Regulation of GABAA receptor membrane trafficking and synaptic localization

Pharmacology & Therapeutics 123 (2009) 17–31

Contents lists available at ScienceDirect

Pharmacology & Therapeutics j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h a r m t h e r a

Associate editor: K.W. Roche

Regulation of GABAA receptor membrane trafficking and synaptic localization I. Lorena Arancibia-Cárcamo, Josef T. Kittler ⁎ Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London, WC1E 6BT, UK

a r t i c l e

i n f o

Keywords: Endocytosis Epilepsy Phosphorylation Ubiquitination Palmitoylation Inhibition

a b s t r a c t Synaptic inhibition plays a key role in regulating neuronal excitability and information processing in the brain. The strength of synaptic inhibition is therefore an important determinant of both cellular and network activity levels in the central nervous system (CNS). γ-aminobutyric acid type A (GABAA) receptors are the major sites for fast inhibitory neurotransmission in the CNS and alterations in their trafficking, synaptic accumulation and function play a key role in regulating neuronal excitability. Synaptic receptor number is determined by the trafficking of GABAA receptors to and away from inhibitory synapses and by their stability and localization at the inhibitory postsynaptic domain. Here we discuss advances that have led to an improved understanding of the mechanisms that regulate the delivery and stabilization of GABAA receptors at inhibitory synapses and address the role of GABAA receptor trafficking, GABAA receptor associated proteins and post-translational modifications in regulating this process. © 2009 Elsevier Inc. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of the assembly, localisation, membrane trafficking and membrane dynamics of GABAA receptors and the role of GABAA receptor associated proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Regulation of GABAA receptor trafficking and synaptic receptor number by receptor post-translational modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. The inhibitory postsynaptic domain and its role in regulating the membrane stability and subcellular localization of GABAA receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Cell adhesion complexes at the inhibitory synapse . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Diffusion properties of cell surface GABAA receptors . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The majority of fast inhibitory neurotransmission in the central nervous system (CNS) is mediated by the activation of GABAA receptors, while the structurally unrelated GABAB receptors (which are G protein coupled receptors) mediate a slower component of GABA action (for a detailed review of GABAB receptor function see Bettler & Tiao, 2006). GABAA receptors are hetero-pentameric ligand gated ion channels and are members of the ‘Cys-loop’ ligand gated ion Abbreviations: ER, Endoplasmic reticulum; CNS, central nervous system; GABA, gamma aminobutyric acid; GABAA, GABA type A; TM, trans-membrane domain; Y2H, yeast two hybrid; UBL, ubiquitin like; UBA, ubiquitin associated; BIG2, Brefeldin A inhibited GDP/GTP exchange factor 2; GABARAP, GABAA receptor associated protein; NSF, N-ethymalemide sensitive factor; PKC, protein kinase C; BDNF, brain derived neurotrophic factor. ⁎ Corresponding author. E-mail address: [email protected] (J.T. Kittler). 0163-7258/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2009.03.012

.

17

.

17 19

.

17 22

. . . . .

17 23 25 17 27 17 27 17 19 27

channel family of receptors, which also include the closely related GABAC receptors (which mediate fast GABAergic transmission in the retina) in addition to nicotinic acetylcholine receptors (nACh receptors), glycine receptors and serotonin (5HT3 ) receptors (reviewed in Connolly & Wafford, 2004). Activation of GABAA receptor leads to the opening of an integral channel permeable to chloride and bicarbonate ions. When chloride is low intracellularly (as in most adult neurons), this results in a rapid chloride influx into the cell, resulting in cell hyperpolarisation, moving the membrane potential away from the spike threshold for action potential generation. Thus, GABAA receptors play a key role in regulating cell and network activity in the brain. In addition, GABAA receptors are drug targets for a number of naturally occurring and synthetic compounds. A range of compounds, several of which are clinically relevant therapeutic agents, including ethanol, benzodiazepines, barbiturates and some anaesthetics, can allosterically modulate GABAA receptors (for reviews see Macdonald & Olsen, 1994; Franks, 2008; Mody, 2008). GABAA

18

I.L. Arancibia-Cárcamo, J.T. Kittler / Pharmacology & Therapeutics 123 (2009) 17–31

Fig. 1. GABAA receptor trafficking and associated proteins. GABAA receptors are assembled from individual subunits in the ER where the chaperones BiP and Calnexin assist in quality control. Unassembled GABAA receptor subunits that are to be targeted for ER associated degradation are ubiquitinated and degraded in the proteasome. Plic can interact with GABAA receptors thereby inhibiting their targeting for proteasomal degradation. Assembled pentameric GABAA receptors exit the ER and bind the guanidine exchange factor BIG2 in the Golgi. Here they also interact with the palmitoylase transferase GODZ and GABARAP. GABARAP interacts with NSF, as does the GABAA receptor β subunit, and this association may facilitate transport of the receptor complexes to the cell surface. GABAA receptors are inserted at extrasynaptic sites and can diffuse along the plasma membrane in and out of synaptic domains. At synapses they are stabilized by an interaction with the scaffolding protein Gephyrin. The interaction of the GABAA receptor intracellular loops with the μ2 subunit of AP2 is important for GABAA receptor internalization. GABAA receptors are delivered by a clathrin mediated pathway to early endosomes where they can be targeted for degradation in the lysosome or for recycling upon binding of Huntington associated protein (HAP1).

I.L. Arancibia-Cárcamo, J.T. Kittler / Pharmacology & Therapeutics 123 (2009) 17–31

receptors are also regulated by several endogenous modulators including neurosteroids, protons and Zn2+ (Macdonald & Olsen, 1994; Belelli & Lambert, 2005; Hosie et al., 2007). Like other members of the Cys-loop receptor family of ligand gated ion channels, GABAA receptors are polytopic type I membrane proteins. Each subunit shares a common subunit structure comprising a large extended N-terminal domain with potential glycosylation sites, four highly conserved transmembrane domains (TM1–4) and a large intracellular loop domain between TM3 and TM4 which protrudes into the cytoplasm (Connolly & Wafford, 2004). Electron microscopic and biochemical studies of the nACh receptor, support a model whereby members of this family are pentameric in structure where subunits are arranged around a central aqueous pore (Unwin, 1993). It is assumed that for all Cys-loop receptors, the subunit's α-helical TM2 domain lines the central water-filled pore, while TM1, TM3, and TM4 form the interface with lipids and isolate TM2 from a hydrophobic environment (Connolly & Wafford, 2004). The crystal structure of the soluble acetylcholine-binding protein, which shares approximately 20% sequence homology with nACh, GABAA/C and glycine receptors, suggests that the N-terminal domain of the Cys-loop receptor family consists of an alpha helix and a sandwich of antiparallel β sheets with conserved residues positioned in order to stabilize the protomers, whereas residues at their interfaces are highly variable (Brejc et al., 2001; Smit et al., 2001). The acetylcholine-binding protein structure has further contributed to our understanding of the structure– function parameters for Cys loop receptor of the family (Brejc et al., 2001; Smit et al., 2001), for example improving our understanding of binding of ligands and modulators of GABAA receptor function (for

19

example Zn2+ and neurosteroid binding) (Absalom et al., 2003; Chen et al., 2004; Smart et al., 2004; Hosie et al., 2006; Hosie et al., 2007). Sequences within the N-termini of GABAA receptor subunits are the primary sites for inter-subunit contact and oligomerisation. In contrast, the intracellular domain between TM domains 3 and 4 of GABAA receptor subunits is the main location for protein–protein interactions between the receptor and regulatory proteins in the cytosol important for regulating GABAA receptor activity, trafficking and localization. Furthermore, subunit intracellular domains are also a key site for receptor regulation by post-translational modifications of the receptor including palmitoylation, ubiquitination and phosphorylation (Kittler & Moss, 2003; Jacob et al., 2008). 2. Regulation of the assembly, localisation, membrane trafficking and membrane dynamics of GABA A receptors and the role of GABAA receptor associated proteins It has become clear over the last few years that GABAA receptors can be viewed as relatively dynamic entities. The number of synaptic GABAA receptors is in part determined by the exchange of synaptic and extrasynaptic surface receptors while the number of surface receptors is determined by the rates of receptor assembly, maturation through the secretory pathway and plasma membrane insertion, in addition to the rates of receptor cycling between surface and intracellular compartments (Fig. 1). Similarly the membrane dynamics of surface receptors are determined by the relationship of the receptors with inhibitory synaptic scaffold proteins, adhesion proteins and other resident components of the inhibitory synaptic domain (Fig. 2).

Fig. 2. The inhibitory postsynaptic domain. Adhesion molecules including neurexin–neuroligin (NL) 2/3 complexes and the dystrophin glycoprotein complex are found enriched at inhibitory synapses where they are thought to be of major importance in the specification and validation of inhibitory synapses and the correct apposition of inhibitory presynaptic terminals. GABAA receptors (blue) are confined to inhibitory synapses by scaffold molecules such as gephyrin (slate). Gephyrin can interact with a number of proteins enriched at inhibitory post-synaptic domains as well as cytoskeletal elements such as tubulin and actin. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

20

I.L. Arancibia-Cárcamo, J.T. Kittler / Pharmacology & Therapeutics 123 (2009) 17–31

Several proteins have been demonstrated to interact directly with GABAA receptors via the intracellular domain and TM4. The membrane trafficking of GABAA receptors appears to be tightly regulated by these GABAA receptor associated proteins (Fig. 1). In contrast to several of the inhibitory postsynaptic domain constituents which are highly enriched at inhibitory synapses, many of the GABAA receptor associated proteins so far identified are only marginally localized to synaptic sites. Instead they are thought to be important for receptor transport and maturation through the secretory pathway or for regulating endocytosis, post-endocytic sorting and recycling of receptors (Fig. 1). 2.1. GABAA receptor associated proteins and receptor transport, maturation and stability in the secretory pathway A total of 16 genes encoding GABAA receptor subunits have been identified in the mammalian nervous system based on amino acid sequence homology: six α subunits, three β subunits, three γ subunits, in addition to δ, ε, π and θ (reviewed in Macdonald & Olsen, 1994; Moss & Smart, 2001; Connolly & Wafford, 2004). Despite the potential for a bewildering heterogeneity of receptor structure, a number of studies have revealed that only a limited number of receptor subunit combinations may exist on the neuronal cell surface (Moss & Smart, 2001; Sieghart & Sperk, 2002). In the brain, GABAA receptor molecular heterogeneity is in part restricted by regional and temporal selectivity in subunit expression (for reviews see Fritschy & Mohler, 1995; Pirker et al., 2000; Sieghart & Sperk, 2002). The heterogeneity of native GABAA receptors is further restricted by a number of assembly rules (Connolly & Wafford, 2004). Receptor oligomerisation occurs within the endoplasmic reticulum (ER) with assembly domains within subunit N-termini of α, β and γ mediating the oligomerisation of these subunits into heteromeric channels (reviewed in Bollan et al., 2003; Connolly & Wafford, 2004). Expression studies in cell lines have revealed that individual GABAA receptor subunits are mostly retained in the ER and are rapidly degraded when expressed alone. Co-expression of α and β subunits results in the production of functional GABA gated chloride channels (sensitive to picrotoxin, bicuculline, barbiturates and Zn2+) in agreement with the binding site for GABA lying at the interface of α and β subunits (Connolly & Wafford, 2004). The majority of native receptors also contain a γ2 subunit, which confers benzodiazepine sensitivity and zinc insensitivity and is critical for synaptic targeting (Kittler & Moss, 2001; Bollan et al., 2003). The above results together with biochemical experiments suggest that the most prevalent GABAA receptor subunit composition in the brain consists of α, β and γ subunits with a majority of receptors containing 2α, 2β and 1γ subunit isoform (Moss & Smart, 2001; Bollan et al., 2003; Fritschy & Brunig, 2003; Luscher & Keller, 2004). A number of receptor-associated proteins have been implicated in the GABAA receptor cell surface transport and maturation through the secretory pathway. GABAA receptor subunits associate with BiP and Calnexin in the ER, two chaperone molecules that assist in protein quality control in the ER (Connolly et al., 1996; Gorrie et al., 1997; Kleizen & Braakman, 2004). Interestingly, the interaction with calnexin is increased in GABAA receptor subunit mutations that lead to familial epilepsy syndromes (Bradley et al., 2008). Other proteins that are also implicated in GABAA receptor subunit stability at the ER include the ubiquitin related protein Plic-1 (also called ubiquilin). Plic-1 was identified to interact with GABAA receptors from yeast two hybrid (Y2H) screens with receptor α and β subunit intracellular domains and is implicated in receptor transport and maturation through the secretory pathway (Bedford et al., 2001). Plic-1 contains a ubiquitin like (UBL) domain at its N-terminus and a ubiquitin associated domain (UBA) at its C-terminus (Kleijnen et al., 2000). Plic-1 interacts with all GABAA receptor α (1–6) and β (1–3) subunit intracellular domains via its UBA domain (Bedford et al., 2001).

