GABA_A receptor trafficking and its role in the dynamic modulation of neuronal inhibition

Abstract

γ-Aminobutyric acid type A (GABA_A) receptors mediate the majority of fast synaptic inhibition in the mammalian brain, controlling activity both at the network and cellular level. The diverse functions of GABA in the central nervous system are matched not just by the heterogeneity of GABA_A receptors, but also the complex trafficking mechanisms and protein-protein interactions that generate and maintain appropriate receptor cell surface localization. In this review, we discuss recent progress in our understanding of the dynamic regulation of GABA_A receptor 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 receptor trafficking occur.

1. Introduction

Synaptic inhibition in the brain is largely mediated via γ-aminobutyric acid (GABA). The fast inhibitory actions of GABA are mediated via the activation of GABA_A receptors (GABA_ARs) in the brain^1,2 and via GABA_C receptors in the retina³, whereas the slow prolonged actions of this transmitter are mediated via metabotropic G-protein coupled GABA_B receptors^4,5. GABA_ARs are also clinically relevant drug targets for anti-convulsant, anxiolytic, and sedative-hypnotic agents. Moreover, deficits in the functional expression of GABA_ARs are critical in epilepsy, anxiety disorders, cognitive deficits, schizophrenia, depression, and substance abuse. Understandably, there has been considerable interest in comprehending the cellular mechanisms that regulate their accumulation on the neuronal plasma membrane.

Molecular studies have demonstrated that GABA_ARs are members of a ligand-gated ion channel superfamily, whose members include nicotinic acetylcholine, glycine and 5-hydroxy-tryptamine 3 (5-HT₃) receptors^6,7. Members of this superfamily are heteropentamers that are assembled from a range of homologous subunits that share a common structure: a large N-terminal extracellular domain and 4 transmembrane domains (TMs) with a large intracellular domain between TM 3 and 4 (Fig. 1A). To date, 18 GABA_AR subunits have been identified. Based on sequence homology, these are divided into 7 subunit classes, each of which has multiple members; α(1–6), β(1–3), γ(1–3), δ, ε(1–3), θ, π. GABA_AR structural diversity is further increased via the alterative splicing of some receptor mRNAs. However, most GABA_ARs are composed of 2α, 2β and 1γ (or 1δ) subunit² (Fig. 1B). GABA_ARs with different subunit composition have different physiological and pharmacological properties, are differentially expressed throughout the brain, and targeted to different subcellular regions. For instance, receptors composed of α(1,2,3 or 5) subunits together with β and γ subunits form benzodiazepine-sensitive receptors that are largely synaptically located and mediate the majority of phasic inhibition in the brain² (with the notable exception of extrasynaptically-localized α5-containing receptors^8,9) (Fig. 1C). In contrast, those composed of α(4/6)βδ subunits form a specialized population of predominantly extrasynaptic receptor subtypes that mediate tonic inhibition and are insensitive to benzodiazepine modulation⁹. In addition, GABA_ARs at presynaptic sites also exist¹⁰.

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.

Here we will address how neurons regulate the assembly, membrane trafficking, synaptic accumulation and function of these distinct GABA_AR subtypes, and the relevance of these emerging regulatory processes for the efficacy of neuronal inhibition in both health and disease.

2. Controlling GABA_AR assembly

GABA_ARs are assembled from their component subunits in the endoplasmic reticulum (ER). This process plays a critical role in determining the diversity of GABA_ARs that are expressed on the neuronal cell surface, because exit from the ER is dependent upon proteins reaching “conformation maturity” and misfolded proteins are retro-translocated from this organelle for degradation in the proteasome.

Limiting diversity via selective oligomerization

Many different subunit combinations are theoretically possible, however studies reveal that only a limited number of these combinations can actually exit the ER and access the neuronal cell surface. Most studies agree that most GABA_ARs expressed on the surface of neurons are composed of 2α, 2β, and 1γ subunit (the γ subunit can be replaced by δ, ε or π depending on the neuron type and subcellular localization of the receptor)^2,11. Most homomeric subunits, and αγ and βγ heteromers, are retained in the ER and degraded (for a review, see12). Thus, the expression and assembly of these subunits must be carefully regulated in the ER, via mechanisms that involve classical ER-resident chaperones, such as heavy chain binding protein and calnexin¹³.

Sequences in the N-terminus of GABA_AR subunits control receptor oligomerization, thus promoting the assembly of particular subunit combinations¹². Oligomerization of individual GABA_A receptor subunits into heteromers occurs within 5 minutes after translation¹⁴. However, it is inefficient, with less than 25% of translated subunits being assembled into heteromeric receptors¹⁴. GABA_AR subunit-deficient mice have provided insights into the preferential assembly of select GABA_ARs in vivo. For example, a loss of the δ subunit from the plasma membrane of cerebellar granule cells is observed in α6 knock-out mice¹⁵. Similarly, there is a decrease in the α4 subunit levels in the forebrain of δ subunit-deficient mice, whereas the levels of α1 subunits remain unchanged^16,17. This indicates that the δ subunit preferentially assembles with α4 and α6 subunits. There is also a compensatory increase in γ2 subunit levels in δ subunit-deficient mice^16,17, suggesting that γ2 associates with the α4 subunit in the absence of the δ subunit. These findings suggest that subunits compete to find their preferential oligomerization partners in the ER. However, the details of these processes remain to be determined.

