Physiol Rev 91: 1009–1022, 2011 doi:10.1152/physrev.00015.2010

THE DYNAMIC MODULATION OF GABAA TRAFFICKING AND ITS ROLE IN REGULATING THE PLASTICITY OF INHIBITORY SYNAPSES

Mansi Vithlani, Miho Terunuma, and Stephen J. Moss

Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts; and Department of Neuroscience, Physiology, and Pharmacology, University College London, London, United Kingdom

Vithlani M, Terunuma M, Moss SJ. The Dynamic Modulation of GABAA Receptor Trafficking and Its Role in Regulating the Plasticity of Inhibitory Synapses. Physiol Rev 91: 1009–1022, 2011; doi:10.1152/physrev.00015.2010.—Inhibition in the adult mammalian (CNS) is mediated by ␥-aminobutyric acid (GABA).

The fast inhibitory actions of GABA are mediated by GABA type A receptors (GABAARs); Lthey mediate both phasic and tonic inhibition in the brain and are the principle sites of action for anticonvulsant, anxiolytic, and -hypnotic agents that include , ,

neurosteroids, and some general anesthetics. GABAARs are heteropentameric ligand-gated ion channels that are found concentrated at inhibitory postsynaptic sites where they mediate phasic inhibition and at extrasynaptic sites where they mediate tonic inhibition. The efficacy of inhibition and

thus neuronal excitability is critically dependent on the accumulation of specific GABAAR subtypes at inhibitory synapses. Here we evaluate how neurons control the number of GABAARs on the neuronal plasma membrane together with their selective stabilization at synaptic sites. We then go on to examine the impact that these processes have on the strength of synaptic inhibition and behavior.

I. INTRODUCTION 1009 slow and prolonged inhibitory responses (10). GABAARs

II. GABAA RECEPTOR STRUCTURE 1010 are clinically relevant drug targets for anticonvulsant, an- III. SUBUNIT COMPOSITION OF SYNAPTIC... 1011 xiolytic, and sedative-hypnotic agents including benzodi-

IV. FUNCTIONAL SIGNIFICANCE OF GABAA... 1011 azepines, barbiturates, neurosteroids, certain classes of

V. MEMBRANE TRAFFICKING OF GABAA... 1012 general anesthetics, and alcohol. These allosteric com-

VI. EXOCYTOSIS OF GABAA RECEPTORS... 1012 pounds bind to discrete sites distinct from the agonist

VII. ENDOCYTOSIS OF GABAA RECEPTORS... 1014 binding site to either positively or negatively modulate

VIII. POSTENDOCYTIC GABAA... 1015 receptor function (69). IX. POSTSYNAPTIC TARGETING AND ... 1015

X. MODULATION OF GABAA RECEPTOR... 1017 The classic GABAAR-mediated hyperpolarization of the XI. CONCLUDING REMARKS 1019 is attributed to the direct activation of an integral and the resultant influx of chloride I. INTRODUCTION along its electrochemical gradient. The concentration-re- sponse curve exhibits positive cooperativity consistent with Inhibition in the adult mammalian central nervous system the presence of at least two GABA binding sites on the (CNS) is mediated by ␥-aminobutyric acid (GABA). It is receptor complex (5). Following brief exposure to high estimated that at least one-third of all CNS neurons utilize (millimolar) concentrations of GABA (released from pre- GABA as their primary . Most of these synaptic vesicles), activation of GABAARs located at synap- GABAergic neurons are interneurons and therefore are tic sites transiently moves the membrane potential away uniquely able to alter the excitability of local circuits within from the spike threshold required for gen- a given brain region. GABA exerts its powerful inhibitory eration underlying what is known as “phasic” inhibition. In influence by acting on two distinct classes of receptors based contrast, low (submicromolar) concentrations of ambient on their electrophysiological and pharmacological proper- GABA in the extracellular space can persistently activate ties. Ionotropic GABA type A receptors (GABAARs) are spatially and temporally less-restricted extrasynaptic recep- fast-acting ligand-gated chloride channels (69), while tors to generate a persistent or “tonic” inhibitory state (29). metabotropic GABAB receptors are coupled indirectly via G Given the critical role of GABAARs in controlling neuronal proteins to either calcium or potassium channels to produce excitability and animal behavior, it is of fundamental im-

1009 GABA TYPE A RECEPTOR TRAFFICKING

portance to understand the mechanisms used by neurons to nel (FIGURE 1), which in the case of the GABAAR is perme- regulate their function. able to chloride and, to a lesser extent, bicarbonate anions (81). Each subunit is predicted to share a common mem-

Emerging evidence indicates that activity-dependent changes brane topology encompassing a large extracellular NH2- in the number of postsynaptic GABAARs represent one of terminal ligand-binding domain and four transmembrane the most powerful mechanisms underlying the functional (TM) 1–4 domains. A major cytoplasmic loop lies between plasticity of GABAergic synapses. Moreover, deficits in the TM3 and TM4 and is the most divergent part of the se- functional expression of GABA Rs have been implicated in A quence among the GABAAR subfamily. This intracellular the pathogenesis of a wide range of neurological and psy- domain (ICD) is the target for a number of posttranslational chiatric diseases including , anxiety, depression, modifications that can directly affect channel function schizophrenia, and substance abuse. A significant amount and/or mediate the interaction with cytosolic proteins im- of research has thus focused on detailing the cellular and portant for regulating receptor localization and/or mem- molecular mechanisms that regulate the stability of brane trafficking. GABAARs on the cell surface and their accumulation at the inhibitory postsynapse. Comparative modeling of the GABAAR based on the 4-Å resolution model of the Torpedo nACh receptor for the first

II. GABAA RECEPTOR STRUCTURE time provided an insight into the three-dimensional organi- zation of this ligand-gated ion channel (27). The extracel- GABA Rs belong to the superfamily of Cys-loop ligand- lular domain of each receptor subunit comprises a variable A ␤ gated ion channels that comprises nicotinic acetylcholine NH2-terminal domain and two -folded sheets that form a receptors (nAChRs), strychnine-sensitive glycine receptors, twisted sandwich connected by a signature disulfide bridge. ϩ Ϫ and 5-hydroxytryptamine type-3 receptors (5-HT3Rs). Each subunit has a principal ( ) and a complementary ( ) Members of this family are heteropentameric glycoproteins side. The GABA binding sites are located in solvent-acces- composed of homologous subunits that specifically recog- sible pockets at the two ␤ϩ␣Ϫ subunit interfaces and that nize one another and assemble around an intrinsic ion chan- of benzodiazepines at the ␣ϩ␥Ϫ interface (26). The TM

FIGURE 1 GABAA receptor (GABAAR) structure. A: transmembrane topology of the GABAAR. Each receptor

subunit is composed of a large extracellular ligand-binding NH2-terminal region that is also the site of action of various drugs followed by four hydrophobic transmembrane TM1–4 ␣-helices and a short, barely extruding COOH terminus. Each receptor subunit also contains a large intracellular domain (ICD) between TM3 and TM4 that mediates the majority of protein-protein interactions and is subject to a number of posttranslational

modifications. B: transverse view of the assembly of GABAAR subunits to form an ion channel. TM2 faces the lumen of the channel ion pore, and TM4 is anchored in the lipid membrane. TM1 and TM3 interact with the neighboring subunits.