Immunofluorescence and immunoelectron microscopic studies demonstrated that Plic-1 is mainly expressed in intracellular compartments where it colocalises with GABAA receptors at subsynaptic membranes (Bedford et al., 2001). Blockade of the interaction between Plic-1 and GABAA receptors using interfering peptides results in reduced GABAA receptor cell surface expression whereas Plic-1 overexpression results in an increased GABAA receptor surface expression with no effect on receptor internalization rates (Bedford et al., 2001; Saliba et al., 2008). Plic-1 proteins regulate ubiquitin dependent protein degradation in the proteasome by their ability to bind ubiquitin ligases and components of the proteasome (Kleijnen et al., 2000, 2003) suggesting that Plic-1 modulates GABAA receptor surface numbers by inhibiting proteasomal degradation of the receptor (see also ubiquitination of GABAA receptors section below). In agreement with this, Plic-1 increases both the stability of ER resident GABAA receptors and the number of poly-ubiquitinated GABAA receptor subunits (Bedford et al., 2001; Saliba et al., 2008). Plic-1 also selectively increases the rates of receptor insertion into the surface membrane (Saliba et al., 2008). Several additional Plic family members, Plic-2–Plic-4, in addition to Plic-1, have been identified. Plic-2 has recently been demonstrated to be a negative regulator of G protein-coupled receptor endocytosis (while Plic-1 was not), which is dependent on the Plic-2 UBL domain (N'Diaye et al., 2008). Via their UBL domains, Plic proteins can interact with UIM containing endocytic adaptors such as eps15 and epsin, with preferential binding by Plic-2 over Plic-1 (Regan-Klapisz et al., 2005; Heir et al., 2006; N'Diaye et al., 2008). Plic-2 can also interact with GABAA receptors (Bedford et al., 2001), although whether Plic-2 (or other Plic members) also regulate GABAA receptor function remains to be determined. Similarly, whether there is a role for Plic-2 and eps15/epsin in regulating GABAA receptor endocytosis in addition to receptor surface insertion remains unknown. Interestingly Plic proteins have also more recently been implicated in the degradation of nicotinic acetylcholine receptor α3 subunits (Ficklin et al., 2005) which are also members of the Cys loop family of ligand gated ion channels, suggesting that the Plic protein family may have a general role in regulating the trafficking and stability of ligand gated ion channels. The guanine nucleotide exchange factor, brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2) has also been implicated in receptor transport and maturation through the secretory pathway. BIG2 can catalyse the GDP–GTP exchange on ARF GTPases, and was identified (from a Y2H screen) to interact with GABAA receptor β(1–3) subunit intracellular domains (Charych et al., 2004b). BIG2 is primarily localized to the trans Golgi network, where it co-localises with GABAA receptors and has also been localised in vesicle-like structures along dendrites and near post-synaptic sites (Charych et al., 2004b). Interestingly, co-expression of BIG2 with the GABAA receptor β3 subunit results in an increase in β3 exit from the ER, suggesting that BIG2 may be involved in the post-Golgi vesicular trafficking of GABAA receptors (Charych et al., 2004b). BIG2 has also been demonstrated to play a role in the post-endocytic sorting of transferrin receptor (Shen et al., 2006) suggesting that BIG2 could also be similarly involved in the endocytosis or endocytic sorting of GABAA receptors, although this has not yet been demonstrated. A number of proteins have been identified to interact directly with GABAA receptor γ-subunits and are also suggested to be important for the surface transport of GABAA receptors and their targeting to synapses. The 17 kDa GABAA receptor associated protein (GABARAP) interacts with GABAA receptor γ subunits both in vitro and in vivo (Wang et al., 1999) and is a member of a family of homologous small microtubule binding proteins that also includes GABARAP like 1 (GBRL1, GEC1) GABARAP like 2 (GBRL2, GATE-16) and the light chain3A and B (LC3A, LC3B) subunits of MAP-1A and 1B. Immunocytochemical and immunoelectron microscopy studies have revealed that GABARAP colocalizes with GABAA receptors primarily at the Golgi compartment and in subsynaptic cisternae (Wang et al., 1999; Kittler

I.L. Arancibia-Cárcamo, J.T. Kittler / Pharmacology & Therapeutics 123 (2009) 17–31

et al., 2001). Immunofluorescence and electrophysiological studies demonstrate that GABARAP is important for the membrane trafficking of GABAA receptors to the plasma membrane and for inhibitory plasticity in the cerebellum (Leil et al., 2004; Boileau et al., 2005; Chen et al., 2005, 2007; Kawaguchi & Hirano, 2007). Coexpression of GABARAP and GABAA receptors in COS-7 cells and neurons results in an increase in the levels of GABAA receptors expressed at the cell surface as well as an increase of GABA mediated currents in oocytes (Leil et al., 2004; Boileau et al., 2005; Chen et al., 2005, 2007). In addition, it has also been suggested that GABARAP can regulate receptor kinetic properties (Everitt et al., 2004; Luu et al., 2006). Several GABARAP associated proteins have been identified that are implicated in GABARAP dependent regulation of GABAA receptor function and membrane trafficking. GABARAP interacts directly with N-ethylmaleimide sensitive factor (NSF), a membrane fusion and trafficking factor that has also been implicated in the membrane trafficking of several neurotransmitter receptors including GABAB receptors and AMPA receptors (Nishimune et al., 1998; Pontier et al., 2006). More recently it has been demonstrated that NSF can interact directly with GABAA receptor β-subunit intracellular domains and that co-expression of NSF with GABAA receptors reduces cell surface GABAA receptor number (Goto et al., 2005). GABARAP may exist in a tripartite complex with GABAA receptors and NSF or alternatively NSF may serve to regulate GABARAP dependent trafficking of GABAA receptors in a manner analogous to that of NSF dependent regulation of PICK and/or AP2 dependent regulation of AMPA receptor trafficking and cell surface stability (Hanley et al., 2002; Lee et al., 2002). Intriguingly, as with the overlapping AMPA receptor GluR2 subunit interaction sites for NSF and AP2, the interaction domain within GABAA receptor β-subunits for AP2 and NSF similarly overlaps although the significance of this observation currently remains unclear (Nishimune et al., 1998; Lee et al., 2002; Goto et al., 2005; Kittler et al., 2005). Y2H screens and in vitro binding studies have demonstrated that two other proteins localized to inhibitory postsynaptic domains, GRIP1 and gephyrin, interact with GABARAP suggesting that GABARAP may also be important for the synaptic transport of other components of the inhibitory postsynaptic domain in addition to GABAA receptors (Kneussel et al., 2000; Kittler et al., 2004a). GABARAP has also been shown to associate with PRIP-1 (Phospholipase-C related inactive protein type 1), which competes with the γ2 subunit for GABARAP binding (Kanematsu et al., 2002). There are two PRIP genes in mammals, PRIP-1 and PRIP2. Both PRIP-1 knockout mice and PRIP-1/PRIP-2 double knockout mice have impaired trafficking of γ2 subunit containing GABAA receptors to the cell surface, resulting in altered modulation of GABAA receptors by pharmacological agents such as diazepam (Kanematsu et al., 2002; Mizokami et al., 2007). PRIP-1/PRIP-2 can also directly interact with GABAA receptor β-subunits, and appear to regulate both receptor phosphorylation levels (via recruitment of phosphatases to the receptor) and receptor trafficking (Terunuma et al., 2004; Kanematsu et al., 2006; Mizokami et al., 2007). In contrast to cellular and molecular studies of GABARAP dependent functional regulation of GABAA receptors, gene deletion studies have shown that, in vivo, GABARAP is not essential for post synaptic trafficking of GABAA receptors (O'Sullivan et al., 2005). Since the closely related GBRL1 also associates directly with the GABAA receptor γ2 subunit, it remains unclear if interaction with GBRL1 may compensate for GABARAP function in the GABARAP KO animals. An SOS-recruitment Y2H approach identified the protein GODZ (Golgi-specific DHHC zinc finger domain protein) as another γ2 subunit binding partner. The interaction is mediated by a 14-amino acid cysteine rich domain conserved in the intracellular loops of all three GABAA receptor γ subunits (Keller et al., 2004). GODZ is primarily localized to the Golgi where there is partial overlap in localization with the γ2 subunit in HEK293 cells but not in neurons (Keller et al., 2004). GODZ and its paralogue SERZ beta are palmitoyl transferases that are important for GABAA receptor transport to the

21

cell surface by regulating palmitoylation of the γ2 subunit (see also palmitoylation section below) and GODZ function is required for the normal assembly and function of GABAergic synapses (Keller et al., 2004; Fang et al., 2006). 2.2. Role of GABAA receptor associated proteins in GABAA receptor endocytosis and endocytic sorting The number of surface and synaptic GABAA receptors can also be regulated by their internalization from the cell membrane (Kittler et al., 2000). Internalization of GABAA receptors is primarily thought to occur via a clathrin and dynamin dependent mechanism (Kittler et al., 2000; Barnes, 2001). Dynamin independent receptor internalization of GABAA receptors heterologously expressed in cell lines has also been reported, as has dynamin and caveolin-dependent GABAA receptor endocytosis (Cinar & Barnes, 2001; Bradley et al., 2008). GABAA receptor β- and γ-subunits intracellular domains can interact directly with the μ2 subunit of the clathrin adaptor protein AP2, which facilitates the recruitment of these receptors into the endocytic pathway (Kittler et al., 2000). GABAA receptor β-subunits interact directly with AP2 via an atypical basic patch binding motif (Kittler et al., 2005; Smith et al., 2008) with homology to similar domains identified in synaptotagmin 1 (Haucke et al., 2000), AMPA receptors (Lee et al., 2002; Kastning et al., 2007) and alpha 1b adrenergic receptors (Diviani et al., 2003). In the case of GABAA receptor β-subunits, this AP2 binding motif (residues 401 KTHLRRRSSQLK412 in the β3-subunit) also contains the major site for phosphorylation by serine and threonine kinases (Kittler & Moss, 2003) and interestingly the interaction with AP2 is negatively regulated by serine phosphorylation (Kittler et al., 2005) (see phosphorylation section below). Blocking GABAA receptor internalization, by either targeting dynamin function or by blocking GABAA receptor β-subunit interaction with AP2 dramatically increases synaptic GABAA receptor mediated currents, highlighting the importance of dynamin and AP2 dependent receptor internalization for regulating synaptic receptor number and inhibitory synapse strength (Kittler et al., 2000, 2005). Clathrin dependent GABAA receptor internalization is also dependent on a di-leucine type motif within the GABAA β2 subunit (Herring et al., 2005) but whether this motif also mediates direct interaction with endocytic adaptor proteins and whether receptor endocytosis via this motif can be regulated, or is a constitutive mechanism remains unknown. GABAA receptors can also bind directly with high affinity to the μ2 subunit of AP2 via a tyrosine type Yxxθ motif (where x represents any amino acid and θ an amino acid with a bulky hydrophobic side chain) within the γ2 subunit intracellular loop (residues 365YGYECL370) (Kittler et al., 2008). Structural studies of the γ2 subunit YGYECL-peptide co-crystalized with AP2 revealed that both Y365 and Y367 within this motif, in addition to L370, were important for the interaction. Y365 acts as an additional specificity determinant of the γ2-AP2 interaction by binding into a third pocket on μ2-AP2 providing an explanation for the high affinity of this motif for AP2. Furthermore, the structure suggested that γ2 subunit containing GABAA receptors may be internalized as dimers or multimers, and it will be interesting to further investigate this possibility (Kittler et al., 2008). Dialysis of a function blocking peptide to the γ2 subunit specific AP2 binding motif significantly increased mIPSC amplitude, similar to what was previously observed upon blockade of the β3 subunit AP2 interaction. Co-dialysis of peptides targeting both the β3 and γ2 AP2 interaction mechanisms had an additive effect on mIPSC amplitude suggesting that these two mechanisms can act either separately or in concert to regulate synaptic receptor number (Kittler et al., 2008). Thus, synaptic GABAA receptor number can be controlled by at least two mechanisms for AP2-dependent receptor recruitment into the internalization pathway, one of which is γ2subunit-selective. In addition to direct binding of the receptor to AP2, the GABAA receptor associated proteins GABARAP and PRIP-1 have been demonstrated to interact with clathrin heavy chain and AP2