Activity-dependent GABA_AR ubiquitination

The ER is responsible for the retention and degradation of misfolded or unassembled subunits and, accordingly, homomeric unassembled GABA_ARs subunits have been shown to be degraded in this organelle^14,18. ER-associated degradation (ERAD) involves protein ubiquitination and degradation via the ubiquitin-proteasome system (UPS)¹⁹. GABA_AR subunits have recently been shown to be ubiquitinated in an activity-dependent manner²⁰. Chronic blockade of neuronal activity dramatically increased the levels of GABA_AR ubiquitination within the ER, resulting in decreased insertion at the plasma membrane²⁰. Correspondingly, increasing the level of neuronal activity resulted in a decrease in the level of GABA_AR ubiquitination and an enhancement of receptor cell surface expression²⁰. Thus, neuronal activity can regulate the ubiquitination of GABA_ARs in the ER, affecting their rate of degradation via the UPS. This may be one mechanism neurons use to homeostatically regulate synaptic inhibition.

The fate of ubiquitinated GABA_ARs is also likely to be modulated via their association with the ubiquitin-like proteins Plic-1 and Plic-2¹⁸, which have been demonstrated to block the degradation of ubiquitinated substrates²¹. Plic-1 binds to the intracellular domain of α and β GABA_AR subunits via its ubiquitin-associated (UBA) domain¹⁸. Plic-1 increases the half-life of GABA_ARs, resulting in an increase in the number of receptors available for insertion into the plasma membrane¹⁸. Plic-1 does not affect the rate of receptor endocytosis¹⁸, thus it appears to function solely in the secretory pathway by stabilizing and/or inhibiting the degradation of GABA_ARs by the UPS, thereby facilitating receptor accumulation at inhibitory synapses.

3. Facilitating GABA_AR trafficking

After assembly in the ER, transport-competent GABA_ARs are trafficked to the Golgi apparatus and segregated into vesicles for transport to, and insertion into, the plasma membrane. Our understanding of these processes remains rudimentary, but it is becoming clear that they are facilitated by a number of receptor-associated proteins (Fig. 2), which are described in the following sections.

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.

GABARAP and NSF

GABA receptor-associated protein (GABARAP) interacts with the intracellular domain of GABA_AR γ subunits in vitro and in vivo²². It also binds to microtubules²³ and to N-ethylmaleimide-sensitive factor (NSF)²⁴, a protein involved in intracellular vesicular fusion events²⁵. GABARAP is concentrated in the Golgi apparatus and in intracellular vesicles, but is not present at GABAergic synapses^22,24,26, suggesting that its main role is in the intracellular transport of GABA_ARs. Overexpressing GABARAP with GABA_ARs results in increased cell surface receptor expression, possibly as a result of enhanced intracellular receptor trafficking^27,28,29. This effect can be abolished by a mutation that disrupts the addition of phospholipids to GABARAP³⁰, which apparently increases its membrane association, and is thus critical for GABARAP to control GABA_AR trafficking³⁰. Analysis of GABARAP knock-out mice did not reveal any alterations in synaptic γ2-containing GABA_AR numbers³¹; however, this may reflect redundancy, given the existence of other GABARAP homologs that can interact with GABA_ARs³². Recently, it was demonstrated that GABARAP is necessary for increasing cell surface GABA_AR expression after N-methyl-D-aspartate (NMDA) receptor activation³³, suggesting that it may have a role in the regulated delivery of GABA_ARs to the surface after activity, rather than in the maintenance of basal receptor levels.

NSF has also been found to bind directly to GABA_AR β subunits³⁴. NSF and GABARAP may act together to promote the forward trafficking of GABA_ARs from the Golgi apparatus. Indeed, the subcellular distribution of both GABA_ARs and NSF is disturbed when the lipid modification of GABARAP is prevented in neurons, resulting in less GABA_ARs being trafficked to the plasma membrane³⁰. However, another study found that overexpression of NSF significantly reduced GABA_AR cell surface numbers in both heterologous systems and in neurons³⁴. This is opposite to the effect observed on GABA_ARs when GABARAP is overexpressed^27,28,29, and is also opposite to NSF’s role in enhancing α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor surface expression^35,36. This may indicate that NSF has additional functions in the endocytic pathway, however further studies are required to understand the exact mechanism of how NSF regulates GABA_AR numbers.

