors (GABA(A)Rs) mediate most fast synaptic inhibition in the mammalian brain, controlling activity at both the network and the cellular levels. The diverse functions of GABA in the CNS are matched not just by the heterogeneity of GABA(A)Rs, but also by the complex trafficking mechanisms and protein-protein interactions that generate and maintain an appropriate receptor cell-surface localization. In this Review, we discuss recent progress in our understanding of the dynamic regulation of GABA(A)R composition, trafficking to and from the neuronal surface, and lateral movement of receptors between synaptic and extrasynaptic locations. Finally, we highlight a number of neurological disorders, including epilepsy and schizophrenia, in which alterations in GABA(A)R trafficking occur.

Figure 1. GABA_A receptor structure and neuronal localization

(A) GABA_A receptors are members of the ligand-gated ion channel superfamily. Receptor subunits consist of four hydrophobic transmembrane (TM1–4) domains, where TM2 is believed to line the pore of the channel. The large extracellular N-terminus is the site for the binding of the neurotransmitter GABA, as well as containing binding sites for psychoactive drugs, such as benzodiazepines (BZ). Each receptor subunit also contains a large intracellular domain between TM3 and TM4, which is the site for various protein interactions as well as the site for various post-translational modifications that modulate receptor activity. (B) Five subunits from 7 subunit subfamilies (α,β,γ,δ,ε,θ,π) assemble to form a heteropentameric chloride-permeable channel. Despite the extensive heterogeneity of GABA_A receptor subunits, the majority of GABA_A receptors expressed in the brain consist of 2α, 2β, and 1γ subunit, where the γ subunit can be replaced by δ, ε or π. Binding of the neurotransmitter GABA occurs at the interface between the α and β subunits and triggers the opening of the channel, allowing the rapid influx of chloride ions. BZ-binding occurs at the interface between α(1,2,3 or 5) and γ subunits and potentiates GABA-induced chloride flux. (C) GABA_A receptors composed of α(1–3) subunits together with β and γ subunits are thought to be primarily synaptically localized, whereas α5βγ receptors are located largely at extrasynaptic sites. Receptors composed of the aforementioned subunits are benzodiazepine-sensitive. In contrast, receptors composed of α(4,6)βδ are benzodiazepine-insensitive, and are localized at extrasynaptic sites.

Figure 2. Trafficking of GABA_A receptors

GABA_A receptor subunits are synthesized and assembled into pentameric structures in the endoplasmic reticulum (ER). This process is carefully regulated in the ER. The fate of GABA_A receptor subunits can be modulated by ubiquitination and subsequent ER-associated degradation via the proteasome. Ubiquitinated GABA_A receptor subunits can also be modulated via their association with Plic-1. Plic-1 facilitates GABA_A receptor accumulation at the synapse by preventing the degradation of ubiquitinated GABA_A receptors. Exit into the Golgi network and subsequent trafficking to the plasma membrane are also facilitiated by a number of GABA_A receptor-associated proteins. GABARAP associates with the γ2 subunit of GABA_A receptors and aids in the trafficking of receptors from the Golgi network to the plasma membrane. NSF and BIG2 are also localized to the Golgi network, where they bind to β subunits of GABA_A receptors and modulate receptor trafficking. Palmitoylation of γ subunits occurs in the Golgi apparatus as a result of an association with the palmitoyltransferase, GODZ, and is a critical step in the delivery of GABA_A receptors to the plasma membrane. GRIF proteins play a role in the trafficking of GABA_A receptors to the membrane. PRIP proteins also play essential roles in the trafficking of GABA_A receptors, as well as in modulating the phosphorylation state of GABA_A receptors.

Figure 3. Dynamic regulation of receptor lateral mobility at the GABAergic synapse

GABA_A receptors are inserted into the plasma membrane at extrasynaptic sites, where they can then diffuse into synaptic sites. Lateral diffusion (black arrows) within the plasma membrane allows for continual exchange between diffuse receptor populations and synaptic or extrasynaptic receptor clusters, with anchoring molecules tethering or corralling moving receptors. The synaptic localization of α2-containing GABA_A receptors is maintained by direct binding to gephyrin, which binds to microtubules and actin interactors such as the GDP/GTP exchange factor collybistin, Mena/ VASP (vasodilator-stimulated phosphoprotein) and profilins 1 and 2,. No direct interaction between gephyrin and the γ2 subunit has been demonstrated. However, gephyrin depletion increases γ2 cluster mobility, and loss of the γ2 subunit results in post-synaptic sites devoid of gephyrin. This suggests an unidentified intermediary interactor or a post-translational modification that could link γ2-containing receptors and gephyrin. Alternatively, clustering of γ2-containing receptors might occur via an independent mechanism. Gephyrin also displays local lateral movements (red double arrow), and removal or addition by microtubule dependent trafficking, contributing additional mechanisms to regulate synaptic transmission. Extrasynaptic localization of α5-containing GABA_A receptors is controlled by binding to activated radixin, which directly binds F-actin.

Figure 4. Regulation of GABA_A receptor endocytosis and post-endocytic sorting

Clathrin-dependent endocytosis is the major internalization mechanism for neuronal GABA_A receptors. The intracellular loops of β and γ subunits interact with the clathrin adaptor protein 2 (AP2) complex. Binding of the μ2 subunit is inhibited by phosphorylation of the AP2-interacting motifs in GABA_A receptor subunits, increasing cell surface receptor levels and enhancing the efficacy of inhibitory synaptic transmission. Once endocytosed in clathrin-coated vesicles (CCV), the vesicles are uncoated and fuse with early or sorting endosomes, resulting in GABA_A receptors being subsequently recycled to the plasma membrane or degraded in lysosomes. Huntingtin associated protein-1 (HAP1) interacts with β subunits and promotes receptor recycling to the plasma membrane. PKA and PKC regulate phosphorylation of serine residues 408/9 in the AP2-binding motif of β3 subunits, while tyrosine residues 365/7 in the γ2 subunit are phosphorylated by Src kinase.

Figure 5. Dysregulation in GABA_A receptor trafficking in neurological disease

(A) Repetitive, non-abating seizures that lead to status epilepticus result in a decrease in the phosphorylation of GABA_A receptor β subunits by PKC. This leads to an increased associated with the clathrin adaptor AP2, followed by increased internalization via clathrin-mediated endocytosis. Decreased numbers of synaptic GABA_A receptors lead to reduced synaptic inhibition (ie. increased excitatory drive and a lower seizure threshold) as well as decreased benzodiazepine sensitivity. (B) Alcohol-induced plasticity in GABA_A receptors involves changes in both synaptic and extrasynaptic GABA_A receptor populations. After alcohol administration, there is an increased internalization of δ-containing extrasynaptic GABA_A receptors. There is also an increased internalization of α1-containing synaptic GABA_A receptors via clathrin-dependent internalization. Insertion of distinct GABA_A receptor populations at synaptic sites (ie. α4βγ2) have been hypothesized to serve a compensatory role at inhibitory synapses, however, these receptors differ in their physiological functions from normal synaptic GABA_A receptor populations, as well as being benzodiazepine-insensitive.