22

I.L. Arancibia-Cárcamo, J.T. Kittler / Pharmacology & Therapeutics 123 (2009) 17–31

respectively (Kanematsu et al., 2007; Mohrluder et al., 2007), however the relationship between these molecules for regulating receptor downmodulation are still not fully understood. Internalized GABAA receptors can be either targeted for degradation in the lysosome or rapidly recycled back to the cell surface (Kittler et al., 2004b). From a Y2H screen with a β-subunit intracellular domain a direct interaction of the intracellular loop of GABAA receptor β-subunits with the huntingtin associated protein 1 (HAP1) was identified (Kittler et al., 2004b). HAP1 expression was found to block the targeting of GABAA receptors for degradation in lysosomes and to facilitate the recycling of receptors back to the cell surface and synapses (Kittler et al., 2004b). Interestingly, decreasing HAP1 levels in the hypothalamus using siRNA leads to reduced levels and activity of GABAA receptors and altered animal feeding suggesting that HAP1 dependent GABAA receptor trafficking may underlie important aspects of normal animal behavior (Kittler et al., 2004b; Sheng et al., 2006). Recently, the γ2 subunit has been found to interact with calciummodulating cyclophilin ligand (CAML), dependent on both the γ2 subunit intracellular domain and the fourth transmembrane domain of the subunit (Yuan et al., 2008). Knockdown of CAML by shRNAi results in neurons with reduced GABA-evoked currents and GABAergic synaptic function. Interestingly, while CAML co-localises with γ2 subunits in ER vesicles, the main trafficking defect of disrupted CAML function appears to be a reduction in the endocytic recycling of receptors (Yuan et al., 2008). 2.3. Other GABAA receptor associated proteins Several other GABAA receptor associated proteins have been identified. The GABAA receptor interacting factor 1 GRIF-1 was identified from a yeast two-hybrid screen with the intracellular domain of the GABAA receptor β2 subunit and the related TRAK1 is also proposed to interact with GABAA receptor complexes (Beck et al., 2002; Gilbert et al., 2006). GRIF-1 was found to interact with GABAA receptors in vitro and in cell lines (Beck et al., 2002). The role of GRIF-1 with respect to GABAA receptor function remains unclear. However, the demonstration that GRIF-1 can also interact with the potassium channel Kir2.1 to regulate its trafficking (Grishin et al., 2006), suggests that GRIF-1 may also be involved in the trafficking of GABAA receptors. Intriguingly GRIF-1 and TRAK1 have also been implicated in the trafficking of a number of organelles in both cell lines and neurons including mitochondria and endosomes (Brickley et al., 2005; Kirk et al., 2006; MacAskill et al., 2009), suggesting that this protein family may have diverse functions within cells. Immunopurification approaches have also identified an interaction between GABAA receptors and the multifunctional protein gC1qR but the role of this interaction remains unknown (Schaerer et al., 2001). A number of other GABAA receptor associated proteins are proposed to link GABAA receptors to the cytoskeleton. GABARAP and gephyrin are both tubulin binding proteins suggesting they may link GABAA receptors to microtubules, although it remains unclear for GABARAP where in the cell this linkage may be occurring, since GABARAP does not appear to be highly expressed at synapses (Kittler et al., 2001). In addition, GABARAP has also been shown to interact with microfilaments (Wang & Olsen, 2000). The ERM family member radixin was identified by yeast two hybrid to interact with extrasynaptic GABAA receptor α5 subunits. Radixin's ability to bind to actin suggests that it may act to link α5 subunit containing GABAA receptors to the actin cytoskeleton and thus to be important for the formation of some populations of extrasynaptic GABAA receptors (Loebrich et al., 2006). Modulation of GABAA receptor function is in part achieved by the phosphorylation of GABAA receptor subunits (extensively reviewed in Brandon et al., 2002; Kittler & Moss, 2003; Song & Messing, 2005). GABAA receptors are substrates for a number of protein kinases and phosphatases (see the following section). Several proteins involved

in regulating the phosphorylation state of GABAA receptors interact directly with subunit intracellular domains. These include the receptor for activated C kinase (RACK1) in addition to protein kinase C (PKC) itself and the protein kinase A (PKA) anchoring protein AKAP150, which all interact directly with GABAA receptor β subunits to regulate receptor phosphorylation (Brandon et al., 1999, 2003). In addition, dephosphoryation of receptors is mediated by the protein phosphatases PP1 and PP2A, which interact with β subunit intracellular domains while calcineurin can interact in an activity dependent manner with the γ2 subunit intracellular domain (Wang et al., 2003a; Jovanovic et al., 2004; Terunuma et al., 2004). 3. Regulation of GABAA receptor trafficking and synaptic receptor number by receptor post-translational modifications 3.1. Phosphorylation A large number of studies on GABAA receptor phosphorylation have implicated this process in altering channel kinetics, channel open time, rate of desensitization and sensitivity to pharmacological agents (reviewed in Brandon et al., 2002; Kittler & Moss, 2003; Song & Messing, 2005). Direct receptor phosphorylation has also been implicated in regulating receptor trafficking. The large intracellular domains of GABAA receptor β subunits contain a number of sites for phosphorylation including conserved serine residues (S409 in β1, S410 in β2, S408/409 in β3) that can be phosphorylated by a number of kinases both in vitro and in vivo including PKA, PKC, and AKT. Phosphorylation of S410 in GABAA receptor β2 subunits by insulin dependent AKT activation mediates an increase of surface GABAA receptor number and trafficking to synaptic sites to potentiate the inhibitory synaptic response (Wang et al., 2003b). In addition, brain derived neurotrophic factor (BDNF) induced PKC mediated phosphorylation of the β3 subunit results in a transient enhancement of GABAA receptor function followed by a lasting depression of miniature inhibitory post-synaptic currents (mIPSCs) in both cultured hippocampal and cortical neurons (Jovanovic et al., 2004). This BDNFdependent modulation correlated with an increase in surface GABAA receptor number (Jovanovic et al., 2004). In the case of β-subunits, the domain that mediates interaction with the clathrin adaptor AP2 overlaps with these important sites for β-subunit phosphorylation (Kittler & Moss, 2003) and interestingly phosphorylation negatively regulates AP2 binding. In the β3 subunit, dephosphorylation of S408/S409 unmasks the basic patch-binding motif for AP2, enhancing the endocytosis of selected GABAA receptor subtypes (Kittler et al., 2005; Terunuma et al., 2008). Blocking this internalization pathway with a dynamin inhibitory peptide or a peptide that selectively blocks interaction of β-subunits with AP2 inhibits D3 dopamine receptor dependent downmodulation of inhibitory synapse strength in the striatum (Chen et al., 2006). D3 dopamine receptor dependent downmodulation of synaptic inhibition in CA1 hippocampal pyramidal cells, leading to facilitated hippocampal long term potentiation, is also dependent on enhanced GABAA receptor endocytosis and is similarly blocked by function blocking dynamin peptides (Swant et al., 2008). Thus, phospho-dependent GABAA receptor trafficking may be an important control point for regulating cell and network activity in the brain. Furthermore, enhanced pathological surface GABAA receptor downmodulation due to dephosphorylation of S408/S409 during status epilepticus can also be blocked by targeting this phospho-dependent endocytosis pathway with a selective AP2 blocking peptide, restoring the efficacy of synaptic inhibition in status epilepticus (Terunuma et al., 2008). Selectively blocking GABAA receptor interaction with AP2 or enhancing receptor phosphorylation may provide novel therapeutic strategies to ameliorate SE or other nervous system diseases where downmodulation of surface GABAA receptor number leads to pathological disinhibition. This suggests that phospho-dependent alterations in

I.L. Arancibia-Cárcamo, J.T. Kittler / Pharmacology & Therapeutics 123 (2009) 17–31

AP2-dependent GABAA receptor endocytosis may be a critical mechanism underlying functional modulation of inhibition during synaptic plasticity and in pathology. The γ2 subunit is also a substrate for phosphorylation by serine/ threonine kinases (e.g. PKC) at S327 as well as S343 in γ2L (Kittler & Moss, 2003). The dephosphorylation of the γ2 subunit at S327 in hippocampal neurons by calcineurin has been shown to result in a downregulation of GABAA receptor function (Wang et al., 2003a) although it remains unclear if this effect is dependent on GABAA receptor trafficking. In addition, the γ2 subunit can be phosphorylated by Src family kinases on tyrosine residues Y365 and Y367 (Kittler & Moss, 2003). Biochemical and structural studies have revealed that the interaction of AP2 via the γ2 subunit specific tyrosine motif is inhibited by phosphorylation of the Y365 and Y367 residues within the motif. Since a significant proportion of GABAA receptor γ2 subunits are tyrosine phosphorylated at these sites under resting conditions (Brandon et al., 2001), this would prevent the high affinity AP2 binding site from constitutively recruiting GABAA receptors into the endocytic pathway under resting conditions. However, conditions that would promote tyrosine de-phosphorylation of the receptor would be expected to promote receptor endocytosis. It will be interesting to determine which phosphatases dephosphorylate this site in vivo and whether the rapid internalization of γ2 subunit containing GABAA receptors during status epilepticus (Naylor et al., 2005; Goodkin et al., 2008), is mediated by tyrosine de-phosphorylation of the GABAA receptor γ2 subunit AP2 binding site. GABAA receptor interactions with AP2 can be controlled by phosphorylation at two sites within the receptor, one on β-subunits and one on the γ2 subunit. The reason for two phospho-dependent AP2 interaction mechanisms remains unclear, but since both mechanisms are regulated by different kinase families (i.e. serine/threonine or tyrosine kinase for β- and γ2 respectively) this suggests that this may have evolved to allow for tight regulation of AP2-dependent synaptic receptor number by multiple signaling cascades that converge at the level of receptor phosphorylation (Kittler et al., 2008). Much remains to be determined regarding phospho-dependent regulation of GABAA receptor trafficking. Of particular interest will be to better understand the role of signaling pathways for regulating receptor trafficking and synaptic inhibition in vivo and the contribution this plays in regulating network function and animal behavior. For example, it will be interesting to determine the role of GABAA receptor phosphorylation in regulating state-dependent bidirectional modification of somatic inhibition in neocortical pyramidal cells, which was recently shown to be dependent on rapid activity-dependent GABAA receptor trafficking via calcium channel activation (Kurotani et al., 2008). The use of knock-in mice with mutations in key GABAA receptor phosphorylation sites, a strategy that has been successfully applied to elucidating the role of phosphorylation in regulating AMPA receptor trafficking and synaptic plasticity, will be an important step (Lee et al., 2000; Lee et al., 2003; Hu et al., 2007). 3.2. Palmitoylation Palmitoylation is a post-translational modification that involves the attachment of the fatty acid palmitate to cysteine residues and is involved in the membrane targeting and subcellular trafficking of various proteins including AMPA receptors, GABAA receptors and the neuronal scaffold proteins PSD-95 and GRIP (DeSouza et al., 2002; ElHusseini et al., 2002; Smotrys & Linder, 2004). The γ2 subunit contains cysteine residues within its intracellular domain that have been shown to serve as substrates for palmitoylation. Mutating the palmitoylation substrates (cysteine residues) within the intracellular loop of the γ2 subunit resulted in a loss of GABAA receptor clusters at the cell surface as did treatment of neurons with the palmitoylation inhibitor 2-BrP (Rathenberg et al., 2004). Thus, palmitoylation of the

23

γ2 subunit appears to be important for correct surface delivery and synaptic targeting of GABAA receptors (Keller et al., 2004; Rathenberg et al., 2004; Fang et al., 2006). Palmitoylation of γ2 subunits is mediated by the receptor associated protein GODZ (and to a lesser extent by a GODZ paralogue, SERZ beta) which interacts directly with the γ2 subunit intracellular domain and is necessary for the formation and normal function of inhibitory synapses (Keller et al., 2004; Fang et al., 2006). The exact mechanisms by which GABAA receptor palmitoylation regulates the trafficking and clustering of GABAA receptors remain unknown. Other GABAA receptor subunits, including γ1, γ3, θ, ε and π also contain cysteine residues within their intracellular domains, although whether they are substrates for palmitoylation remains to be addressed. 3.3. Ubiquitination The regulation of GABAA receptor trafficking and receptor degradation in the ubiquitin proteosome system by the ubiquitin like protein Plic-1 first suggested that GABAA receptors may also be a direct target for ubiquitination (Bedford et al., 2001). In agreement with this, studies have demonstrated that the intracellular loop of the GABAA receptor β3 subunit contains a number of lysine residues that are substrates for ubiquitination (Saliba et al., 2007). Mutation of all 12 lysines within the intracellular loop of the β3 subunit resulted in decreased levels of β3 subunit ubiquitination without compromising the ability of this subunit to assemble into benzodiazepine sensitive heteromeric receptors. Expression of this mutant (β3K12R) in cultured cortical neurons showed increased cell surface expression of GABAA receptors compared to neurons expressing wild type β3 subunits, although the cell surface half life or endocytosis rates of the receptors were not affected. In addition, the rate of insertion at the cell surface of the β3K12R subunit was greater than that of the wild type subunit. Ubiquitination of GABAA receptor β-subunits is dependent on neuronal activity levels (Saliba et al., 2007). Blockade of neuronal activity dramatically increases GABAA receptor ubiquitination levels, which correlates with a loss of surface receptor expression levels and a reduction in the amplitude and frequency of mIPSCs. In contrast, increasing neuronal activity levels has the converse effect, decreasing receptor ubiquitination rates. Thus, activity-dependent ubiquitination of GABAA receptor βsubunits can act specifically within the secretory pathway to regulate GABAA receptor insertion at the cell surface (Saliba et al., 2007). Ubiquitination of GABAA receptor subunits appears to target unassembled subunits within the ER for ER associated degradation (ERAD) by the proteasome (Bedford et al., 2001; Gallagher et al., 2007; Saliba et al., 2007). This mechanism also appears to be important for regulating GABAA receptor subunit stability and degradation under pathological conditions. For example, a form of dominant juvenile myoclonic epilepsy caused by a non-conservative missense mutation A322D in the GABAA receptor α1 subunit leads to subunit misfolding, ubiquitination and rapid ERAD through the ubiquitin proteasome system, dramatically reducing GABAA receptor cell surface expression, which leads to compromised inhibition and epilepsy (Gallagher et al., 2007). 4. The inhibitory postsynaptic domain and its role in regulating the membrane stability and subcellular localization of GABAA receptors Both during synaptogenesis and once synapses have been formed, specific mechanisms must exist to target GABAA receptors from intracellular or extrasynaptic compartments and retain them at inhibitory synapses. Several proteins have been specifically localized to the inhibitory postsynaptic domain and are proposed to play an important role in regulating the formation, maintenance and plasticity of these synapses and influencing surface trafficking,