PRIP proteins

Phospholipase C-related catalytically inactive proteins (PRIP) are inositol 1,4,5-trisphosphate binding proteins³⁷. PRIP-1 is expressed mainly in the brain, whereas PRIP-2 is expressed ubiquitously³⁸. PRIP proteins bind to GABARAP as well as to the intracellular domains of GABA_AR β subunits, and more weakly, γ2 subunits^38,39. These findings prompted the hypothesis that PRIP proteins modulate GABA_ARs by competitively inhibiting GABARAP binding³⁹. However, a more recent study⁴⁰ suggests that PRIP molecules act as bridging proteins between GABARAP and GABA_ARs, facilitating the transport of γ2-containing receptors. This model was derived largely from studies of PRIP-1 and PRIP-2 double knock-out (PRIP-DKO) mice, in which the association between GABA_ARs and GABARAP in neurons was significantly reduced⁴⁰. Furthermore, PRIP-DKO mice have reduced sensitivity to diazepam, suggesting an alteration in γ2-containing GABA_ARs⁴⁰. PRIP-1 knockout mice showed a similar phenotype³⁹. In a complementary approach, peptides were used to disrupt the binding of PRIP-1 to GABA_AR subunits, resulting in a reduction in cell surface expression of γ2-containing GABA_ARs in cultured cell lines and neurons⁴⁰. Thus, PRIP and GABARAP proteins may participate together in the trafficking of GABA_ARs to the synaptic membrane.

PRIP proteins may also regulate GABA_AR function by regulating their phosphorylation. Phosphorylation has been shown to dynamically modulate GABA_AR function, and β subunits are substrates for protein kinase C (PKC) and cAMP-dependent protein kinase A (PKA)⁴¹. Dephosphorylation of GABA_ARs by the protein phosphatase 1α (PP1α) terminates phosphorylation-dependent receptor modulation⁴², and PP1α has been shown to be inactivated by PRIP-1⁴³. PRIP-1 knock-out mice exhibited enhanced PP1α activity, resulting in diminished phosphorylation of GABA_ARs by PKA and subsequent changes in hippocampal neuronal inhibition⁴². Lastly, a recent study has implicated PRIP proteins in the constitutive internalization of recombinant GABA_ARs from the plasma membrane of non-neuronal cells⁴⁴. Thus, PRIP proteins might have a central role in controlling GABA_AR function via at least three distinct mechanisms: the trafficking of GABA_ARs, the modulation of GABA_AR phosphorylation and the internalization of GABA_ARs.

Palmitoylation and GODZ

Palmitoylation involves the covalent attachment of the saturated fatty acid palmitate to a protein and has been shown to play a role in protein trafficking and function at both inhibitory and excitatory synapses⁴⁵. Two groups have demonstrated that cysteine residues located within the intracellular domain of γ subunits are substrates for palmitoylation and that this modification is critical for the delivery of GABA_ARs to synapses^46,47. The Golgi-specific DHHC zinc finger domain protein (GODZ) has been shown to mediate the palmitoyl acyl transfer to these subunits⁴⁶. Of the twenty-three members of the Asp, His, His, Cys-cysteine-rich repeat domain (DHHC-CRD) protein family only GODZ and its close paralog Sertoli cell gene with a zinc finger domain-β (Serz-β) can efficiently palmitoylate the γ2 subunit⁴⁸. Furthermore, studies using dominant-negative GODZ or GODZ-specific RNA interferance (RNAi) have demonstrated that GODZ is the principal palmitolytransferase for GABA_ARs⁴⁸. GODZ is not found at inhibitory synapses, but is enriched in the trans-Golgi network, and is essential for the accumulation of γ2-containing GABA_ARs at synapses and for synaptic inhibitory function^46,48. Therefore, GODZ presumably acts to control GABA_AR trafficking in the secretory pathway and the delivery of these receptors to the plasma membrane⁴⁶.

BIG2

Brefeldin A-inhibited GDP/ GTP exchange factor 2 (BIG2) plays an important role in the vesicular trafficking of GABA_ARs to the plasma membrane. A yeast two hybrid screen showed that it can bind to the intracellular domain of the β3 subunit, and it has since been shown to have high binding affinity for the intracellular loops of all β subunits⁴⁹. In hippocampal neurons BIG2 is largely localized to the trans-Golgi network, but it is also found in trafficking vesicles and at the synaptic plasma membrane⁴⁹. BIG2 has a known role in membrane budding and vesicular transport from the Golgi apparatus⁵⁰. Taken together, this suggests that the main function of BIG2 is in the intracellular trafficking of GABA_ARs through the exocytic pathway to the plasma membrane.

GRIF/TRAK proteins

GABA_AR-interacting factor 1 (GRIF-1) was first described as a protein that interacts with the β2 subunit of GABA_ARs⁵¹. It is a member of the TRAK family of coiled-coil domain proteins that have been implicated in the trafficking of intracellular vesicles. GRIF-1 (also known as TRAK2) and TRAK1 both interact with the microtubule-associated motor protein kinesin^52,53. TRAK1 has also been shown to interact with GABA_ARs⁵⁴, suggesting a role for these proteins in regulating the motor-dependent transport of GABA_ARs. Interestingly, deletion of TRAK1 in mice leads to hypertonia and reduced GABA_AR expression in the brain and motor neurons⁵⁴.

4. Clustering GABA_ARs at synapses

After navigating their way through the secretory pathway, GABA_ARs are inserted into the plasma membrane, where they are able to access inhibitory postsynaptic specializations or extrasynaptic sites, depending on subunit composition (Fig. 3). The mechanisms that facilitate these distinct subcellular fates are described below.