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domain of the receptor is made up of four loosely packed GABAAR subunits. Instead they homo- and hetero-oli- helical bundles resulting in a considerable amount of sol- gomerize with other ␳ subunits to form a family of pharma- vent-accessible space within subunits and at the subunit cologically distinct GABA-gated chloride channels that interfaces. It has been proposed that the intersubunit pock- have been termed GABAC receptors (15). ets form a continuous groove with their extracellular coun- terparts, suggesting that they may not only play a role in the In addition to molecular determination of GABAAR assem- conformational mobility of the receptor but also provide bly, subunit composition is further restricted by the spatial putative drug-binding sites. and temporal pattern of subunit expression. In situ hybrid- ization and immunohistochemical studies have demon- The subunit interfaces contain a number of amino acid strated that each one of the subunits has a distinct regional residues that have been shown to be of key importance in and cellular distribution within the brain. Moreover, differ- the binding and/or efficacy of a number of modulatory ent subunits and/or subunit combinations further dictate drugs. For example, S270 in TM2 and A291 in TM3 in the the subcellular localization of these ligand-gated chloride ␣1 subunit are of importance for the actions of volatile channels and determine their biophysical and pharmaco- anesthetics (57). Similarly, an aspartic acid residue (R265) logical properties. Detailed knowledge of the molecular in the ␤2 and ␤3 subunit isoforms is believed to account for composition and the exact anatomical expression of differ- the subtype-selectivity of etomidate (8). Photoaffinity label- ent GABAAR subtypes are therefore crucial in understand- ing has also revealed that Met-236 in the ␣ subunit (and/or ing the physiological actions of GABA within the brain and the homologous methionines in ␣2, 3, 5), in addition to for developing potentially clinically useful subtype-selective Met-286 in the ␤3 subunit, also participate in etomidate’s drugs that are devoid of the side effects associated with the action at GABAARs (50a). In addition, a single glutamine classical benzodiazepines. residue with the ␣ subunit isoforms (Q241) is critical for ␥ steroid modulation of GABAARs (34). Future experiments In the cerebellum, the 2 subunit has been shown to be a ␦ leading to the improvement in the accuracy of the models component of all postsynaptic GABAARs, whereas the will not only continue to provide a new perspective on subunit is found almost exclusively at extrasynaptic sites ␥ existing data but also pave the way for structure-based drug (58). GABAARs incorporating a 2 subunit together with design. ␣1–3 subunits (␣1–3␤2/3␥2) are thus the predominant re- ceptor subtypes responsible for mediating phasic inhibition. It is important to note, however, that these receptor sub- III. SUBUNIT COMPOSITION OF types are also abundant at perisynaptic and extrasynaptic SYNAPTIC AND EXTRASYNAPTIC sites. This is consistent with recent evidence for the dynamic GABAA RECEPTORS mobility and rapid exchange of ␥2 subunit-containing re- ceptors between extrasynaptic and synaptic receptor pools ␣ To date, 21 GABAAR subunits have been cloned and se- (36, 74). Interestingly, 5-containing GABAAR subtypes quenced from the mammalian CNS. These have been di- are found at both extrasynaptic and synaptic sites where vided into eight classes on the basis of sequence identity they contribute to both phasic and tonic inhibition (1, 19, (87): ␣(1–6), ␤(1–3), ␥(1–3), ␦, ␧, ␲, ␪, and ␳(1–3) (FIGURE 20, 66). The ␣4 and ␣6 subunits form assembled channel 1). Subunit isoforms within a single class share ϳ70% se- complexes with ␦ subunits (␣4␤␦ and ␣6␤␦) that are exclu- quence identity, but between classes this falls to 30–40%. sively extrasynaptic, accounting for tonic conductance in Moreover, alternatively spliced variants of several of these the thalamus and cerebellum, respectively (58). On the subunits have been reported, generating further subunit di- other hand, the majority of phasic signaling in the cerebel- versity and the potential for extensive molecular heteroge- lum is due largely to synaptic ␣6 subunit-containing recep- neity (70). For example, the ␥2 subunit exists in short (␥2S) tors of the ␣6␤2␥2 combination (58). and long (␥2L) forms, which differ in an eight-amino acid insert in the ICD of the ␥2L subunit (47, 86). Despite the IV. FUNCTIONAL SIGNIFICANCE OF plethora of GABAAR subunit isoforms due to the selective oligomerization mediated by receptor assembly, only a lim- GABAA RECEPTOR STRUCTURAL ited number of subunit combinations are in fact expressed HETEROGENEITY on the neuronal plasma membrane (71). The majority of ␣ ␤ ␥ GABAAR subtypes in the brain are composed of 1 2 2, While the precise subcellular localization of different ␣ ␤ ␥ ␣ ␤ ␥ followed by 2 3 2 and 3 3 2 with a likely stoichiome- GABAAR subtypes undoubtedly contributes to their partic- try of 2␣:2␤:1␥, similar to that found for nAChRs (21, 30, ipation in phasic and tonic forms of signaling, this distinc- 46, 76). To a lesser extent, ␦, ␧, and ␲ subunits replace the tion alone is not sufficient to account for their differential ␥ subunit to form -insensitive receptor sub- activation. Subunit composition is a major determinant of types, whereas the ␪ subunit has been shown to replace the the binding and gating properties of the ion channels, and ␤ subunit (71). Conversely, ␳ subunits (which are expressed hence the magnitude of the response following exposure to predominantly in the ) rarely coassemble with other ligand. Studies using recombinant GABAARs have revealed