24

I.L. Arancibia-Cárcamo, J.T. Kittler / Pharmacology & Therapeutics 123 (2009) 17–31

synaptic localization and residency time of GABA A receptors (Fig. 2). 4.1. Gephyrin and the regulation of the synaptic clustering, localization and stability of GABAA receptors at synapses The 93 kDa protein gephyrin, which was initially isolated as a protein co-purifying with glycine receptors (Pfeiffer et al., 1982), was one of the first proteins found enriched at inhibitory synapses (Triller et al., 1987). Using immunofluorescence and electron microscopic approaches, gephyrin has been conclusively shown to accumulate at inhibitory GABAergic (and also glycinergic) synapses and substantial work has focused on elucidating the role gephyrin plays in synaptic organisation (Fritschy et al., 2008). However, there are key differences between gephyrin's relationship with glycine receptors versus GABAA receptors at synapses. A high affinity direct interaction between gephyrin and glycine receptors exists in vivo, mediated by a 14 residue stretch in the glycine receptor β-subunit loop and clustering of glycine receptors appears to be critically dependent on gephyrin (Meyer et al., 1995). In contrast, although it has been demonstrated that gephyrin can be recruited to GABAA receptors, and GABAA receptor γ2 subunit or gephyrin KO experiments have emphasized the relationship between GABAA receptor clustering and gephyrin (see next section) (Essrich et al., 1998; Alldred et al., 2005), an interaction between gephyrin and native GABAA receptors in vivo has so far remained elusive. Recently a direct interaction between gephyrin and the GABAA receptor α2 subunit was demonstrated (Tretter et al., 2008). In this study a hydrophobic 10 amino acid motif identified in the intracellular loop of the α2 subunit was found to be responsible for α2 clustering in cultured hippocampal neurons. Under conditions lacking detergent, a direct interaction between gephyrin and the α2 subunit has been observed in vitro, however under the same conditions GABAA receptor β- and γ-subunits were unable to interact with gephyrin (Tretter et al., 2008). It will be interesting to further elucidate the regulatory mechanisms of this interaction. Whether or not gephyrin can, in addition, interact with other GABAA receptors indirectly via a bridging molecule remains unclear. Furthermore, in contrast to the dependency of gephyrin for glycine receptor clustering, not all subtypes of GABAA receptor are clustered by gephyrin dependent mechanisms. 4.2. Gephyrin cluster formation Gephyrin, which forms clusters at inhibitory synapses, is made up of an N-terminal G-domain linked by a 170 residue central region to a C-terminal E-domain giving it a modular structure (Sola et al., 2001; Kim et al., 2006). Evolutionarily it is thought this structure originates from the fusion of two genes of bacterial origin (Moe and MogA), which are important for molybdenum co-factor biosynthesis and homologous to gephyrin E- and G-domains respectively (Sola et al., 2001; Xiang et al., 2001; Bader et al., 2004). Structural and biochemical studies provide the basis for a model of how gephyrin can form clusters at inhibitory postsynaptic domains (Bedet et al., 2006; Lardi-Studler & Fritschy, 2007). The gephyrin N-terminal G-domain and C-terminal E-domain can form trimers and dimers respectively, leading to the formation of a hexagonal lattice which may allow the sequestration/entrapment of inhibitory receptors (Sola et al., 2004; Kim et al., 2006). Blue Native-PAGE experiments revealed that some affinity purified gephyrin complexes run on the gel as hexamers which may represent dimers of trimers, whereas gephyrin mutants that can no longer oligomerise no longer form hexamers (Saiyed et al., 2007). Gephyrin hexamers may therefore be a natural intermediate in the formation of the gephyrin lattice. In agreement with the above results, interfering with gephyrin dimerisation and trimerisation has been demonstrated to inhibit gephyrin's ability to form clusters and causes disruption of both GABAA receptor and glycine receptor clustering at inhibitory postsynaptic sites in

neurons, confirming the essential role of G and E-domains for clustering gephyrin and inhibitory receptors at synapses. Several splice variants of gephyrin have been identified that could potentially explain its functional diversity (reviewed in Fritschy et al., 2008). Interestingly, a specific role for gephyrin splice variants in regulating GABAA receptor distribution has been proposed while insertion of a gephyrin splice cassette C5' into the gephyrin N-terminal G-domain interferes with N-terminal trimersation (Meier et al., 2000; Rees et al., 2003; Meier & Grantyn, 2004; Bedet et al., 2006; Paarmann et al., 2006; Saiyed et al., 2007; Fritschy et al., 2008). Further investigation of gephyrin splicing in regulating the synaptic recruitment of GABAA receptors is an important goal. 4.3. Gephyrin associated proteins A number of gephyrin interacting partners have been identified including the guanylate exchange factor (GEF) for Cdc42 collybistin (Kins et al., 2000), tubulin (Prior et al., 1992), the motor protein component dynein light chain (DLC) (Fuhrmann et al., 2002), Mena/ VASP (Giesemann et al., 2003), profilin isoforms I and II (Mammoto et al., 1998), RAFT (Sabatini et al., 1999), Pin1 (Zita et al., 2007) and GRIP1 (Yu et al., 2008). Several of the proteins suggest a functional link between gephyrin and tubulin and actin cytoskeleton dependent anchoring and/or transport processes. Gephyrin binding to tubulin may provide a direct link between gephyrin and microtubules. In addition, a link to the actin cytoskeleton is suggested by gephyrin's ability to interact directly with profilin I and profilin II, proteins that can bind actin monomers, and Mena and VASP, which are actin microfilament adaptors (Mammoto et al., 1998; Giesemann et al., 2003; Bausen et al., 2006). Several gephyrin interacting proteins have been found to partially colocalise with gephyrin at inhibitory GABAergic synapses including Mena/VASP, DLC and GRIP1. Collybistin is a member of the Dbl family of GEFs which are composed of tandem Dbl-homology (DH) and pleckstrin-homology domains and can specifically accelerate the GDP-GTP exchange on the small Rho GTPases Cdc42, Rac and Rho (Kins et al., 2000). Collybistin is thought to be specific for Cdc42, a GTPase which can regulate a number of cellular processes including the reorganisation of actin filaments. Collybistin exists in a number of splice variants with three alternatively spliced C-terminal isoforms combined with presence or absence of an N-terminal SH3 domain. Collybistin 2 (the shortest version) can recruit gephyrin to submembranous clusters which can recruit glycine receptors (Kins et al., 2000; Harvey et al., 2004). Since gephyrin can interact with components of the cytoskeleton, collybistin may act in part to regulate gephyrin function and receptor clustering by controlling local actin dynamics around the gephyrin lattice. Interestingly, recent biochemical and structural studies suggest that gephyrin binding may inhibit collybistin GEF activity (Xiang et al., 2006) and therefore regulate Cdc42 signaling activity during inhibitory synapse formation or plasticity. Gephyrin can also interact with dynein light chain 1 and 2 (Fuhrmann et al., 2002). The interaction between gephyrin and dynein light chain allows the recruitment of both gephyrin and glycine receptors to dynein motor complexes for retrograde transport in neurons (Fuhrmann et al., 2002; Maas et al., 2006). Whether a similar gephyrin dependent recruitment of dynein to GABAA receptors occurs, and whether gephyrin also plays a role in the intracellular transport of GABAA receptors has not yet been established. However the recent report that RNAi mediated knockdown of gephyrin has no effect on whole cell GABAA receptor current suggests that gephyrin is unlikely to have a role in steady state receptor transport to the plasma membrane (Yu et al., 2007). GRIP1 (Glutamate receptor interacting protein 1) and its homologue GRIP2 are 7 PDZ domain containing (6 in the case of GRIP2) proteins originally identified to interact with glutamate receptors and to be localized at excitatory synapses. Immunofluorescence and

I.L. Arancibia-Cárcamo, J.T. Kittler / Pharmacology & Therapeutics 123 (2009) 17–31

immunoelctron microscopic studies have demonstrated that several GRIP1 splice variants (GRIP1a, GRIP1b and GRIP1c4–7) also co-localise with gephyrin and GABAA receptors at inhibitory synapses in intact brain (Burette et al., 1999; Dong et al., 1999; Wyszynski et al., 1999; Charych et al., 2004a; Li et al., 2005; Charych et al., 2006). GRIP1 associates with several proteins including the C-termini of AMPA receptor GluR2/3 and 4c subunits, ephrins and their receptors, the proteoglycan NG2, the extracellular matrix protein Fraser syndrome protein 1, GRASPs (for GRIP associated proteins) and liprin α family members (Dong et al., 1997; Dong et al., 1999; Ye et al., 2000; Wyszynski et al., 2002; Stegmuller et al., 2003; Charych et al., 2004a; Takamiya et al., 2004) in addition to several signaling molecules and proteins implicated in cytoskeletal transport processes including kinesin motor proteins and the microtubule associated and γ2 subunit binding protein GABARAP. Interestingly, a direct interaction between gephyrin and GRIP1 was recently identified further supporting an important role for this scaffold at inhibitory synapses (Yu et al., 2008). The role of GRIP1 at inhibitory synapses remains unclear but may be linked to GABAA receptor trafficking. While under resting conditions knockdown of GRIP1 levels does not appear to influence the accumulation of GABAA receptors at inhibitory synapses (Hoogenraad et al., 2005), GRIP1 (in conjunction with GABARAP) does appear to be important for NMDA receptor dependent GABAA receptor trafficking and membrane insertion (Marsden et al., 2007). 4.4. Gephyrin and collybistin dependent regulation of synaptic GABAA receptor recruitment and clustering The exact role and mechanism by which gephyrin regulates the formation and/or maintenance of inhibitory GABAergic synapses and the synaptic recruitment and/or stabilization of GABAA receptors are still to be established. Gephyrin KO mice and gephyrin antisense studies have provided substantial evidence that gephyrin is essential for facilitating the clustering and synaptic localisation of all glycine receptors (Kirsch et al., 1993; Feng et al., 1998; Levi et al., 2004). In agreement with this gephyrin knockout mice die soon after birth and exhibit a rigid hyper-extended posture which is similar to the phenotype of animals treated with the glycine receptor blocker strychnine, suggesting a dramatic disruption of glycinergic transmission (Feng et al., 1998). In contrast, the effects of depleting gephyrin on the function of GABAA receptors and GABAergic synapses are less clear. In cultured hippocampal neurons subjected to either RNAi or antisense mediated gephyrin knockdown there is a dramatic reduction in the density of GABAA receptor clusters containing α2, γ2 and β2/3 subunits (Essrich et al., 1998; Jacob et al., 2005; Yu et al., 2007). In hippocampal neurons cultured from gephyrin knockout mice a complete absence of α2 and γ2 containing clusters was reported in one study (Kneussel et al., 1999) however other studies have demonstrated that although surface and synaptic clusters of α2 and γ2 subunits were significantly reduced many GABAA receptor clusters could still be detected (Levi et al., 2004). In both gephyrin knockout neurons and neurons treated with gephyrin siRNAi, GABAA receptor α1 subunit (and in some studies α5 subunit) clusters were unaffected. This suggests that gephyrin may contribute only partially to aggregation/clustering of GABAA receptors at synapses and that the effect may be subunit specific (Kneussel et al., 2001; Levi et al., 2002, 2004). In agreement with this, GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in gephyrin knockout neurons are present, but have a reduced amplitude and neurons acutely treated with gephyrin RNAi show a significant (approximately 50%) reduction in mIPSC amplitude and an even larger effect on frequency. Thus, while gephyrin is clearly important for GABAA receptor clustering these results also suggest that GABAA receptor clustering mechanisms that are independent of gephyrin must exist and may compensate during inhibitory synapse development in the complete absence of gephyrin in gephyrin knockout neurons (Levi et al., 2004).