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 collybistin⁷², Mena/ VASP (vasodilator-stimulated phosphoprotein)¹⁴⁴ and profilins 1 and 2¹⁴⁴,¹⁴⁵. 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.

Synaptic vs. extrasynaptic GABA_ARs

GABA_ARs on the neuronal cell surface exist as diffuse receptor populations or synaptic or extrasynaptic clusters. Lateral diffusion within the plasma membrane allows for continual exchange between these receptor populations^55,56. GABA_ARs that can bind bungarotoxin have been used to examine the subcellular sites of GABA_AR insertion into the neuronal membrane. These studies have demonstrated that most receptors are delivered to extrasynaptic locations in the plasma membrane. Over time, diffusion and trapping increases the population of synaptic receptors⁵⁷.

Heteromeric GABA_ARs retain distinct cell surface expression patterns dependent on subunit composition. Most surface receptor clusters of γ2 receptor subunits are synaptic, whereas β3-containing GABA_ARs have a higher proportion of diffuse and/or extrasynaptic receptors^55,58. α5-containing receptors are predominantly extrasynaptic^8,9. Other receptor subunits, such as δ, appear as diffuse populations on the neuronal surface^59,60 and are exclusively located outside the synapse at perisynaptic and extrasynaptic locations^57,61. These extrasynaptic α5- and δ- containing GABA_ARs are considered the main receptors mediating tonic inhibition.

Gephyrin-dependent clustering of GABA_ARs

One protein strongly implicated in regulating the clustering of GABA_ARs at inhibitory synapses is the multifunctional protein gephyrin, which was first identified by its association with glycine receptors⁶². Gephyrin binds directly with the intracellular domain of the β subunit of glycine receptors, which stabilizes these proteins at inhibitory synapses in the spinal cord^63–67. Gephyrin is also widely expressed in non-neuronal tissues⁶⁵. In the brain, it is found in neurons, and is enriched at postsynaptic specializations that contain GABA_AR subtypes composed of α(1–3), β(2,3) and γ2 subunits⁶⁸.

Reducing gephyrin expression compromises the accumulation of GABA_AR subtypes containing α2 or γ2 subunits at inhibitory synapses^{55,66,69–71}, although there is no change in overall levels of these subunits⁷¹ and only a modest reduction in the amplitude of miniature inhibitory postsynaptic currents (mIPSCs) or GABA-induced whole-cell currents⁶⁶. In addition, surface clusters of GABA_ARs formed in the absence of gephyrin were three times more mobile than those in control neurons⁵⁵, indicating that gephyrin plays a role in enhancing the confinement of GABA_ARs at synaptic sites. Furthermore, gene knockout of collybistin, an established binding partner for gephyrin^72,73, also leads to loss of synaptic GABA_AR clusters⁷⁴. Together, these results support the concept that gephyrin may act to promote the stability of GABA_AR clusters containing α2/γ2 subunits.

A loss of α3 and β(2/3) subunits were observed in spinal cord neurons of gephyrin knockout mice, whereas there were only minimal changes in α1 or α5 subunits in hippocampal and spinal cord neurons^66,70. These observations suggested the existence of gephyrin-dependent and independent GABA_AR clustering mechanisms. However, the development of compensatory clustering mechanisms in neurons devoid of gephyrin cannot be discounted.

The molecular mechanisms underlying gephyrin-dependent clustering of GABA_ARs remain poorly understood. Evidence suggests that a domain critical for clustering may exist within the γ2subunit, as cultured neurons from γ2 knock-out mice are devoid of both GABA_ARs and gephyrin at postsynaptic sites^69,75. In order to identify such a domain, chimeric α2/γ2 and δ/γ2 receptors have been studied^76,77. These suggest that the intracellular loop and/or transmembrane domain 4 of the γ2 subunit are critical for GABA_AR synaptic clustering^76,77, but whether these domains actually mediate their effects in a mechanism dependent upon gephyrin remains to be established.

Efforts to show gephyrin binding to native GABA_ARs have been unsuccessful⁶³. Similarly, co-expression of gephyrin and α1–3, β1–3 and γ2 GABA_AR subunits in HEK-293 cells revealed only a weak interaction with the β3 subunit⁷⁸. Interestingly, a recent study⁷⁹ identified a 10 amino acid hydrophobic motif within the major intracellular domain of the α2 subunit that is responsible for the targeting of GABA_AR subunits to inhibitory synapses. Critically, this phenomenon is dependent upon gephyrin expression⁷⁹. In addition, this motif was demonstrated to mediate the direct interaction of the intracellular domain of the α2 subunit with gephyrin in in vitro binding assays⁷⁹. However, under the same conditions, minimal binding of gephyrin to the intracellular domains of the γ2 and β3 subunits was evident⁷⁹. The interaction of the α2 intracellular domain with gephyrin was blocked by low concentrations of detergent⁷⁹, thus providing a possible explanation as to why previous studies had not identified such a direct association between gephyrin and GABA_ARs.