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that sensitivity to GABA is defined by the type of ␣ subunit The diverse functions of GABA in the CNS are matched not ␣ ␤ ␦ ␣ ␤ ␦ present. Extrasynaptic 6 3 and 4 3 subunit composi- just by the heterogeneity of GABAARs but also by the com- tions display the highest affinities for GABA and synaptic plex trafficking and clustering mechanisms that generate ␣3␤3␥2 subtypes the lowest (14). For both ␣␤␥ and ␣␤␦ and maintain surface receptor populations accessible to the assemblies, the identity of the ␣ subunit also affects the rates neurotransmitter at inhibitory postsynaptic specializations of activation, deactivation, and desensitization (11, 20, 32, or extrasynaptic sites, depending on their subunit composi- 49, 53, 75). Conversely, in ␣␤3␥2 subunit-containing re- tion. The regulation of these mechanisms by GABAAR-as- ceptors, replacing the ␥2 subunit with a ␦ subunit results in sociated proteins is discussed below. a dramatic reduction in single-channel conductance inde- ␣ pendent of the type of subunit present (31). This supports V. MEMBRANE TRAFFICKING OF the notion that GABA has a high affinity but low efficacy at GABA RECEPTORS ␦ subunit-containing extrasynaptic receptors. The presence A of a ␥2or␦ subunit also influences channel kinetics: ␣␤␦ GABA Rs are not static entities on the neuronal cell surface receptors desensitize more slowly and less extensively than A but are believed to cycle continuously between the plasma ␣␤␥ receptors (12, 33, 64). Together the distinct biophysi- ␥ ␦ membrane and intracellular compartments. The relative cal properties of and subunit-containing receptors are rates of receptor exo- and endocytosis are therefore key wholly consistent with the involvement of these receptor determinants in controlling the size of the postsynaptic pool subtypes in phasic and tonic signaling, respectively. accessible to GABA and GABAergic compounds and thus

the strength of synaptic inhibition. Importantly, GABAARs Differences in subunit composition between synaptic and have recently been reported to be inserted into and removed extrasynaptic receptors are reflected in a differential mod- from the plasma membrane exclusively at extrasynaptic ulation of phasic and tonic signaling by a number of com- sites (13, 74), highlighting the importance of lateral diffu- pounds of therapeutic importance. The most frequently sion for their postsynaptic specialization. cited example of this is the role of the ␣ subunit in defining

receptor affinity for benzodiazepines. GABAARs incorpo- rating either an ␣4or␣6 subunit renders receptors insensi- VI. EXOCYTOSIS OF GABAA RECEPTORS tive to functional modulation by benzodiazepines (9), as TO THE PLASMA MEMBRANE does elimination or substitution of the ␥2 subunit. This difference can be attributed solely to the presence of a con- GABAARs can be delivered to the cell surface either as served arginine residue in ␣4 and ␣6 subunits, which in newly assembled channel complexes via a de novo secretory ␣1–3 and ␣5 subunits is a histidine (88). Thus benzodiaz- pathway or reinserted following internalization. The oli- epine site ligands selective for ␣1–3 subunits largely influ- gomerization of GABAAR subunits into channel complexes ence phasic signaling, whereas those selective for ␣5 sub- is believed to occur in the endoplasmic reticulum (ER). Ev- units are capable of primarily modulating tonic conduc- idence suggests that this assembly process plays a critical tance. role in determining the diversity of receptor subtypes ex- pressed on the neuronal plasma membrane. Proteins cannot The diverse CNS depressant effects of benzodiazepines have exit the ER until they have achieved their correctly folded ␣ conformation, and misfolded or unassembled proteins are been attributed to specific subunit types of GABAARs (61). A combined molecular genetic and pharmacological retrotranslocated from this organelle for degradation in the proteasome, restricting the number of subunit combina- approach has revealed that ␣1 subunit-containing receptors tions that can access the cell surface (42). Following correct mediate the sedative, amnesic, and, in part, anticonvulsant assembly, GABA Rs are trafficked to the Golgi apparatus actions of diazepam, whereas ␣2 subunits contribute to its A and segregated into vesicles for transport to and insertion anxiolytic and muscle-relaxant effects. Strong pharmaco- into the plasma membrane facilitated by a number of recep- logical evidence for the involvement of the ␣3 subunit in tor-associated proteins (FIGURE 2). anxiety comes from a study by Atack et al. (3a), who ␣ ␣ showed that the 3 subtype-selective inverse agonist ( 3IA) Yeast two-hybrid screens using the ␥2 subunit ICD as bait induces an anxiogenic response in rats. The ␣5 subunit has isolated the first known GABAAR associated protein been implicated in learning and memory following the ob- (GABARAP; Ref. 84), a 17-kDa cytosolic protein belonging servation that a single point mutation (H105R), which pre- to the family of membrane-associated proteins (MAPs) in- vents the interaction of this receptor subtype with diaze- volved in membrane trafficking. This includes the estrogen- pam, abolishes its memory-impairing effects (25). The de- induced protein first isolated in guinea pig endometrial cells velopment of tolerance to the sedative actions of chronic (GEC)-1 (or GABRAP like-1), Golgi-associated transport diazepam is also associated with a downregulation of ␣5 enhancer of 16 kDa (GATE-16, or GABARAP like-2), ␣ subunit-containing GABAARs to which 5-H105R mice Apg8L, and light chain (LC)-3 subunits of MAP1. Interest- are resistant (82). ingly, GABARAP knockout mice do not show differences in

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FIGURE 2 GABAAR trafficking in the secretory pathway is regulated by multiple receptor-associated proteins. GABAARs are assembled within the ER and transport competent combinations to the Golgi. Within the ER, unassembled receptor subunits are subjected to polyubiquitination that targets them for proteasomal degra- dation, a phenomenon that is dependent on the level of neuronal activity. This process is negatively regulated by Plic-1, which binds directly to receptor ␣- and ␤-subunits, prolonging their ER residence times. Within the Golgi, receptors bind to complexes of GABARAP/NSF, facilitating their transport to the plasma membrane. BIG2 is also found within the Golgi and modulates receptor forward trafficking. GODZ is a Golgi resident ␥ palmitoyltransferase that regulates palmitoylation of -subunits, a critical step in the delivery of GABAARs to the plasma membrane. Finally, PRIP proteins also play essential roles in the trafficking of GABAARs and in modulating their phosphorylation state.