25

Importantly it has also been demonstrated that gephyrin clustering at GABAergic synapses is itself dependent on GABAA receptors themselves. In neurons in vivo or cultured from GABAA receptor α1, α3 and γ2 subunit knockout mice gephyrin does not form synaptic clusters but instead forms large intracellular aggregates (Essrich et al., 1998; Alldred et al., 2005; Studer et al., 2006; Lardi-Studler & Fritschy, 2007). In agreement with these results, artificial aggregation of GABAA receptors induces co-clustering of gephyrin (Levi et al., 2004). These results suggest that clustered synaptic GABAA receptors may facilitate recruitment and stabilization of gephyrin at synapses. Additionally, RNAi mediated gephyrin knockdown combined with fluorescence imaging of GFP labelled GABAA receptors revealed that the mobility of GABAA receptor clusters at the cell surface is regulated by gephyrin suggesting that at inhibitory synapses gephyrin may further stabilize newly recruited GABAA receptors by forming a submembranous lattice (Jacob et al., 2005). This would provide a feedback loop to promote and validate the formation of inhibitory postsynaptic specializations. Recent knockout studies on the role of collybistin further support an important role for gephyrin and collybistin in regulating the formation and maintenance of a subset of inhibitory GABAergic specializations and the accumulation of GABAA receptors at synapses. Constitutive ablation of collybistin expression results in a loss of gephyrin and gephyrin dependent GABAA receptor clustering at a subset of inhibitory synapses, most notably in the hippocampus. Surprisingly, there was no significant impairment of gephyrin dependent glycine receptor clustering (Papadopoulos et al., 2007). Altered GABAA receptor clustering in collybistin knockout mice is accompanied by significant changes in hippocampal plasticity, including an enhancement in long-term potentiation and reduction in long term depression, with animals exhibiting increased anxiety and impaired spatial learning. More recently, Cre-lox transgenesis was used to conditionally ablate collybistin in the forebrain during various developmental stages. Inactivation of collybistin during embryonic development blocked gephyrin clustering during synaptogenesis resulting in gephyrin aggregate accumulation in the cell soma (Papadopoulos et al., 2007). Deletion of collybistin during the third postnatal week resulted in a protracted loss of postsynaptic gephyrin clusters and synaptic GABAA receptor γ2 subunits and correlated with the appearance cytoplasmic gephyrin aggregates. Thus collybistin appears to be vitally important for the localisation and maintenance of gephyrin and GABAA receptor clusters at a subset of inhibitory synapses in the hippocampus. 5. Cell adhesion complexes at the inhibitory synapse The exact mechanisms that underlie the precise alignment of presynaptic terminals from innervating neurons with postsynaptic domains containing the cognate neurotransmitter receptor remain unclear. It has become clear however that in addition to postsynaptic scaffold proteins a critical role also exists for cell adhesion molecules that can span the synapse, linking pre and postsynaptic membranes. A number of cell adhesion molecules including cadherins (Benson & Tanaka, 1998), neurexin/neuroligin complexes and dystroglycan complexes have been localised to inhibitory synapses and are proposed to play an important role in their formation and maintenance (Fig. 1). Cell adhesion molecules in cooperation with synaptic scaffolds are likely to play a key role in facilitating the accumulation of surface GABAA receptors at synaptic sites. 5.1. Neurexins and neuroligins The neurexins and their postsynaptic counterparts the neuroligins, are thought to act as key trans-synaptic organizing molecules at both inhibitory and excitatory synapses (Ushkaryov et al., 1992). A number of neurexin binding partners have been identified including neurexophilins, dystroglycan and neuroligins, (Craig & Kang, 2007).

26

I.L. Arancibia-Cárcamo, J.T. Kittler / Pharmacology & Therapeutics 123 (2009) 17–31

Neurexins and neuroligins form cell adhesion complexes that span the synaptic cleft, with neurexins localised on the presynaptic side and the neuroligins located in the postsynaptic domain (Craig & Kang, 2007). Neurexins are also proposed to interact with postsynaptic dystroglycan complexes (Sugita et al., 2001). When neuroligins are expressed in a human embryonic kidney (HEK) cell line they can induce glutamatergic or GABAergic axons (in part depending on the neurexin) from co-cultured neurons to form functional release sites onto these non-neuronal cells (Scheiffele et al., 2000) suggesting that neurexin/neuroligin complexes may be important for the formation or maintenance of synaptic connections. Complimentary co-culture experiments, where neurexins were presented on the surface of beads or expressed alone in non-neuronal cells revealed these proteins could trigger postsynaptic differentiation and clustering of postsynaptic receptors and scaffolds in contacting dendrites (Graf et al., 2004; Craig & Kang, 2007; Huang & Scheiffele, 2008). Three vertebrate genes encode for neurexins (Neurexin I, II and III) each containing two independent promoters that allow the formation of either α- or β-neurexins. Neurexins are neuro-specific cell surface proteins that contain a large extracellular N-terminal domain, transmembrane region and short intracellular C-terminus. Neurexins can be spliced at five different sites (three in α- and two in βneurexins) allowing for the expression of hundreds of alternatively spliced isoforms (Craig & Kang, 2007; Huang & Scheiffele, 2008). The α-neurexins N-terminal domain contains six laminin-neurexin-sex hormone-binding globulin (LNS) domains and three EGF-like regions whereas the shorter β-neurexins contain only a single LNS domain, however both α- and β-neurexins bind to neuroligins. α- and βneurexins have a relatively short intracellular domain which can bind to a number of presynaptic interacting proteins including synaptotagmin, CASK, syntenin and Mint (Craig & Kang, 2007; Huang & Scheiffele, 2008). The four main neuroligin isoforms in mammals (neuroligin 1–4) comprise a large extracellular neurexin binding domain similar in structure to acetylcholinesterase and which may also allow neuroligins to homo-multimerise. Neuroligins also have a relatively short intracellular domain containing a PDZ domain ligand that is believed to link them to several PDZ domain containing postsynaptic proteins including PSD-95 and S-SCAM family members (Meyer et al., 2004; Craig & Kang, 2007). The importance of neuroligins for the formation of inhibitory synapses was suggested by the specific localization of neuroligin-1 and neuroligin-2 to excitatory and inhibitory synapses, respectively (Varoqueaux et al., 2004). Recently, neuroligin-3 has been localized to both inhibitory and excitatory synapses (Budreck & Scheiffele, 2007). Interestingly, mutant mice carrying an autism associated point mutation in neuroligin-3 exhibit enhanced inhibitory synaptic function (Tabuchi et al., 2007). When presented alone on the surface of HEK cells or beads neurexin is sufficient to induce the localised clustering of glutamatergic and GABAergic postsynaptic receptors and scaffolds (Graf et al., 2004). In the converse experiment, neuroligin-1 induces clustering of excitatory postsynaptic components while neuroligin-2 redistributes both excitatory and inhibitory postsynaptic proteins. The presynaptic contacts induced by neuroligins co-expressed with either AMPA receptors or GABAA receptors and co-cultured with neurons produce miniature excitatory or inhibitory postsynaptic currents that exhibit many of the release characteristics of normal synapses (Fu et al., 2003; Dong et al., 2007). Knockdown (by RNA interference) of neuroligins-1, -2 or -3 in hippocampal cultures reduced the density of inhibitory and excitatory synapses identified by immunofluorescence with inhibitory and excitatory presynaptic markers (Chih et al., 2005) and also resulted in a large reduction in the amplitude and frequency of mIPSCs providing additional evidence for a critical role of neuroligins as inhibitory synaptogenic molecules (Chih et al., 2005). The synaptogenic activity of neurexins/neuroligins appears to be in part dependent on alternative splicing in splice site B of the neuroligin

AChE-domain and at site 4 in β-neurexins. It has been proposed that neuroligin lacking a splice insert in splice B together with β-neurexins containing an S4 insert together selectively promote differentiation of GABAergic synapses, whereas β-neurexins lacking S4 but containing splice insert B selectively promote differentiation of glutamatergic synapses (Boucard et al., 2005; Chih et al., 2006; Craig & Kang, 2007). Recently it has been shown that α-neurexin synaptogenic activity appears to be almost exclusively GABAergic (Kang et al., 2008). Loss of all three alpha-neurexins in triple alpha-neurexin knockout mice results in a 50% decrease in inhibitory synapses in the brainstem (Missler et al., 2003). Gene deletion studies have revealed that in vivo neuroligin function appears essential for synaptic function and animal survival but does not appear to be required for synapse formation per se (Varoqueaux et al., 2006; Craig & Kang, 2007; Taniguchi et al., 2007). Recent work suggests that rather than inducing the differentiation of new synapses, neurexin/neuroligin complexes may act to specify and validate already formed synapses by an activity dependent mechanism (Chubykin et al., 2007). In this model, activity dependent postsynaptic signaling mechanisms could converge on either neuroligin-1 or neuroligin-2 to validate the formation of excitatory versus inhibitory synapses, respectively (Chubykin et al., 2007; Huang & Scheiffele, 2008). Indeed, in neuroligin-2 knockout mice, the number of symmetric inhibitory synapses (as determined by electron microscopy) is unaltered suggesting that neuroligin-2 deletion may impair the function of inhibitory synapses without decreasing their numbers (Blundell et al., 2008). This decrease in inhibitory synaptic function in neuroligin-2 deficient mice correlates with a marked increase in anxiety like behaviour (Blundell et al., 2008). 5.2. Dystrophin glycoprotein complex (DGC) and dystrobrevin A number of immuno-localisation and functional studies have implicated the dystrophin–glycoprotein complex (DGC) in the formation and function of a subset of inhibitory synapses (Knuesel et al., 1999; Knuesel et al., 2000; Brunig et al., 2002; Levi et al., 2002). In nonneuronal cells the DGC, a large multi protein complex containing at least 10 proteins (e.g. dystrophin, dystroglycan, utrophin, syntrophins, sarcoglycans and dystrobrevins), links the extracellular matrix to the intracellular cytoskeleton (Blake et al., 2002). A major component of the DGC is dystroglycan, a large protein composed of an extracellular α-subunit and transmembrane β-subunit, which are bound to each other on the cell surface and which are both derived from proteolytic cleavage of a single pre-cursor protein. Through its extracellular αsubunit domain dystroglycan can bind matrix proteins such agrin, laminin and perlcan in addition to neurexins. Through the intracellular domain of the β-subunit, dystroglycan can bind utrophin and a large N400 kDa cytoskeletal protein of the α-actinin/β-spectrin family called dystrophin which itself can bind dystrobrevins, syntrophin and the actin cytoskeleton (Blake et al., 2002). Mutations in many of the genes encoding components of the DGC (including dystrophin, α-, β-, γ- and δ-sarcoglycan, α-dystrobrevin, laminin α2, and four enzymes that glycosylate dystroglycan) lead to muscular dystrophies in human or animal models (Blake et al., 2002). In the CNS, a number of groups have demonstrated that DGC components (including α- and β-dystroglycan, long and short forms of dystrophin, syntrophin and α- and β-dystrobrevin) are localized to inhibitory postsynaptic domains (Levi et al., 2002). Both dystrophin mutant mdx mice and mice deleted for DGC components present a reduction in GABAA receptor clusters (but not gephyrin) (Knuesel et al., 1999). A significant reduction in the amplitude and frequency of mIPSCs in Purkinje cells from mdx mutant mice has also been reported (Kueh et al., 2008). Furthermore, cerebellar Purkinje cells from double α- and β-dystrobrevin knockout mice have depleted synaptic dystrophin levels and reduced GABAA receptor clusters leading to altered sensorimotor behaviours (Grady et al., 2006)