In summary, these results provide strong evidence that gephyrin can bind directly to receptor subtypes containing α2 subunits and regulate their synaptic targeting, but the relevance of this mechanism for receptor subtypes containing other α subunit variants remains to be evaluated. Significantly, a large number of gephyrin splice variants have been identified⁸⁰ and the synaptic localization and function of gephyrin can be regulated by both activity^81,82 and phosphorylation⁸³. It will therefore be of merit to examine the roles that these differing variants of gephyrin play in regulating the synaptic clustering of distinct GABA_AR subtypes.

Gephyrin-independent clustering of GABA_ARs

Gephyrin-independent GABA_AR clustering mechanisms are suggested by the presence of clustered receptors and mIPSCs in gephyrin knockout mice^66,70,83. Recently, radixin, an ERM (ezrin, radixin, moesin)-family member protein, has been identified as a specific interactor for the intracellular domain of the α5 subunit⁸⁴. ERM proteins exist in an inactive conformation and are activated by phosphatidylinositol-4,5-bisphosphate (PIP₂) binding and subsequent phosphorylation of the C-terminus (for a review, see85). In neurons, depletion of radixin dramatically decreased α5-containing GABA_AR clustering, although total cell surface levels of the α5 subunit remained unchanged⁸⁴. Radixin appears to directly link the α5 subunit to the actin cytoskeleton, as activated radixin is capable of binding both the α5 subunit and F-actin⁸⁴. The apparent radixin binding domain within the α5 subunit is a highly conserved region that is also found in α1–3 subunits, differing only in the last two amino acids in the α2 subunit. Further work clearly remains in elucidating the mechanism of radixin-dependent GABA_AR anchoring.

5. Endocytosis and post-endocytic sorting of GABA_ARs

GABA_AR endocytosis

GABA_ARs undergo extensive endocytosis in both heterologous and neuronal systems. Although a clathrin-independent endocytic pathway has been demonstrated in heterologous cells⁸⁶, clathrin-dependent endocytosis appears to be the major internalization mechanism for neuronal GABA_ARs⁸⁷ (Fig. 4), with approximately 25% of β3-containing cell surface GABA_ARs being internalized within 30 minutes⁸⁸. Blocking clathrin-dependent endocytosis results in reduced GABA_AR internalization^87,89,90 and a large increase in mIPSC amplitude⁸⁷, consistent with an increase in cell surface receptor levels^87,89,90.

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.

The clathrin adaptor protein 2 (AP2) complex plays a critical role in recruiting membrane-associated proteins into clathrin-coated pits. AP2 is composed of four distinct subunits (α, β2, μ2 and σ2 subunits; reviewed in⁹¹). GABA_ARs in the brain are intimately associated with AP2 via a direct binding of β1–3 and γ2 GABA_AR subunits to the μ2 subunit of this complex⁸⁷.

Within the β2 GABA_AR subunit, a dileucine motif has been identified that is important for clathrin-dependent GABA_AR internalization in heterologous cells⁸⁹. In addition, an atypical AP2 binding motif within the intracellular domains of GABA_AR β subunits has been identified⁹². Intriguingly, this binding motif contains the major sites of phosphorylation for PKA and PKC, and phosphorylation of these sites reduces binding to the μ2 subunit of AP2⁹². A peptide corresponding to the AP2 binding motif in the β3 subunit binds to AP2 with high affinity only when dephosphorylated⁹². Furthermore, this peptide enhanced mIPSC amplitude and whole cell GABA_AR currents.

More recently, another AP2 binding motif, centered around tyrosines 365/7 in the GABA_AR γ2 subunit, has been identified⁹³. These tyrosine residues are the principal sites for phosphorylation by Src kinase⁹⁴. A peptide containing residues Y365/7 exhibits very high affinity for the μ2 subunit, and the affinity of this interaction is dramatically decreased via phosphorylation of these sites⁹³. Introduction of the non-phosphorylated γ2 peptide into neurons produced a large increase in the mIPSC amplitude, and was observed to increase the number of cell surface GABA_ARs. Intriguingly, co-dialysis of neurons with both the non-phosphorylated β3 and γ2 subunit peptides produced an additive effect on mIPSC amplitudes⁹³.

Together, these results provide direct evidence that phosphorylation of GABA_AR subunits at distinct AP2 binding sites can regulate the cell surface stability of GABA_ARs and the strength of synaptic inhibition. Moreover they also provide a mechanism by which neurotransmitter and/or growth factor signaling pathways that regulate the activity of protein kinases/ phosphatases^41,95–97; could influence the efficacy of synaptic inhibition by controlling the stoichiometry of GABA_AR phosphorylation and, thus, their endocytosis.