␥ punctate staining of 2 subunit-containing GABAARs and GABARAP-mediated exocytosis of GABAARs is necessary lack an overt behavioral phenotype (59). GABARAP may for potentiation of inhibitory transmission by NMDA re- thus be functionally redundant. Nevertheless, the notion ceptor activation, suggesting that GABARAP may have a that GABARAP is involved in the trafficking of GABAARs role in the regulated delivery of GABAARs to the plasma to the plasma membrane (FIGURE 2) is supported by the membrane after activity rather than in the maintenance of finding that overexpression of GABARAP in heterologous basal surface receptor levels (52). expression systems and in cultured hippocampal neurons results in an increase in the cell surface expression of ␥2 Evidence in support of a function of GABARAP as a traf- subunit-containing GABAARs (50). In Xenopus oocytes ex- ficking factor includes the identification of phospholipase C ␣ ␤ ␥ pressing 1 2 2 subunit-containing GABAARs, this was (PLC)-related catalytically inactive protein (PRIP)-1, a 130- accompanied by an increase in GABAAR-mediated synaptic kDa protein that is believed to competitively inhibit the ␥ ␥ inhibition, an effect requiring the 2 subunit- and microtu- binding of the 2 subunit of GABAARs to GABARAP (39). bule-binding motifs as well as intact polymerized microtu- PRIP1 knockout mice show impairments in GABAAR mod- bules (23). More recently, it has been demonstrated that ulation by benzodiazepines and zinc sensitivity, indicating

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reduced activation of ␥2 subunit-containing receptors (39). posed that BIG2 may play a role in the transport of newly

These findings suggest that PRIP1 may play a role in the assembled GABAARs to the postsynaptic plasma membrane regulation of GABAAR trafficking by GABARAP, ensuring and also be involved in receptor recycling (67, 68). Other that only mature ␣␤␥ receptor complexes are delivered to GABAAR-associated proteins implicated in the forward the plasma membrane (FIGURE 2). In addition, PRIP1 has trafficking of GABAARs include the GABAAR interacting ␤ been shown to directly bind to the ICD of GABAAR 1–3 factor (GRIF)-1 (6) and the multifunctional protein gC1qR subunits, serving as an adaptor protein for the protein phos- (65). However, their functional significance remains un- phatase (PP)-1␣, and as such has been implicated in the clear.

phospho-dependent modulation of GABAAR functional ex- pression (73). Recently, a second PRIP isoform (PRIP2) has VII. ENDOCYTOSIS OF GABAA RECEPTORS been identified that, like PRIP1, binds both GABARAP and FROM THE PLASMA MEMBRANE PP1␣, suggesting a central role for all PRIP isoforms in GABAARs have been shown to be localized in clathrin- modulating GABAAR functional expression (80). coated pits, suggesting that they undergo clathrin-mediated endocytosis, a process that is further dependent on dynamin GABAARs also interact with the protein that links the in- tegrin-associated protein with the cytoskeleton (Plic)-1. for endocytic vesicle formation. The clathrin adaptor pro- tein (AP)-2 is a central component in the formation of these Plic1 is a 67-kDa protein with a ubiquitin-like (UBL) NH2- terminal domain and a ubiquitin-associated (UBA) COOH- vesicles, forging a link between membrane proteins and clathrin, which forms the outer layer of the coat. AP2 is a terminal domain (45). It is able to bind ubiquitin ligases and ϳ components of the proteasome. As such, it is thought to heterotetrameric complex composed of two large ( 100 kDa) ␣ and ␤2 subunits, a medium (50 kDa) ␮2 subunit, interfere with ubiquitin-dependent proteolysis of proteins and a small (19 kDa) ␴2 subunit, commonly referred to as (45). Yeast two-hybrid screens and glutathione S-trans- adaptins. ferase (GST) affinity purification assays have shown that Plic1 interacts with the ICD of ␣1–3,6 and ␤1–3 (but not ␥2 GABA Rs are intimately associated with AP2 in the brain or ␦) subunits of GABA Rs (7), indicating that Plic-1 func- A A through a direct binding of the ␤1–3 and ␥2 GABA R sub- tion may be relevant for the majority of receptor subtypes A units (41). In the GABA R ␤2 subunit, a dileucine AP2-␤2 expressed in the brain. This interaction has been demon- A adaptin-binding motif (L343L344) has been identified. This strated to be of significance in mediating the functional motif is important for clathrin-mediated endocytosis in expression of GABA Rs in human embryonic kidney A HEK-293 cells and in cortical slices (32, 33). It is also pres- (HEK-293) cells and in hippocampal slices as revealed using ent in the ICDs of receptor ␤1 and ␤3 subunits, but evidence dominant negative peptides (7). In a recent study by Saliba suggests that it is not involved in the interaction of these et al. (63), the authors showed that Plic1 increases the ac- subunits with the AP2 complex (39). In addition, an atypi- ␤ cumulation of GABAAR 3 subunits on the cell surface in a cal AP2-binding motif conserved within the ICD of all manner independent of their rates of internalization (FIG- ␤ GABAAR subunit isoforms has been identified (KTHLR URE 2). These findings suggest that Plic1 selectively modu- RRSSQLK in the ␤3 subunit) (39). This motif, which is lates the secretory pathway. In accordance with this, Plic1 enriched in lysine and arginine residues, also incorporates was found to significantly increase the half-life of polyubiq- the major sites of phosphorylation for cAMP-dependent ␤ uitinated GABAAR 3 subunits in the ER and to facilitate protein kinase (PKA) and calcium/phospholipid-dependent their insertion into the plasma membrane (63). By increas- protein kinase (PKC) within this class of receptor subunits: ing the residence times of unassembled subunits in the ER, S409 in ␤1, S410 in ␤2, and S408/9 in ␤3 (56). In vitro Plic1 may also increase subunit maturation and production binding experiments have shown that phosphorylation of heteromeric receptors. Plic1 regulation of the ubiquitin- and/or mutation of these residues confers a reduction in dependent proteasomal degradation of GABA Rs may thus ␤ ␮ A binding of the GABAAR subunits to the 2 adaptin of the provide a dynamic mechanism for regulating the efficacy of AP2 complex (FIGURE 2; Refs. 35, 39). Moreover, neurons inhibitory synaptic transmission. ␤ S408/9A expressing GABAARs incorporating fluorescent 3 subunits exhibit reduced endocytosis and enhanced func- The 190-kDa brefeldin A-inhibited guanine nucleotide ex- tional expression at both synaptic and extrasynaptic sites ␤ change factor (BIG)-2 was also identified as a GABAAR (35). Intriguingly, while analyzing the cell surface distribu- subunit interacting protein in a yeast two-hybrid screen tions of wild-type and S408/9A mutant ␤3 subunit-contain-

(22). BIG2 is concentrated mainly in the trans-Golgi net- ing GABAARs, the authors noted an apparent increase in work but is also found in vesicle-like structures along den- long, filopodia-like spines on neurons expressing the latter. drites and at the postsynaptic plasma membrane (FIGURE 2; This deficit in spine maturity was reversed by pharmacolog-

Ref. 22). Interestingly, coexpression of BIG2 with the ical blockade of GABAARs. Regulating the efficacy of syn- ␤ ␤ GABAAR 3 subunit results in an increase in 3 exit from aptic inhibition by modulating GABAAR membrane traf- the ER, suggesting that BIG2 is involved in the post-Golgi ficking may thus play a critical role in regulating spinogen-

vesicular trafficking of GABAARs (22). It is currently pro- esis and synaptic plasticity (35).