I.L. Arancibia-Cárcamo, J.T. Kittler / Pharmacology & Therapeutics 123 (2009) 17–31

suggesting that motor deficits observed in muscular dystrophy patients may also reflect alterations of synaptic inhibition in the CNS (Grady et al., 2006). The DGC forms a stoichiometric complex with α- and β-neurexin in the CNS (Sugita et al., 2001). In a hippocampal cell culture model of inhibitory synapse formation where there is insufficient inhibitory terminal inervation, gephyrin and GABAA receptors are mis-targeted and can be found opposed to glutamatergic terminals (Rao et al., 2000; Brunig et al., 2002; Christie et al., 2002; Studler et al., 2002). In contrast components of the DGC are not mis-targeted to glutamatergic synapses due to reduced inhibitory presynaptic input or in the absence of GABAA receptor γ2 subunits suggesting independent targeting mechanisms (Sugita et al., 2001; Brunig et al., 2002). S-SCAM (synaptic scaffolding molecule) is a PDZ domain scaffold with 5 or 6 PDZ domains, a guanylate kinase domain and two WW domains and interacts with β-dystroglycan via its WW domains. Interestingly, S-SCAM can be found localised to inhibitory synapses (Sumita et al., 2007) and can also interact with neuroligin-2 via its WW domains and second PDZ domain to form a tripartitie complex of S-SCAM, β-dystroglycan and neuroligin-2 suggesting that S-SCAM may function as a postsynaptic linker at inhibitory synapses between the DGC/neurexin complex and the neuroligin/neurexin complex. Neuroligin 2 and the DGC can be found localized to inhibitory synapses in the absence of GABAA receptors and gephyrin at cerebellar synapses of α1 subunit knockout mice (Patrizi et al., 2008). Thus at some synapses the DGC and neuroligins may act in concert as inhibitory synaptogenic molecules. It currently remains less clear whether cell adhesion molecules also play a role in regulating the surface and intracellular trafficking of GABAA receptors but these results suggest that neuroligin 2 (and potentially the DGC) could act to recruit GABAA receptors to synapses and increase their residency time there (Dong et al., 2007; Huang & Scheiffele, 2008; Patrizi et al., 2008). Aggregating neuroligin-2 heterologously co-expressed with GABAA receptors results in co-aggregation of GABAA receptors suggesting that these proteins may form tight surface complexes (Dong et al., 2007). This could be mediated in part by intracellular interactions between GABAA receptors and adhesion molecules via scaffolds or via extracellular interactions between the extracellular domains of adhesion molecules and the GABAA receptor (Dong et al., 2007; Huang & Scheiffele, 2008). If high affinity interactions between GABAA receptors and neuroligins do exist at synapses, this could significantly affect GABAA receptor surface diffusion properties. It will also be intriguing to determine whether under some circumstances, neuroligins or DGC components can co-assemble with GABAA receptors within intracellular compartments and be co-trafficked to the plasma membrane and synapses. 6. Diffusion properties of cell surface GABAA receptors Lateral diffusion within the plane of the plasma membrane has been demonstrated for several neurotransmitter receptors (Triller & Choquet, 2008). Studies using GABAA receptors containing an electrophysiological or fluorescent tag suggest that significant lateral mobility of GABAA receptors in the plasma membrane does occur (Jacob et al., 2005; Thomas et al., 2005). Using a fluorescent bungarotoxin labeling approach it was also demonstrated that synaptic GABAA receptors are recruited from their extrasynaptic counterparts (Bogdanov et al., 2006), in agreement with electrophysiological tagging studies (Thomas et al., 2005). Furthermore, fluorescent recovery after photobleaching (FRAP) imaging of GFP tagged GABAA receptors demonstrated that extrasynaptic GABAA receptors have higher levels of lateral mobility than their synaptic counterparts (Jacob et al., 2005). In agreement with these observations, single-particle tracking (SPT) studies with antibodies to the γ2 subunit extracellular domains have more recently demonstrated that single GABAA receptors at mixed GABAergic/glycinergic synapses in

27

motor neuron cultures can rapidly exchange between synaptic and extrasynaptic compartments (Levi et al., 2008). Periods of fast GABAA receptor mobility (diffusion coefficient of 0.2 μm2 s− 1) correspond to Brownian movement in the lipid bilayer, whereas GABAA receptors can also be confined at postsynaptic sites with a diffusion coefficient of less than 0.03 μm2 s− 1 (Levi et al., 2008). Thus, like glycine receptors (Dahan et al., 2003), GABAA receptor movements display interspersed periods of low and high diffusion rates in the synaptic and extrasynaptic membrane, respectively (Levi et al., 2008; Renner et al., 2008). This retention of GABAA receptors at inhibitory postsynaptic domains most likely results from the association of GABAA receptors with gephyrin clusters. While this has not yet been conclusively demonstrated for single receptors as has been done for glycine receptors, combined RNAi mediated gephyrin knockdown and FRAP imaging of GFP tagged GABAA receptors demonstrate that gephyrin plays a specific role in limiting the mobility of GABAA receptor clusters (Jacob et al., 2005). The fact that this synaptic confinement of GABAA receptors is reversible suggests that any postsynaptic gephyrin cluster can behave as both donor and/or acceptor sites for GABAA receptors as for glycine receptors (Renner et al., 2008). Interestingly, compared to glycine receptors, GABAA receptors are found to have a weaker confinement at synaptic sites, to explore a larger area of the synaptic membrane and to escape synapses more easily. This may be due to the weaker apparent affinities of GABAA receptors versus glycine receptors for gephyrin binding (Fritschy et al., 2008; Tretter et al., 2008). The dynamic regulation of interactions between GABAA receptors and scaffold molecules is likely to be a key mechanism for regulating synaptic receptor number during synaptic plasticity, or in homeostatic mechanisms setting the level of inhibition to that of excitation. 7. Concluding remarks GABAA receptors play a key role in regulating neuronal excitability and information processing in the brain. A number of studies over the last two decades have used molecular, biochemical and genetic approaches to further our understanding of the biology of these receptors. The trafficking of GABAA receptors from their assembly in the ER to the cell surface is a tightly regulated process highly dependent on several binding proteins. Trafficking of receptors through the secretory pathway as well as regulated endocytosis and endosomal sorting is key to regulating the number of synaptic receptors and the strength of synaptic inhibition. In addition, a number of proteins found enriched at inhibitory postsynaptic domains have been recently identified to be of major significance in the specification and validation of inhibitory synapses and are also likely to play key roles in regulating GABAA receptor residency time at synapses. Finally, the post-translational modification of GABAA receptors has been shown to play a role in modifying receptor function and trafficking, also contributing to alterations in synaptic strength. However, much work lies ahead to establish a detailed model of how the varying processes of receptor membrane dynamics and the formation of inhibitory postsynaptic domains are coordinated during development and in the adult, to regulate GABAergic transmission and to better understand how these processes may be disrupted in disease states.

References Absalom, N. L., Lewis, T. M., Kaplan, W., Pierce, K. D., & Schofield, P. R. (2003). Role of charged residues in coupling ligand binding and channel activation in the extracellular domain of the glycine receptor. J Biol Chem 278(50), 50151−50157. Alldred, M. J., Mulder-Rosi, J., Lingenfelter, S. E., Chen, G., & Luscher, B. (2005). Distinct gamma2 subunit domains mediate clustering and synaptic function of postsynaptic GABAA receptors and gephyrin. J Neurosci 25(3), 594−603. Bader, G., Gomez-Ortiz, M., Haussmann, C., Bacher, A., Huber, R., & Fischer, M. (2004). Structure of the molybdenum-cofactor biosynthesis protein MoaB of Escherichia coli. Acta Crystallogr D Biol Crystallogr 60(Pt 6), 1068−1075.

28

I.L. Arancibia-Cárcamo, J.T. Kittler / Pharmacology & Therapeutics 123 (2009) 17–31

Barnes, E. M., Jr. (2001). Assembly and intracellular trafficking of GABAA receptors. Int Rev Neurobiol 48, 1−29. Bausen, M., Fuhrmann, J. C., Betz, H., & O'Sullivan, G. A. (2006). The state of the actin cytoskeleton determines its association with gephyrin: role of ena/VASP family members. Mol Cell Neurosci 31(2), 376−386. Beck, M., Brickley, K., Wilkinson, H. L., Sharma, S., Smith, M., Chazot, P. L., et al. (2002). Identification, molecular cloning, and characterization of a novel GABAA receptorassociated protein, GRIF-1. J Biol Chem 277(33), 30079−30090. Bedet, C., Bruusgaard, J. C., Vergo, S., Groth-Pedersen, L., Eimer, S., Triller, A., et al. (2006). Regulation of gephyrin assembly and glycine receptor synaptic stability. J Biol Chem 281(40), 30046−30056. Bedford, F. K., Kittler, J. T., Muller, E., Thomas, P., Uren, J. M., Merlo, D., et al. (2001). GABA (A) receptor cell surface number and subunit stability are regulated by the ubiquitin-like protein Plic-1. Nat Neurosci 4(9), 908−916. Belelli, D., & Lambert, J. J. (2005). Neurosteroids: endogenous regulators of the GABA(A) receptor. Nat Rev Neurosci 6(7), 565−575. Benson, D. L., & Tanaka, H. (1998). N-cadherin redistribution during synaptogenesis in hippocampal neurons. J Neurosci 18(17), 6892−6904. Bettler, B., & Tiao, J. Y. (2006). Molecular diversity, trafficking and subcellular localization of GABAB receptors. Pharmacol Ther 110(3), 533−543. Blake, D. J., Weir, A., Newey, S. E., & Davies, K. E. (2002). Function and genetics of dystrophin and dystrophin-related proteins in muscle. Physiol Rev 82(2), 291−329. Blundell, J., Tabuchi, K., Bolliger, M. F., Blaiss, C. A., Brose, N., Liu, X., et al. (2008). Increased anxiety-like behavior in mice lacking the inhibitory synapse cell adhesion molecule neuroligin 2. Genes Brain Behav 8(1), 114−126. Bogdanov, Y., Michels, G., Armstrong-Gold, C., Haydon, P. G., Lindstrom, J., Pangalos, M., et al. (2006). Synaptic GABAA receptors are directly recruited from their extrasynaptic counterparts. EMBO J 25(18), 4381−4389. Boileau, A. J., Pearce, R. A., & Czajkowski, C. (2005). Tandem subunits effectively constrain GABAA receptor stoichiometry and recapitulate receptor kinetics but are insensitive to GABAA receptor-associated protein. J Neurosci 25(49), 11219−11230. Bollan, K., Robertson, L. A., Tang, H., & Connolly, C. N. (2003). Multiple assembly signals in gamma-aminobutyric acid (type A) receptor subunits combine to drive receptor construction and composition. Biochem Soc Trans 31(Pt 4), 875−879. Boucard, A. A., Chubykin, A. A., Comoletti, D., Taylor, P., & Sudhof, T. C. (2005). A splice code for trans-synaptic cell adhesion mediated by binding of neuroligin 1 to alphaand beta-neurexins. Neuron 48(2), 229−236. Bradley, C. A., Taghibiglou, C., Collingridge, G. L., & Wang, Y. T. (2008). Mechanisms involved in the reduction of GABAA receptor alpha1-subunit expression caused by the epilepsy mutation A322D in the trafficking-competent receptor. J Biol Chem 283 (32), 22043−22050. Brandon, N. J., Delmas, P., Hill, J., Smart, T. G., & Moss, S. J. (2001). Constitutive tyrosine phosphorylation of the GABA(A) receptor gamma 2 subunit in rat brain. Neuropharmacology 41(6), 745−752. Brandon, N. J., Jovanovic, J. N., Colledge, M., Kittler, J. T., Brandon, J. M., Scott, J. D., et al. (2003). A-kinase anchoring protein 79/150 facilitates the phosphorylation of GABA (A) receptors by cAMP-dependent protein kinase via selective interaction with receptor beta subunits. Mol Cell Neurosci 22(1), 87−97. Brandon, N., Jovanovic, J., & Moss, S. (2002). Multiple roles of protein kinases in the modulation of gamma-aminobutyric acid(A) receptor function and cell surface expression. Pharmacol Ther 94(1–2), 113−122. Brandon, N. J., Uren, J. M., Kittler, J. T., Wang, H., Olsen, R., Parker, P. J., et al. (1999). Subunit-specific association of protein kinase C and the receptor for activated C kinase with GABA type A receptors. J Neurosci 19(21), 9228−9234. Brejc, K., van Dijk, W. J., Klaassen, R. V., Schuurmans, M., van Der Oost, J., Smit, A. B., et al. (2001). Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 411(6835), 269−276. Brickley, K., Smith, M. J., Beck, M., & Stephenson, F. A. (2005). GRIF-1 and OIP106, members of a novel gene family of coiled-coil domain proteins: association in vivo and in vitro with kinesin. J Biol Chem 280(15), 14723−14732. Brunig, I., Suter, A., Knuesel, I., Luscher, B., & Fritschy, J. M. (2002). GABAergic terminals are required for postsynaptic clustering of dystrophin but not of GABA(A) receptors and gephyrin. J Neurosci 22(12), 4805−4813. Budreck, E. C., & Scheiffele, P. (2007). Neuroligin-3 is a neuronal adhesion protein at GABAergic and glutamatergic synapses. Eur J Neurosci 26(7), 1738−1748. Burette, A., Wyszynski, M., Valtschanoff, J. G., Sheng, M., & Weinberg, R. J. (1999). Characterization of glutamate receptor interacting protein-immunopositive neurons in cerebellum and cerebral cortex of the albino rat. J Comp Neurol 411(4), 601−612. Charych, E. I., Li, R., Serwanski, D. R., Li, X., Miralles, C. P., Pinal, N., et al. (2006). Identification and characterization of two novel splice forms of GRIP1 in the rat brain. J Neurochem 97(3), 884−898. Charych, E. I., Yu, W., Li, R., Serwanski, D. R., Miralles, C. P., Li, X., et al. (2004). A four PDZ domain-containing splice variant form of GRIP1 is localized in GABAergic and glutamatergic synapses in the brain. J Biol Chem 279(37), 38978−38990. Charych, E. I., Yu, W., Miralles, C. P., Serwanski, D. R., Li, X., Rubio, M., et al. (2004). The brefeldin A-inhibited GDP/GTP exchange factor 2, a protein involved in vesicular trafficking, interacts with the beta subunits of the GABA receptors. J Neurochem 90(1), 173−189. Chen, Z. W., Chang, C. S., Leil, T. A., Olcese, R., & Olsen, R. W. (2005). GABAA receptorassociated protein regulates GABAA receptor cell-surface number in Xenopus laevis oocytes. Mol Pharmacol 68(1), 152−159. Chen, Z. W., Chang, C. S., Leil, T. A., & Olsen, R. W. (2007). C-terminal modification is required for GABARAP-mediated GABA(A) receptor trafficking. J Neurosci 27(25), 6655−6663. Chen, Z., Dillon, G. H., & Huang, R. (2004). Molecular determinants of proton modulation of glycine receptors. J Biol Chem 279(2), 876−883.