GABA_AR recycling and lysosomal degradation

Once endocytosed, most internalized GABA_ARs recycle back to the plasma membrane over short time frames; however over longer time periods they are targeted for lysosomal degradation⁸⁸. Clearly the fate of internalized GABA_ARs will therefore play a critical role in controlling cell surface receptor levels and hence the efficacy of synaptic inhibition. Huntingtin associated protein-1 (HAP1)⁹⁸ is a GABA_AR associated protein that binds the intracellular loop of β subunits in vitro and in vivo⁸⁸. HAP1 is a cytoplasmic protein with several central coil-coiled domains that are likely to regulate protein-protein interactions. Overexpression of HAP1 in neurons inhibits GABA_AR degradation and consequently increases receptor recycling⁸⁸. Furthermore, HAP1 overexpression increased steady state surface levels of GABA_ARs and produced a 63% increase in mIPSC amplitude, showing a dramatic functional effect of increased surface receptor number⁸⁸. The mechanism underlying post-endocytic GABA_AR sorting remains to be elucidated, and HAP1’s specific role in this process is also an area of active research. The impact of HAP1 regulation of GABA_ARs was recently shown in the hypothalamus, where down-regulation of HAP1 levels resulted in decreased GABA_AR levels, causing decreased food intake and loss of body weight⁹⁹. An unresolved issue is whether HAP1 acts to promote recycling of GABA_ARs or prevents their lysosomal degradation.

6. Compromised GABA_AR trafficking in disease

The significance of the aforementioned mechanisms for maintaining homeostatic synaptic inhibition is highlighted by the multiple neurological and psychiatric diseases in which GABA_AR dysfunction has been suggested. These include epilepsy¹⁰⁰, anxiety disorders², Huntington’s disease¹⁰¹, Angelman syndrome¹⁰², fragile X syndrome¹⁰³, schizophrenia¹⁰⁴, and drug abuse¹⁰⁵. In this section, we highlight recent findings related to a few of these disorders.

Epilepsy

The epileptic state represents a dramatic change in balance between excitatory and inhibitory activity. Studies have shown that seizure activity results in altered GABA_AR trafficking and/or subunit expression in animal models of status epilepticus (SE) and temporal lobe epilepsy (TLE), as well as in patients^100,106. These changes involve both up- and down-regulation, depending on the particular GABA_AR subunit in question, as well as on the time-point studied in relation to the evolution of the chronic seizure state.

SE is a life-threatening state in which seizures occur unremittingly¹⁰⁷. Decreases in synaptic GABA_ARs, resulting from enhanced endocytosis, have been observed in animals in which SE has been experimentally induced^108–110. The loss of these synaptic receptor populations, which are normally benzodiazepine-sensitive, may explain the rapid development of pharmacoresistance in patients with SE and also explain the non-terminating nature of these seizures. A recent study showed decreased phosphorylation of β3 GABA_AR subunits during SE, resulting in an increased association of receptors with the clathrin adaptor AP2¹¹⁰ (Fig. 5A). Enhancing GABA_AR subunit phosphorylation, or selectively blocking subunit binding to AP2, increased GABA_AR surface expression levels and normalized synaptic inhibition in hippocampal slices derived from mice with SE¹¹⁰. Thus, novel therapeutic strategies for SE may one day be based on preventing or reversing this aberrant internalization of GABA_ARs.

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.

Altered GABA_AR expression has also been observed in animal models of TLE, however these have generally shown increases in the expression of synaptic GABA_ARs, at least in the dentate gyrus¹¹¹. Corresponding increases in the expression of GABA_AR-associated proteins, such as gephyrin, and in the size and density of postsynaptic GABA_AR clusters have also been demonstrated¹¹². This suggests that novel GABAergic synapses form, which could occur as a result of the aberrant sprouting of GABAergic axons¹⁰⁶. Extrasynaptic GABA_AR subunits (α4 and δ) have also been reported to be increased in granule cells of the dentate gyrus in rat models of TLE^113–115, however, one study in mice has observed decreases in δ subunit expression in these cells¹¹⁶. Analyses of hippocampal tissue from patients with TLE reveal alterations in GABA_AR subunit expression patterns that are similar to those observed experimentally^117,118.

Human genetic studies have provided further evidence that abnormal GABA_AR function contributes to epilepsy disorders. Multiple distinct mutations in the γ2^119–121, α1^122,123 and δ subunits¹²⁴ have been identified in patients with epilepsy. While the exact mechanisms by which each of these mutations contribute to seizure disorders remain to be fully elucidated, deficits in the assembly, trafficking and function of recombinant mutant receptors have been described^{119,120,125–127}. For example, a missense mutation that occurs in the α1 subunit (A322D) leads to subunit retention in the ER, followed by ubiquitin-dependent degradation¹²⁸, resulting in overall lower levels of α1-containing GABA_ARs at the cell surface.