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␮ More recently, a tyrosine-based AP2- 2 adaptin-binding targeting of GABAARs to inhibitory synapses dependent on ␥ 365 367 motif in the GABAAR 2 subunit (Y GY ECL) has been the molecular motor-KIF5 (80). identified that is also conserved in the ␥1 and ␥3 subunits (40). These tyrosine residues are the principal sites for phos- IX. POSTSYNAPTIC TARGETING AND phorylation by Fyn and Src kinase (13, 36, 57). Utilizing CLUSTERING OF GABAA RECEPTORS nonphosphorylated and phosphorylated peptides corre- sponding to Y365 and Y367, the authors showed that The highly selective subcellular localization of GABAAR this high-affinity interaction is phospho-dependent (40). subtypes implies that subunit composition plays a major Introduction of the nonphosphorylated ␥2 peptide into role in the postsynaptic targeting and clustering of these neurons induced a large increase in the miniature inhib- receptors. While the exact molecular mechanisms that gov- itory postsynaptic current (mIPSC) amplitude that was ern the accumulation of GABAARs at inhibitory synapses accompanied by an increase in the number of receptors are not yet fully understood, it involves a number of recep- on the cell surface (40). tor-associated proteins and cytoskeletal elements that are concentrated at postsynaptic densities (PSDs).

The physiological significance of GABAAR endocytosis has been further evaluated by creating a knock-in mouse in The inhibitory synaptic marker gephyrin is a 93-kDa sub- which Y365 and Y367 in the ␥2 subunit have been mutated synaptic scaffolding protein that was originally implicated to phenylalanine, significantly increasing the affinity for in regulating the postsynaptic clustering of glycine receptors AP2-␮2. Homozygotes for these mutations die in utero. in the spinal cord by directly binding to the receptor ␤ Heterozygotes are viable and do not exhibit any gross subunit (55). More recently, data derived from gephyrin behavioral deficits. Consistent with the roles of Y365/7F knockout mice and knockdown experiments using anti- in mediating high-affinity binding to AP2-␮2 Y365/7F, sense oligonucleotides or shRNAi have revealed that reduc- knock-in mice have elevated total and cell surface expres- ing gephyrin expression also leads to an extensive loss in the ␥ sion levels. In the hippocampus this results in an increase punctate staining of GABAARs incorporating a 2 subunit in the size of inhibitory synapses and efficacy of synaptic together with ␣2 (but not ␣1) subunits (23, 34, 46, 51). This inhibition that is specific to CA3. This increase in inhibi- suggests the existence of both gephyrin-dependent and -in- tion correlates with a specific deficit in spatial memory in dependent GABAAR clustering mechanisms. Gephyrin clus- Y365/7F mice, a behavioral paradigm dependent on ters are absent in ␥2 subunit knockout mice. The remaining ␣ ␤ CA3. Modifying GABAAR endocytosis clearly has behav- and subunit-containing receptors show diffuse staining ioral consequences. (28). Furthermore, transfection of ␥2 subunit-deficient neu- rons with chimeric ␣2/␥2 constructs revealed that the TM4 of the ␥2 subunit is sufficient for targeting these receptors to VIII. POSTENDOCYTIC GABAA postsynaptic sites, but that both the TM4 and ICD of the ␥2 RECEPTOR SORTING subunit were necessary for recruiting gephyrin to the syn- apse and rescuing GABAergic inhibitory synaptic function The fate of internalized receptors is another determinant of sur- (2). A role for this receptor-associated protein in stabilizing

face receptor levels. Following internalization, GABAARs are ei- previously clustered GABAARs at the cell surface, rather ther rapidly recycled back to the neuronal plasma mem- than their specialization at inhibitory synapses, has thus brane or, over longer time frames, are targeted for lyso- been proposed. Concurrent with this, postsynaptic

zomal degradation, an endocytic sorting decision that is GABAARs have been shown to be three times more mobile regulated by the Huntingtin-associated protein (HAP)-1. in cells where gephyrin expression has been impaired as Yeast two-hybrid screens have revealed that HAP1 interacts measured by fluorescence recovery after photobleaching ␤ ␣ ␥ with the GABAAR 1 subunit (FIGURE 3) but not the 1, 2, (34). It is emerging that the rate of lateral mobility of ␦ or subunits (44). Overexpression of HAP1 in cultured GABAARs plays a central role in determining the number of neurons has been shown to increase the number of receptors these receptors at synaptic sites, a process revealed by the on the cell surface at steady state, an increase that correlates use of single particle tracking. This approach has illustrated with a dramatic increase in the mean amplitude of mIPSCs that there is rapid exchange between extrasynaptic and syn-

but that does not affect the frequency or kinetics of these aptic populations of GABAARs and that this process is sub- events (44). However, whether HAP1 promotes GABAAR ject to modulation via the activity of protein phosphatase recycling or prevents receptor lysozomal degradation is an 2B (PP2B). This calcium-dependent mechanism activated unresolved issue. Nevertheless, the importance of HAP1- via NMDA receptors leads to an increase in the lateral

dependent regulation of GABAAR trafficking is clear, as mobility of GABAARs and a reduction in the size of inhib- evidenced by a study in which selective suppression of hy- itory synapses, a process that favors neuronal depolariza- pothalamic HAP1 by siRNA induced a decrease in feeding tion (4). behavior in mice that was attributed to reduced surface

expression and activity of GABAARs (69). More recently it Gephyrin is believed to anchor postsynaptic GABAARs to was established that HAP1 plays a role in regulating the the plasma membrane by cross-linking the receptor mole-

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FIGURE 3 Clathrin-mediated endocytosis. The receptors cluster in specialized sites at the plasma membrane known as clathrin-coated pits, which invaginate and pinch off to form clathrin-coated vesicles (CCVs), a process that is dependent on dynamin. The clathrin adaptor protein (AP)-2 is a central component in the formation of these vesicles, forging a link between membrane proteins and clathrin that forms the outer layer of the coat. The vesicles subsequently lose their coat and fuse together to form an early endosome. Internalized receptors are then either subject to rapid recycling or are targeted for lysosomal degradation, an endocytic sorting decision that is regulated by the Huntingtin-associated protein (HAP)-1.

cule to the tubulin and actin cytoskeleton. Gephyrin binds the otherwise intracellular gephyrin, where it directly inter-

with high affinity to tubulin and as such is viewed as a bona acts with and immobilizes specific GABAAR subtypes on fide microtubule-binding protein MAP (61). Gephyrin also the plasma membrane (30, 38). In addition, the dystrophin mediates an indirect interaction with the cytoskeleton by glycoprotein complex (DGC) and neuroligin (NL)-2 bridge binding to LC1 and LC2 of the dynein motor proteins and the synaptic cleft by interacting with presynaptic ␤-neur- the microfilament-associated proteins belonging to the exin, promoting GABAergic synaptogenesis (78). Mena/VASP family (27). The membrane-associated protein collybistin II is a guanine-nucleotide exchange factor (GEF) Although gephyrin is found to be concentrated at GABAergic that binds to and so permits the subsynaptic localization of synapses in both hippocampal and cortical neurons (where