Chen, G., Kittler, J. T., Moss, S. J., & Yan, Z. (2006). Dopamine D3 receptors regulate GABAA receptor function through a phospho-dependent endocytosis mechanism in nucleus accumbens. J Neurosci 26(9), 2513−2521. Chih, B., Engelman, H., & Scheiffele, P. (2005). Control of excitatory and inhibitory synapse formation by neuroligins. Science 307(5713), 1324−1328. Chih, B., Gollan, L., & Scheiffele, P. (2006). Alternative splicing controls selective transsynaptic interactions of the neuroligin–neurexin complex. Neuron 51(2), 171−178. Christie, S. B., Miralles, C. P., & De Blas, A. L. (2002). GABAergic innervation organizes synaptic and extrasynaptic GABAA receptor clustering in cultured hippocampal neurons. J Neurosci 22(3), 684−697. Chubykin, A. A., Atasoy, D., Etherton, M. R., Brose, N., Kavalali, E. T., Gibson, J. R., et al. (2007). Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2. Neuron 54(6), 919−931. Cinar, H., & Barnes, E. M., Jr. (2001). Clathrin-independent endocytosis of GABA(A) receptors in HEK 293 cells. Biochemistry 40(46), 14030−14036. Connolly, C. N., Krishek, B. J., McDonald, B. J., Smart, T. G., & Moss, S. J. (1996). Assembly and cell surface expression of heteromeric and homomeric gamma-aminobutyric acid type A receptors. J Biol Chem 271(1), 89−96. Connolly, C. N., & Wafford, K. A. (2004). The Cys-loop superfamily of ligand-gated ion channels: the impact of receptor structure on function. Biochem Soc Trans 32(Pt3), 529−534. Craig, A. M., & Kang, Y. (2007). Neurexin–neuroligin signaling in synapse development. Curr Opin Neurobiol 17(1), 43−52. Dahan, M., Levi, S., Luccardini, C., Rostaing, P., Riveau, B., & Triller, A. (2003). Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking. Science 302 (5644), 442−445. DeSouza, S., Fu, J., States, B. A., & Ziff, E. B. (2002). Differential palmitoylation directs the AMPA receptor-binding protein ABP to spines or to intracellular clusters. J Neurosci 22(9), 3493−3503. Diviani, D., Lattion, A. L., Abuin, L., Staub, O., & Cotecchia, S. (2003). The adaptor complex 2 directly interacts with the alpha 1b-adrenergic receptor and plays a role in receptor endocytosis. J Biol Chem 278(21), 19331−19340. Dong, H., O'Brien, R. J., Fung, E. T., Lanahan, A. A., Worley, P. F., & Huganir, R. L. (1997). GRIP: A synaptic PDZ domain-containing protein that interacts with AMPA receptors. Nature 386(6622), 279−284. Dong, N., Qi, J., & Chen, G. (2007). Molecular reconstitution of functional GABAergic synapses with expression of neuroligin-2 and GABAA receptors. Mol Cell Neurosci 35(1), 14−23. Dong, H., Zhang, P., Liao, D., & Huganir, R. L. (1999). Characterization, expression, and distribution of GRIP protein. Ann N Y Acad Sci 868, 535−540. El-Husseini Ael, D., Schnell, E., Dakoji, S., Sweeney, N., Zhou, Q., Prange, O., et al. (2002). Synaptic strength regulated by palmitate cycling on PSD-95. Cell 108(6), 849−863. Essrich, C., Lorez, M., Benson, J. A., Fritschy, J. M., & Luscher, B. (1998). Postsynaptic clustering of major GABAA receptor subtypes requires the gamma 2 subunit and gephyrin. Nat Neurosci 1(7), 563−571. Everitt, A. B., Luu, T., Cromer, B., Tierney, M. L., Birnir, B., Olsen, R. W., et al. (2004). Conductance of recombinant GABA (A) channels is increased in cells co-expressing GABA(A) receptor-associated protein. J Biol Chem 279(21), 21701−21706. Fang, C., Deng, L., Keller, C. A., Fukata, M., Fukata, Y., Chen, G., et al. (2006). GODZmediated palmitoylation of GABA(A) receptors is required for normal assembly and function of GABAergic inhibitory synapses. J Neurosci 26(49), 12758−12768. Feng, G., Tintrup, H., Kirsch, J., Nichol, M. C., Kuhse, J., Betz, H., et al. (1998). Dual requirement for gephyrin in glycine receptor clustering and molybdoenzyme activity. Science 282(5392), 1321−1324. Ficklin, M. B., Zhao, S., & Feng, G. (2005). Ubiquilin-1 regulates nicotine-induced upregulation of neuronal nicotinic acetylcholine receptors. J Biol Chem 280(40), 34088−34095. Franks, N. P. (2008). General anaesthesia: from molecular targets to neuronal pathways of sleep and arousal. Nat Rev Neurosci 9(5), 370−386. Fritschy, J. M., & Brunig, I. (2003). Formation and plasticity of GABAergic synapses: physiological mechanisms and pathophysiological implications. Pharmacol Ther 98(3), 299−323. Fritschy, J. M., Harvey, R. J., & Schwarz, G. (2008). Gephyrin: where do we stand, where do we go? Trends Neurosci 31(5), 257−264. Fritschy, J. M., & Mohler, H. (1995). GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits. J Comp Neurol 359(1), 154−194. Fu, Z., Washbourne, P., Ortinski, P., & Vicini, S. (2003). Functional excitatory synapses in HEK293 cells expressing neuroligin and glutamate receptors. J Neurophysiol 90(6), 3950−3957. Fuhrmann, J. C., Kins, S., Rostaing, P., El Far, O., Kirsch, J., Sheng, M., et al. (2002). Gephyrin interacts with Dynein light chains 1 and 2, components of motor protein complexes. J Neurosci 22(13), 5393−5402. Gallagher, M. J., Ding, L., Maheshwari, A., & Macdonald, R. L. (2007). The GABAA receptor alpha1 subunit epilepsy mutation A322D inhibits transmembrane helix formation and causes proteasomal degradation. Proc Natl Acad Sci U S A 104(32), 12999−13004. Giesemann, T., Schwarz, G., Nawrotzki, R., Berhorster, K., Rothkegel, M., Schluter, K., et al. (2003). Complex formation between the postsynaptic scaffolding protein gephyrin, profilin, and Mena: a possible link to the microfilament system. J Neurosci 23(23), 8330−8339. Gilbert, S. L., Zhang, L., Forster, M. L., Anderson, J. R., Iwase, T., Soliven, B., et al. (2006). Trak1 mutation disrupts GABA(A) receptor homeostasis in hypertonic mice. Nat Genet 38(2), 245−250. Goodkin, H. P., Joshi, S., Mtchedlishvili, Z., Brar, J., & Kapur, J. (2008). Subunit-specific trafficking of GABA(A) receptors during status epilepticus. J Neurosci 28(10), 2527−2538.

I.L. Arancibia-Cárcamo, J.T. Kittler / Pharmacology & Therapeutics 123 (2009) 17–31 Gorrie, G. H., Vallis, Y., Stephenson, A., Whitfield, J., Browning, B., Smart, T. G., et al. (1997). Assembly of GABAA receptors composed of alpha1 and beta2 subunits in both cultured neurons and fibroblasts. J Neurosci 17(17), 6587−6596. Goto, H., Terunuma, M., Kanematsu, T., Misumi, Y., Moss, S. J., & Hirata, M. (2005). Direct interaction of N-ethylmaleimide-sensitive factor with GABA(A) receptor beta subunits. Mol Cell Neurosci 30(2), 197−206. Grady, R. M., Wozniak, D. F., Ohlemiller, K. K., & Sanes, J. R. (2006). Cerebellar synaptic defects and abnormal motor behavior in mice lacking alpha- and beta-dystrobrevin. J Neurosci 26(11), 2841−2851. Graf, E. R., Zhang, X., Jin, S. X., Linhoff, M. W., & Craig, A. M. (2004). Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins. Cell 119(7), 1013−1026. Grishin, A., Li, H., Levitan, E. S., & Zaks-Makhina, E. (2006). Identification of gammaaminobutyric acid receptor-interacting factor 1 (TRAK2) as a trafficking factor for the K+ channel Kir2.1. J Biol Chem 281(40), 30104−30111. Hanley, J. G., Khatri, L., Hanson, P. I., & Ziff, E. B. (2002). NSF ATPase and alpha-/betaSNAPs disassemble the AMPA receptor–PICK1 complex. Neuron 34(1), 53−67. Harvey, K., Duguid, I. C., Alldred, M. J., Beatty, S. E., Ward, H., Keep, N. H., et al. (2004). The GDP–GTP exchange factor collybistin: an essential determinant of neuronal gephyrin clustering. J Neurosci 24(25), 5816−5826. Haucke, V., Wenk, M. R., Chapman, E. R., Farsad, K., & De Camilli, P. (2000). Dual interaction of synaptotagmin with mu2- and alpha-adaptin facilitates clathrincoated pit nucleation. EMBO J 19(22), 6011−6019. Heir, R., Ablasou, C., Dumontier, E., Elliott, M., Fagotto-Kaufmann, C., & Bedford, F. K. (2006). The UBL domain of PLIC-1 regulates aggresome formation. EMBO Rep 7 (12), 1252−1258. Herring, D., Huang, R., Singh, M., Dillon, G. H., & Leidenheimer, N. J. (2005). PKC modulation of GABAA receptor endocytosis and function is inhibited by mutation of a dileucine motif within the receptor beta 2 subunit. Neuropharmacology 48(2), 181−194. Hoogenraad, C. C., Milstein, A. D., Ethell, I. M., Henkemeyer, M., & Sheng, M. (2005). GRIP1 controls dendrite morphogenesis by regulating EphB receptor trafficking. Nat Neurosci 8(7), 906−915. Hosie, A. M., Wilkins, M. E., da Silva, H. M., & Smart, T. G. (2006). Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites. Nature 444(7118), 486−489. Hosie, A. M., Wilkins, M. E., & Smart, T. G. (2007). Neurosteroid binding sites on GABA (A) receptors. Pharmacol Ther 116(1), 7−19. Hu, H., Real, E., Takamiya, K., Kang, M. G., Ledoux, J., Huganir, R. L., et al. (2007). Emotion enhances learning via norepinephrine regulation of AMPA-receptor trafficking. Cell 131(1), 160−173. Huang, Z. J., & Scheiffele, P. (2008). GABA and neuroligin signaling: Linking synaptic activity and adhesion in inhibitory synapse development. Curr Opin Neurobiol 18 (1), 77−83. Jacob, T. C., Bogdanov, Y. D., Magnus, C., Saliba, R. S., Kittler, J. T., Haydon, P. G., et al. (2005). Gephyrin regulates the cell surface dynamics of synaptic GABAA receptors. J Neurosci 25(45), 10469−10478. Jacob, T. C., Moss, S. J., & Jurd, R. (2008). GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition. Nat Rev Neurosci 9(5), 331−343. Jovanovic, J. N., Thomas, P., Kittler, J. T., Smart, T. G., & Moss, S. J. (2004). Brain-derived neurotrophic factor modulates fast synaptic inhibition by regulating GABA(A) receptor phosphorylation, activity, and cell-surface stability. J Neurosci 24(2), 522−530. Kanematsu, T., Fujii, M., Mizokami, A., Kittler, J. T., Nabekura, J., Moss, S. J., et al. (2007). Phospholipase C-related inactive protein is implicated in the constitutive internalization of GABAA receptors mediated by clathrin and AP2 adaptor complex. J Neurochem 101(4), 898−905. Kanematsu, T., Jang, I. S., Yamaguchi, T., Nagahama, H., Yoshimura, K., Hidaka, K., et al. (2002). Role of the PLC-related, catalytically inactive protein p130 in GABA(A) receptor function. Embo J 21(5), 1004−1011. Kanematsu, T., Yasunaga, A., Mizoguchi, Y., Kuratani, A., Kittler, J. T., Jovanovic, J. N., et al. (2006). Modulation of GABA(A) receptor phosphorylation and membrane trafficking by phospholipase C-related inactive protein/protein phosphatase 1 and 2A signaling complex underlying brain-derived neurotrophic factor-dependent regulation of GABAergic inhibition. J Biol Chem 281(31), 22180−22189. Kang, Y., Zhang, X., Dobie, F., Wu, H., & Craig, A. M. (2008). Induction of GABAergic postsynaptic differentiation by alpha-neurexins. J Biol Chem 283(4), 2323−2334. Kastning, K., Kukhtina, V., Kittler, J. T., Chen, G., Pechstein, A., Enders, S., et al. (2007). Molecular determinants for the interaction between AMPA receptors and the clathrin adaptor complex AP-2. Proc Natl Acad Sci U S A 104(8), 2991−2996. Kawaguchi, S. Y., & Hirano, T. (2007). Sustained structural change of GABA(A) receptorassociated protein underlies long-term potentiation at inhibitory synapses on a cerebellar Purkinje neuron. J Neurosci 27(25), 6788−6799. Keller, C. A., Yuan, X., Panzanelli, P., Martin, M. L., Alldred, M., Sassoe-Pognetto, M., et al. (2004). The gamma2 subunit of GABA(A) receptors is a substrate for palmitoylation by GODZ. J Neurosci 24(26), 5881−5891. Kim, E. Y., Schrader, N., Smolinsky, B., Bedet, C., Vannier, C., Schwarz, G., et al. (2006). Deciphering the structural framework of glycine receptor anchoring by gephyrin. Embo J 25(6), 1385−1395. Kins, S., Betz, H., & Kirsch, J. (2000). Collybistin, a newly identified brain-specific GEF, induces submembrane clustering of gephyrin. Nat Neurosci 3(1), 22−29. Kirk, E., Chin, L. S., & Li, L. (2006). GRIF1 binds Hrs and is a new regulator of endosomal trafficking. J Cell Sci 119(Pt 22), 4689−4701. Kirsch, J., Wolters, I., Triller, A., & Betz, H. (1993). Gephyrin antisense oligonucleotides prevent glycine receptor clustering in spinal neurons. Nature 366(6457), 745−748.