Drug abuse

Considerable evidence exists to support a role for GABA_ARs in mediating the addictive properties of drugs of abuse^105,129. In particular, chronic use of alcohol or benzodiazepines, which are both allosteric modulators of GABA_ARs, can lead to drug tolerance, dependence and withdrawal symptoms following drug cessation. Changes in the mRNA and protein expression of various GABA_AR subunits have been documented after alcohol and benzodiazepine administration in both cultured neurons and animal models^130,131. However, the mechanisms responsible for these alterations have only recently begun to be elucidated. Significant alterations in the surface expression and composition of both synaptic and extrasynaptic GABA_AR populations have been observed after a single intoxicating dose of alcohol in rats¹³² (Fig. 5B). These changes were found to be persistent after chronic alcohol administration and withdrawal^132,133. This long-term plasticity in GABA_ARs is likely to involve changes in the phosphorylation of GABA_AR subunits and alterations in the endocytosis of specific GABA_AR subtypes. For example, the association of PKC with GABA_AR subunits is altered after chronic ethanol exposure¹³⁴. Increased associations between clathrin adaptor proteins and α1 subunits have also been demonstrated¹³⁵, suggesting that enhanced clathrin-mediated endocytosis of α1-containing GABA_ARs contributes to changes in GABA_AR trafficking after chronic alcohol use (Fig. 5B). Interestingly, the well-documented phenomenon of cross-tolerance to benzodiazepines after chronic alcohol use¹³⁶ suggests that similar mechanisms may be responsible for tolerance to both of these drugs. Thus, understanding tolerance-inducing alterations in GABA_AR trafficking should not only advance our understanding of the disease process that leads to alcoholism, but also improve the development of drugs to treat insomnia and anxiety disorders without causing tolerance.

Schizophrenia

Altered expression of several proteins involved in GABAergic transmission have been reported in studies of postmortem tissue from subjects with schizophrenia. Significant reductions in the mRNA levels of GAD-67 (one of the major GABA synthesizing enzymes) and the GABA membrane transporter (GAT-1) have been observed in a subpopulation of interneurons in the prefrontal cortex (PFC) of schizophrenic subjects^137,138. In addition, a compensatory upregulation of α2-containing GABA_ARs in the axon initial segment of pyramidal neurons has been demonstrated¹³⁷. Reduced GABAergic signaling between these affected interneurons and pyramidal cells has been postulated to contribute to cognitive deficits associated with schizophrenia¹⁰⁴.

The in vivo analysis of animal models will help to determine the extent to which aberrant GABAergic plasticity contributes to the pathophysiology of schizophrenia. For example, mice lacking the α3 GABA_AR subunit showed select deficits in prepulse inhibition (PPI), which could be normalized by treatment with the antipsychotic haloperidol¹³⁹. Deficits in PPI have been associated with a number of psychiatric disorders, including schizophrenia, and are a measure of a diminished ability for sensorimotor information processing¹⁴⁰. There was a dramatic loss of synaptic GABA_ARs and gephyrin clusters in the thalamic reticular nucleus¹⁴¹ (one of the main regions in the brain where the α3 subunit is normally expressed¹⁴²) of α3 subunit knockout mice, resulting in an absence of functional inhibitory receptors throughout development in this critical brain region. Deficits in PPI have also been observed in mutant mice in which there is a selective reduction in hippocampal α5-containing GABA_ARs¹⁴³. Together, these findings suggest that sensorimotor gating is highly sensitive to an imbalance in inhibitory neurotransmission, and that hypofunction of select GABA_AR populations can lead to a schizophrenia-related cognitive impairment. Pharmacological interventions to increase GABA_AR function and/or trafficking of relevant GABA_AR subpopulations may help to alleviate some of the symptoms of schizophrenia and other psychiatric disorders.

7. Conclusions and outlook

Fast inhibitory GABAergic synaptic transmission is a principal determinant of neuronal excitability. This process is dependent upon the delivery of individual GABA_AR subtypes, which are endowed with unique physiological and pharmacological properties, to their appropriate synaptic or extrasynaptic sites, where they mediate phasic and tonic inhibition, respectively.

The synthesis and assembly of GABA_ARs in the ER is an important control point in determining receptor diversity on the plasma membrane. Results from knock-out mice have illustrated preferential receptor subunit partnerships, but how this process is orchestrated remains to be determined. It is becoming apparent that subunits within the ER are subject to activity-dependent ubiquitination, which decreases their stability and half-life and limits the rate of insertion of newly synthesized receptors into the plasma membrane. It will be exciting to determine whether the various GABA_AR subunits are differentially ubiquitinated, as this may allow neuronal activity to shape the number and pharmacological properties of GABA_ARs on target cells. Modulating receptor palmitoylation, or their binding to accessory proteins as they passage through the Golgi apparatus, may further refine our understanding of how these processes shape the diversity of GABA_ARs on the plasma membrane.

GABA_ARs exhibit high rates of diffusion at the cell surface, which facilitates their delivery to synaptic sites, or entry into coated pits for removal via clathrin-dependent endocytosis. It is emerging that endocytosis is regulated via phosphorylation-dependent mechanisms; more specifically, receptor binding to clathrin-associated proteins can be negatively modulated via the phosphorylation of serine or tyrosine residues within specific GABA_AR subunits. This could allow cell-signaling pathways that regulate GABA_AR phosphorylation to also influence their cell surface stability. The relevance of these processes awaits the development of knock-in mouse lines in which the phosphorylation residues within individual AP2 binding motifs have been ablated. However, it is interesting to note that dephosphorylation of GABA_ARs and their enhanced endocytosis may be responsible for compromised synaptic inhibition during status epilepticus. Furthermore, the fate of endocytosed receptors is another determinant of steady-state cell surface expression levels. However our understanding of processes that control the recycling and lysosomal degradation of GABA_ARs remains rudimentary.