1016 Physiol Rev • VOL 91 • JULY 2011 • www.prv.org VITHLANI, TERUNUMA, AND MOSS expression is relatively low), in initial studies by the palmitoyltransferase Golgi-specific DHHC zinc fin- using standard biochemical methods it failed to copurify with ger domain protein (GODZ). Palmitoylation enhances the any of the GABAAR subunits. Hence, an intermediate linker pro- hydrophobicity of proteins and contributes to their mem- tein was postulated. Given its ability to directly bind the ␥2 subu- brane association. As such it has been shown to be involved nit ICD and gephyrin, the prime candidate was GABARAP. in the postsynaptic clustering and subcellular trafficking of However, subsequent analysis revealed that GABARAP is a number of proteins, including AMPA receptors (31) and not clustered at gephyrin-rich postsynaptic specializations the neuronal scaffold proteins PSD-95 and glutamate recep- but instead is mainly localized in intracellular compart- tor-interacting protein (GRIP)-1 (20, 74). It is unique in that ments including the ER, Golgi apparatus and, to some ex- it is the only reversible lipid modification and thus allows tent, subsynaptic secretory vesicles (43, 84). This is consis- the cell to dynamically regulate the location of specific pro- tent with a role for this protein in intracellular trafficking teins. In a yeast two-hybrid screen, the 34-kDa protein ␥ rather than postsynaptic clustering. GODZ was identified as a GABAAR 2 subunit-interacting protein that recognizes a 14-amino acid cysteine-rich do- ␥ In a recent study Tretter et al. (77) for the first time provided main conserved in the ICD of all 1–3 subunits, NH2-ter- evidence that gephyrin binds directly to the ␣2 subunit ICD minal to the GABARAP binding site (60). Analysis of Cys- in vitro. This interaction was blocked by low concentra- Ala mutant ␥2 constructs in transfected COS-7 cells re- tions of detergent, providing a possible explanation as to vealed that the ␥2 subunit is palmitoylated at all four why previous attempts to identify a direct association be- cysteines within the GODZ binding domain (60). Mutation tween gephyrin and GABAARs were unsuccessful. How- of these cysteine residues resulted in a loss of GABAAR ever, under the same conditions, only very weak binding to clusters at the cell surface, as did drug-induced global inhi- ␤3 and ␥2 subunits was observed (77), suggesting that ␣ bition of palmitoylation by Br-palmitate (60). Likewise, dis- subunit isoforms may play a predominant role in the syn- rupting GODZ function or expression levels using domi- aptic accumulation of GABAARs via their ability to directly nant negative or RNAi approaches resulted in a significant bind gephyrin. reduction in the amplitude of mIPSCs attributed to a de-

crease in postsynaptic GABAAR number (24). From these The 81-kDa actin-binding protein radixin, a member of the studies it is evident that palmitoylation can dynamically ezrin/radixin/moesin protein family, has recently been regulate the efficacy of neuronal inhibition by controlling ␣ shown to directly link the 5 subunit to the cytoskeleton via the accumulation of GABAARs at the postsynaptic mem- a radixin-binding motif conserved within the ICD of ␣1–3 brane, although the exact mechanism by which this is and ␣5 subunits, differing only by two amino acids in the ␣2 achieved is unknown. subunit (52). In neurons, both depletion of radixin and replacement of the radixin actin-binding motif dramatically ␣ B. Ubiquitination decreased 5 subunit-containing GABAAR clusters without altering total surface expression levels. Radixin also showed limited colocalization with postsynaptic gephyrin, The regulation of GABAAR trafficking by the ubiquitin- ␣ consistent with previous reports that GABAAR 5 subunits related protein Plic1 suggests that GABAARs may also be localize mainly at extrasynaptic sites (14, 52). However, the direct targets for modification by the polypeptide ubiquitin. mechanisms responsible for the formation of radixin-de- The covalent attachment of one or more copies of the 76- pendent extrasynaptic GABAAR clusters remain to be elu- amino acid ubiquitin monomer to lysine residues of target cidated. proteins is referred to as ubiquitination. Monoubiquitina- tion is reversible and serves as an active signal in diverse intracellular trafficking pathways, including as a trigger for X. MODULATION OF GABA RECEPTOR A endocytosis. In contrast, polyubiquitination is required for FUNCTION BY POSTTRANSLATIONAL the translocation of proteins from the ER back into the MODIFICATIONS cytosol, where they are degraded by the proteasome. Activ- ity-dependent polyubiquitination of GABA R ␤3 subunits The cell surface stability of GABA Rs is further regulated A A has been shown to reduce the stability of newly translated by posttranslational modifications such as palmitoylation, and assembled receptors in the ER via a mechanism depen- ubiquitination, and phosphorylation. These modifications dent on the activity of the proteasome (62). have been implicated in altering the biophysical and pharmacological properties of these ligand-gated ion Coincident with a loss of cell surface expression levels, channels (3). chronic blockade of neuronal activity by tetrodotoxin (TTX) treatment reduced both the amplitude and frequency A. Palmitoylation of mIPSCs (62). TTX had no effect on the enhanced func- ␤ tional expression of GABAARs incorporating 3 subunits Palmitoylation is the covalent attachment of the saturated in which all 12 lysine residues within the ICD of this subunit fatty acid palmitate to cysteine residues of a given protein had been mutated to arginines (␤3K12R). These mutations

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did not alter GABAAR cell surface half-life or internaliza- combined with site-directed mutagenesis revealed that the tion rates but did significantly enhance receptor insertion consensus motif for PKA-induced phosphorylation con- ␤ into the plasma membrane (62). served within the ICDs of GABAAR 1–3 subunits is RRRXSQLK, where S is a serine at position 409 in ␤1 and ␤3 subunits and at position 410 in the ␤2 subunit (55) and C. Phosphorylation X represents either an alanine residue in ␤1 and ␤2 subunits or a serine residue at position 408 in the ␤3 subunit. How- Protein phosphorylation is fundamental to the activity of ever, in contrast to in vitro findings, analysis of recombinant cellular signaling networks. It is achieved through protein GABAARs in HEK-293 cells has demonstrated that PKA- kinase-catalyzed transfer of a phosphate group to serine, ␤ threonine, and/or tyrosine residues of a given protein sub- induced phosphorylation of the 3 subunit occurs at both ␤ strate that can be reversed in a dephosphorylation reaction S408 and S409 (rather than at S409 alone), whereas the 2 catalyzed by protein phosphatases. GABA Rs are well-es- subunit is not a substrate for this kinase in heterologous A expression systems (54). Interestingly, PKA was shown to tablished phosphoproteins. Diverse studies on GABAAR phosphorylation have implicated this process in altering depress GABA-activated currents in HEK-293 cells express- ␤ ␤ channel gating, conductance and/or kinetics, sensitivity of ing 1 subunit-containing GABAARs, whereas in 3 sub- the receptors to pharmacological agents, protein-protein unit-expressing cells, a potentiation was observed (54). The interactions, and membrane trafficking (83). Hence, the co- latter was attributed to the presence of two juxtaposed ␤ ordinated activity of kinases and phosphatases plays a piv- serine residues (S408 and S409) in 3, as selective muta- otal role in controlling neuronal excitability. tion of S408 to alanine (to structurally resemble the ␤ GABAAR 1 subunit at this site) converted this potenti- Studies in heterologous expression systems have demon- ation to a depression (54). On the other hand, PKA had no effect on ␤2 subunit-expressing cells (54), which can strated that GABAAR function, depending on the subtype analyzed, can be differentially modulated by phosphoryla- be accounted for by the selective recruitment of PKA to ␤ ␤ ␤ tion of key residues within the ICDs of receptor ␤1–3 and GABAAR 1 and 3, but not 2, subunits via the A-ki- ␥2 subunits by a number of kinases, including PKA, PKC, nase anchoring protein (AKAP) of 79 (rat)/150 (human) calcium/calmodulin-dependent kinase II (CaMKII), protein kDa (18). kinase B (PKB; also known as Akt), cGMP-dependent pro- tein kinase (PKG), and tyrosine kinases of the Src family AKAPs have also been shown to interact directly with PP2B (summarized in TABLE 1). This is best illustrated by the and PKC in addition to PKA (24, 44); however, the target-

differential modulation of GABAAR subtypes by PKA, de- ing of PP2B and PKC to GABAARs by AKAP remains to be pending on the identity of the ␤ subunit. In vitro studies investigated. Interestingly, PP2B has been shown to bind using purified bacterially expressed GST fusion proteins directly to the ICD of the ␥2 subunit, thereby dephospho-

Table 1 GABAAR phosphorylation sites

Protein Kinase Phosphorylation Heterologous cell Subunit Site In vitro lines Primary neurons

␤1 S384 CaMKII S409 PKA, PKC, CaMKII, PKG PKA,PKC ␤2 S410 PKA, PKC, Akt, CaMKII, PKG PKC, Akt Akt ␤3 S383 CaMKII S408 PKC PKA,PKC PKA,PKC S409 PKA, PKC, CaMKII, PKG PKA,PKC PKA,PKC ␥2 S327 PKC S343 PKC, CaMKII S348 CaMKII T350 CaMKII Y365 Src Src Y367 Src Src

PKA, cAMP-dependent protein kinase; PKC, calcium/phospholipid-dependent protein kinase; CaMKII, calcium/ calmodulin-dependent kinase II; PKB, protein kinase B (also known as Akt); PKG, cGMP-dependent protein kinase; Src, and tyrosine kinases of the Src family. [Adapted from Brandon et al. (18).]

1018 Physiol Rev • VOL 91 • JULY 2011 • www.prv.org VITHLANI, TERUNUMA, AND MOSS rylating the receptor and inducing long-term depression whole cell GABA-activated currents recorded from rat cul- (LTD) of synaptic inhibition in the CA1 region of the hip- tured cerebellar granule neurons and from recombinant pocampus, a mechanism that is believed to contribute to GABAARs expressed in neuroblastoma-glioma hybrid learning and memory (85). PKC is recruited to the GABAAR (NG108–15) cells. These findings imply the contribution of via the anchoring protein receptor for activated C kinase some essential neuronal factor (35). (RACK)-1. Like PKA, it has different effects on receptor ␤ function depending on subunit composition. In agreement In addition to binding PKC and the GABAAR subunits, with the findings in vitro, all of the ␤ subunit isoforms were RACK1 also binds the tyrosine kinase Src to facilitate the found to be a substrate for this kinase in HEK-293 cells (17, phosphorylation of the ␥2 subunit on Y365 and Y367 (16, 48, 54). Functional studies employing site-directed mu- 43, 56). Coexpression of Src with recombinant ␣1␤1␥2 tagenesis have demonstrated a downregulation of receptor subunit-containing GABAARs has been shown to increase activity in response to phorbol esters mediated by PKC- whole cell GABA-activated currents in HEK-293 cells, an induced phosphorylation of ␤1 S409, ␤2 S410, ␥2S S327, effect abolished by site-specific mutagenesis of both of these and ␥2L S327/343. However, the use of a constitutively tyrosine residues to phenylalanines (56). Interestingly, mu- active catalytic domain of PKC (PKM) produced results tation of these sites led to increased phosphorylation of the ␤ contradicting those obtained with phorbol esters. In trans- 1 subunit at Y384 and Y386, a subunit that exhibits rela- fected mouse fibroblasts, PKM enhanced the response to tively low stoichiometry of phosphorylation in response to GABA in a manner that was dependent on the phosphory- Src compared with wild-type control (56). In primary neu- lation of the ␤1 subunit at S409 and the ␥2L subunit at S327 ronal cultures, intracellular application of sodium vana- and S343 (51). The reason for this discrepancy remains date, a potent tyrosine phosphatase inhibitor, enhanced unresolved, but it has been speculated that different PKC benzodiazepine-sensitive GABAAR function, suggesting isoforms may produce different responses (72). In contrast, high endogenous tyrosine kinase and phosphatase activity brain-derived neurotrophic factor (BDNF)-induced PKC- under basal conditions (56). Consistent with this, studies mediated phosphorylation of the ␤3 subunit has been using phospho-antibodies have revealed that Y365/7 are principally phosphorylated via the activity of Fyn and Src. shown to transiently increase the amplitude of mIPSCs in Significantly, Fyn is able to bind specifically to phospho- cultured neurons, an effect paralleled by an increase in Y367, providing a mechanism for the recruitment of this GABA R cell surface stability (37). Furthermore, the initial A tyrosine kinase to GABA Rs (16, 38). Studies with phos- enhancement observed in receptor activity was followed by A pho-antibodies have revealed significant variations in the a prolonged depression that was attributed to PP2A-medi- stoichiometry of Y367 phosphorylation in the brains of ated dephosphorylation of the ␤3 subunit (37). rodents, suggesting input-specific control of phosphoryla- tion; however, the signaling pathways that regulate the It is of importance to note that activation of PKC can have phosphorylation of the ␥2 subunit remain to be defined. In varying effects on GABA R functional expression. This di- A keeping with their roles in mediating high-affinity binding versity of effects may result from variations in the experi- to AP2, phosphorylation of Y365/7 increases the cell sur- mental protocols used: for example, variations in tempera- face stability of GABAARs and enhances the amplitude and ture, the identity of experimental system used, or mecha- frequency of mIPSCs (40, 79). nism of kinase activation. In addition, the mechanisms underlying the effect of PKC activation may be distinct, ranging from direct effects dependent on direct receptor XI. CONCLUDING REMARKS phosphorylation to receptor-independent effects mediated via the phosphorylation of other substrates or the direct GABAARs play a central role in mediating neuronal inhibi- effects of phorbol esters on membrane trafficking. Clearly tion and mediating the effects of a broad range of anticonvul- further experimentation is required to address these issues. sant, anxiolytic, and sedative-hypnotic agents. GABAARs medi- ate both phasic and tonic modes of inhibition, phenomena No adaptor protein is known for CaMKII, although the that are mediated via receptor subtypes with distinct molec- kinase is capable of coimmunoprecipitating with GABAARs ular structures. Phasic inhibition is mediated largely by ben- from detergent-soluble mouse brain extracts (K. McAinsh zodiazepine-sensitive receptor subtypes that are highly en- and S.J. Moss, unpublished data). In vitro studies have re- riched at synaptic sites and are principally assembled from vealed that the serine/threonine kinase CaMKII directly phos- ␣1–3, ␤1–3, and ␥2 subunits. A critical determinant of re- ␤ phorylates specific residues within the ICDs of GABAAR and ceptor number at inhibitory synapses is the steady-state cell ␥2 subunits. These include S384/409 in ␤1, S410 in ␤2, and levels of these receptors on the neuronal plasma membrane. S383/409 in ␤3, as well as the ␥2 subunit at S343 (␥2L This is likely determined by the relative rates of receptor only), S348, and T350. Interestingly, the intracellular ap- exo- and endocytosis, processes that are intimately con- plication of purified, active CaMKII failed to modulate the trolled by covalent modifications and interaction with spe- function of GABAARs heterologously expressed in HEK- cific binding partners. While the molecular details of these 293 cells but significantly potentiated the amplitudes of processes are being evaluated, their significance for the ef-