The stabilization of GABA_ARs on the plasma membrane is likely to be facilitated by multiple mechanisms. Extrasynaptic receptors mediate tonic inhibition, and the stabilization of α5-containing receptors at extrasynaptic specializations is facilitated by the actin binding protein radixin. For synaptic receptors, the multifunctional protein gephyrin is strongly implicated in stabilizing receptors containing α2 and γ2 subunits. There is also evidence that α1-containing GABA_ARs, although tightly co-localized with gephyrin, can be maintained at synaptic sites in the absence of gephyrin. Therefore, further studies are required to address the range of GABA_AR subunits that are capable of binding specific gephyrin splice variants, and the roles that these binding motifs play in the accumulation of individual subtypes at inhibitory synapses.

Resolution of these issues will provide key insights into what controls inhibitory synaptic strength and how alterations in these processes result in the development of central nervous system pathologies, ranging from epilepsy to schizophrenia. This information is also likely to lead to the identification of novel therapeutic drug targets that will allow for the pharmacological modulation of individual GABA_AR subtypes.

Glossary

BENZODIAZEPINES

Pharmacologically active molecules with sedative, anxiolytic, amnesic and anticonvulsant effects. They act by binding at the interface between α(1,2,3 or 5) and γ subunits of GABA_A receptors to potentiate the response elicited by GABA.

YEAST TWO-HYBRID SCREEN

System used to determine the existence of direct interactions between two proteins. It involves the expression of two proteins in yeast; the plasmids encoding these proteins are fused to GAL4 DNA-binding and GAL4 activation domains, respectively. If the proteins interact, the resulting complex will drive the expression of a reporter gene, commonly β-galactosidase.

GABAergic PLASTICITY

Changes in local activity that lead to longer-term increases or decreases in inhibitory synaptic strength.

UBIQUITIN-PROTEASOME SYSTEM

Ubiquitin is a 76 amino-acid protein that serves as a tag to mark proteins destined for degradation. Proteins tagged by a polyubiquitin chain are targeted to the proteasome, a large, multimeric barrel-like complex that acts by proteolysis to degrade proteins.

PALMITOYLATION

The covalent attachment of a palmitate (16-carbon saturated fatty acid) to a cysteine residue through a thioester bond.

CLATHRIN

One of the main protein components of the coats formed during membrane endocytosis.

AP2 COMPLEX

Tetrameric complex composed of subunits called adaptins that play an important role in clathrin-dependent membrane endocytosis.

RNA interference (RNAi)

A molecular method in which small interfering RNA sequences are introduced into cells or tissues, and subsequently decrease the expression of target genes.

MINIATURE INHIBITORY POSTSYNAPTIC CURRENT (mIPSC)

The postsynaptic current that results from the activation of synaptic receptors by neurotransmitters (GABA or glycine) that are usually released from a single vesicle.

TONIC INHIBITION

An inhibitory response that is mediated by the activation of extra- or peri-synaptic GABA_A receptors through ambient concentrations of GABA.

Biographies

Tija C. Jacob

Tija C. Jacob obtained her Ph.D. degree from the University of California at Berkeley in the lab of Joshua M. Kaplan, studying neurotransmitter and neuropeptide modulation of behavior in the nematode C. elegans. She was a postdoctoral fellow with Steve J. Moss at University College London, UK, and is currently continuing her training in his lab at the University of Pennsylvania, Philadelphia, USA. Her research interests focus on regulation of GABA_A receptor intracellular trafficking, cell surface mobility, and interactions with the inhibitory scaffold and how these processes are altered by disease or drug treatment.

Stephen J. Moss

Stephen J. Moss obtained his PhD in the laboratory of Eric Barnard at Imperial College London and the MRC-laboratory of Molecular Neurobiology in Cambridge. After postdoctoral training with British Biotechnology in Oxford, Richard Huganir at Johns Hopkins University and Martin Raff at University College London (UCL) he was appointed as a group leader in the MRC laboratory of Molecular Biology at UCL in 1994. In 2000 he was promoted to Professor of Molecular Pharmacology, in the Pharmacology Department at UCL. He joined the University of Pennsylvania as a Professor of Neuroscience in 2003. His research interests centre on the control of synaptic inhibition with particular emphasis on the functional modulation of metabotropic and ionotropic GABA receptors.

Rachel Jurd

Rachel Jurd obtained her Ph.D. at the Institute of Pharmacology and Toxicology, University of Zurich, Switzerland, in the laboratory of Dr. Uwe Rudolph, studying GABA_A receptors and general anaesthetic mechanisms. She then pursued postdoctoral training at the Ernest Gallo Clinic and Research Center, University of California, San Francisco, USA, before joining Dr. Stephen J. Moss’s laboratory at the University of Pennsylvania, Philadelphia, USA, in 2007. Her research focuses on the regulation of GABA_A receptor trafficking by phosphorylation, as well as on the molecular mechanisms underlying drug tolerance.