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ficacy of neuronal inhibition and ultimately for behavior 5. Baumann SW, Baur R, Sigel E. Individual properties of the two functional agonist sites in remains to be established. Our comprehension of how GABA(A) receptors. J Neurosci 23: 11158–11166, 2003.

GABAARs are selectively stabilized at inhibitory synapses is 6. Beck M, Brickley K, Wilkinson HL, Sharma S, Smith M, Chazot PL, Pollard S, Stephen- limited; however, a central role for gephyrin is emerging. son FA. Identification, molecular cloning, and characterization of a novel GABAA re- ceptor-associated protein, GRIF-1. J Biol Chem 277: 30079–30090, 2002. The direct binding of gephyrin to the GABAAR subtypes containing ␣2 subunits has recently been shown (77). The 7. Bedford FK, Kittler JT, Muller E, Thomas P, Uren JM, Merlo D, Wisden W, Triller A, Smart TG, Moss SJ. GABA(A) receptor cell surface number and subunit stability are significance of this interaction for GABAAR subtypes con- regulated by the ubiquitin-like protein Plic-1. Nat Neurosci 4: 908–916, 2001. taining other ␣-subunit isoforms warrants further investi- gation. Gephyrin-independent clustering mechanisms have 8. Belelli D, Lambert JJ, Peters JA, Wafford K, Whiting PJ. The interaction of the general also been postulated, in particular for subtypes containing anesthetic etomidate with the gamma-aminobutyric acid type A receptor is influenced by a single amino acid. Proc Natl Acad Sci USA 94: 11031–11036, 1997. ␣5 subunits. The importance of these emerging processes for other receptor subtypes remains to be demonstrated. 9. Benson JA, Low K, Keist R, Mohler H, Rudolph U. Pharmacology of recombinant gamma-aminobutyric acidA receptors rendered diazepam-insensitive by point-mu- tated alpha-subunits. FEBS Lett 431: 400–404, 1998. The cellular mechanisms that regulate the cell surface accumulation are under active investigation. Future stud- 10. Bettler B, Tiao JY. Molecular diversity, trafficking and subcellular localization of GABAB ies to ascertain the significance of these molecular mech- receptors. Pharmacol Ther 110: 533–543, 2006. anisms in determining the efficacy of neuronal inhibition 11. Bianchi MT, Haas KF, Macdonald RL. Alpha1 and alpha6 subunits specify distinct de- under both normal and pathological conditions are es- sensitization, deactivation and neurosteroid modulation of GABA(A) receptors con- sential. taining the delta subunit. Neuropharmacology 43: 492–502, 2002. 12. Bianchi MT, Macdonald RL. Slow phases of GABA(A) receptor desensitization: struc- ACKNOWLEDGMENTS tural determinants and possible relevance for synaptic function. J Physiol 544: 3–18, 2002.

Address for reprint requests and other correspondence: S. 13. Bogdanov Y, Michels G, Armstrong-Gold C, Haydon PG, Lindstrom J, Pangalos M, Moss, Tufts University, 136 Harrison Ave., Arnold 207, Moss SJ. Synaptic GABAA receptors are directly recruited from their extrasynaptic counterparts. EMBO J 25: 4381–4389, 2006. Boston, MA 02111 (e-mail: [email protected]). 14. Bohme I, Rabe H, Luddens H. Four amino acids in the alpha subunits determine the GRANTS gamma-aminobutyric acid sensitivities of GABAA receptor subtypes. J Biol Chem 279: 35193–35200, 2004.

This work was supported by National Institute of Neurological 15. Bormann J. The “ABC” of GABA receptors. Trends Pharmacol Sci 21: 16–19, 2000. Disorders and Stroke Grants R01NS047478, R01NS048045, 16. Brandon NJ, Delmas P, Hill J, Smart TG, Moss SJ. Constitutive tyrosine phosphorylation R01NS051195, R01NS056359 and R01NS054900 (to S. J. of the GABA(A) receptor gamma 2 subunit in rat brain. Neuropharmacology 41: 745– Moss) and by the Wellcome Trust. M. Terunuma is supported by 752, 2001. a National American Heart Association Senior Scientist Award. 17. Brandon NJ, Delmas P, Kittler JT, McDonald BJ, Sieghart W, Brown DA, Smart TG,

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