29

Kittler, J. T., Arancibia-Carcamo, I. L., & Moss, S. J. (2004). Association of GRIP1 with a GABA(A) receptor associated protein suggests a role for GRIP1 at inhibitory synapses. Biochem Pharmacol 68(8), 1649−1654. Kittler, J. T., Chen, G., Honing, S., Bogdanov, Y., McAinsh, K., Arancibia-Carcamo, I. L., et al. (2005). Phospho-dependent binding of the clathrin AP2 adaptor complex to GABAA receptors regulates the efficacy of inhibitory synaptic transmission. Proc Natl Acad Sci U S A 102(41), 14871−14876. Kittler, J. T., Chen, G., Kukhtina, V., Vahedi-Faridi, A., Gu, Z., Tretter, V., et al. (2008). Regulation of synaptic inhibition by phospho-dependent binding of the AP2 complex to a YECL motif in the GABAA receptor gamma2 subunit. Proc Natl Acad Sci U S A 105(9), 3616−3621. Kittler, J. T., Delmas, P., Jovanovic, J. N., Brown, D. A., Smart, T. G., & Moss, S. J. (2000). Constitutive endocytosis of GABAA receptors by an association with the adaptin AP2 complex modulates inhibitory synaptic currents in hippocampal neurons. J Neurosci 20(21), 7972−7977. Kittler, J. T., & Moss, S. J. (2001). Neurotransmitter receptor trafficking and the regulation of synaptic strength. Traffic 2(7), 437−448. Kittler, J. T., & Moss, S. J. (2003). Modulation of GABAA receptor activity by phosphorylation and receptor trafficking: Implications for the efficacy of synaptic inhibition. Curr Opin Neurobiol 13(3), 341−347. Kittler, J. T., Rostaing, P., Schiavo, G., Fritschy, J. M., Olsen, R., Triller, A., et al. (2001). The subcellular distribution of GABARAP and its ability to interact with NSF suggest a role for this protein in the intracellular transport of GABA(A) receptors. Mol Cell Neurosci 18(1), 13−25. Kittler, J. T., Thomas, P., Tretter, V., Bogdanov, Y. D., Haucke, V., Smart, T. G., et al. (2004). Huntingtin-associated protein 1 regulates inhibitory synaptic transmission by modulating gamma-aminobutyric acid type A receptor membrane trafficking. Proc Natl Acad Sci U S A 101(34), 12736−12741. Kleijnen, M. F., Alarcon, R. M., & Howley, P. M. (2003). The ubiquitin-associated domain of hPLIC-2 interacts with the proteasome. Mol Biol Cell 14(9), 3868−3875. Kleijnen, M. F., Shih, A. H., Zhou, P., Kumar, S., Soccio, R. E., Kedersha, N. L., et al. (2000). The hPLIC proteins may provide a link between the ubiquitination machinery and the proteasome. Mol Cell 6(2), 409−419. Kleizen, B., & Braakman, I. (2004). Protein folding and quality control in the endoplasmic reticulum. Curr Opin Cell Biol 16(4), 343−349. Kneussel, M., Brandstatter, J. H., Gasnier, B., Feng, G., Sanes, J. R., & Betz, H. (2001). Gephyrin-independent clustering of postsynaptic GABA(A) receptor subtypes. Mol Cell Neurosci 17(6), 973−982. Kneussel, M., Brandstatter, J. H., Laube, B., Stahl, S., Muller, U., & Betz, H. (1999). Loss of postsynaptic GABA(A) receptor clustering in gephyrin-deficient mice. J Neurosci 19 (21), 9289−9297. Kneussel, M., Haverkamp, S., Fuhrmann, J. C., Wang, H., Wassle, H., Olsen, R. W., et al. (2000). The gamma-aminobutyric acid type A receptor (GABAAR)-associated protein GABARAP interacts with gephyrin but is not involved in receptor anchoring at the synapse. Proc Natl Acad Sci U S A 97(15), 8594−8599. Knuesel, I., Bornhauser, B. C., Zuellig, R. A., Heller, F., Schaub, M. C., & Fritschy, J. M. (2000). Differential expression of utrophin and dystrophin in CNS neurons: An in situ hybridization and immunohistochemical study. J Comp Neurol 422(4), 594−611. Knuesel, I., Mastrocola, M., Zuellig, R. A., Bornhauser, B., Schaub, M. C., & Fritschy, J. M. (1999). Short communication: altered synaptic clustering of GABAA receptors in mice lacking dystrophin (mdx mice). Eur J Neurosci 11(12), 4457−4462. Kueh, S. L., Head, S. I., & Morley, J. W. (2008). GABA(A) receptor expression and inhibitory post-synaptic currents in cerebellar Purkinje cells in dystrophindeficient mdx mice. Clin Exp Pharmacol Physiol 35(2), 207−210. Kurotani, T., Yamada, K., Yoshimura, Y., Crair, M. C., & Komatsu, Y. (2008). Statedependent bidirectional modification of somatic inhibition in neocortical pyramidal cells. Neuron 57(6), 905−916. Lardi-Studler, B., & Fritschy, J. M. (2007). Matching of pre- and postsynaptic specializations during synaptogenesis. Neuroscientist 13(2), 115−126. Lee, H. K., Barbarosie, M., Kameyama, K., Bear, M. F., & Huganir, R. L. (2000). Regulation of distinct AMPA receptor phosphorylation sites during bidirectional synaptic plasticity. Nature 405(6789), 955−959. Lee, S. H., Liu, L., Wang, Y. T., & Sheng, M. (2002). Clathrin adaptor AP2 and NSF interact with overlapping sites of GluR2 and play distinct roles in AMPA receptor trafficking and hippocampal LTD. Neuron 36(4), 661−674. Lee, H. K., Takamiya, K., Han, J. S., Man, H., Kim, C. H., Rumbaugh, G., et al. (2003). Phosphorylation of the AMPA receptor GluR1 subunit is required for synaptic plasticity and retention of spatial memory. Cell 112(5), 631−643. Leil, T. A., Chen, Z. W., Chang, C. S., & Olsen, R. W. (2004). GABAA receptor-associated protein traffics GABAA receptors to the plasma membrane in neurons. J Neurosci 24(50), 11429−11438. Levi, S., Grady, R. M., Henry, M. D., Campbell, K. P., Sanes, J. R., & Craig, A. M. (2002). Dystroglycan is selectively associated with inhibitory GABAergic synapses but is dispensable for their differentiation. J Neurosci 22(11), 4274−4285. Levi, S., Logan, S. M., Tovar, K. R., & Craig, A. M. (2004). Gephyrin is critical for glycine receptor clustering but not for the formation of functional GABAergic synapses in hippocampal neurons. J Neurosci 24(1), 207−217. Levi, S., Schweizer, C., Bannai, H., Pascual, O., Charrier, C., & Triller, A. (2008). Homeostatic regulation of synaptic GlyR numbers driven by lateral diffusion. Neuron 59(2), 261−273. Li, R. W., Serwanski, D. R., Miralles, C. P., Li, X., Charych, E., Riquelme, R., et al. (2005). GRIP1 in GABAergic synapses. J Comp Neurol 488(1), 11−27. Loebrich, S., Bahring, R., Katsuno, T., Tsukita, S., & Kneussel, M. (2006). Activated radixin is essential for GABAA receptor alpha5 subunit anchoring at the actin cytoskeleton. Embo J 25(5), 987−999.

30

I.L. Arancibia-Cárcamo, J.T. Kittler / Pharmacology & Therapeutics 123 (2009) 17–31

Luscher, B., & Keller, C. A. (2004). Regulation of GABAA receptor trafficking, channel activity, and functional plasticity of inhibitory synapses. Pharmacol Ther 102(3), 195−221. Luu, T., Gage, P. W., & Tierney, M. L. (2006). GABA increases both the conductance and mean open time of recombinant GABAA channels co-expressed with GABARAP. J Biol Chem 281(47), 35699−35708. Maas, C., Tagnaouti, N., Loebrich, S., Behrend, B., Lappe-Siefke, C., & Kneussel, M. (2006). Neuronal cotransport of glycine receptor and the scaffold protein gephyrin. J Cell Biol 172(3), 441−451. MacAskill, A. F., Brickley, K., Stephenson, F. A., & Kittler, J. T. (2009). GTPase dependent recruitment of Grif-1 by Miro1 regulates mitochondrial trafficking in hippocampal neurons. Mol Cell Neurosci 40(3), 301−312. Macdonald, R. L., & Olsen, R. W. (1994). GABAA receptor channels. Annu Rev Neurosci 17, 569−602. Mammoto, A., Sasaki, T., Asakura, T., Hotta, I., Imamura, H., Takahashi, K., et al. (1998). Interactions of drebrin and gephyrin with profilin. Biochem Biophys Res Commun 243(1), 86−89. Marsden, K. C., Beattie, J. B., Friedenthal, J., & Carroll, R. C. (2007). NMDA receptor activation potentiates inhibitory transmission through GABA receptor-associated proteindependent exocytosis of GABA(A) receptors. J Neurosci 27(52), 14326−14337. Meier, J., De Chaldee, M., Triller, A., & Vannier, C. (2000). Functional heterogeneity of gephyrins. Mol Cell Neurosci 16(5), 566−577. Meier, J., & Grantyn, R. (2004). A gephyrin-related mechanism restraining glycine receptor anchoring at GABAergic synapses. J Neurosci 24(6), 1398−1405. Meyer, G., Kirsch, J., Betz, H., & Langosch, D. (1995). Identification of a gephyrin binding motif on the glycine receptor beta subunit. Neuron 15(3), 563−572. Meyer, G., Varoqueaux, F., Neeb, A., Oschlies, M., & Brose, N. (2004). The complexity of PDZ domain-mediated interactions at glutamatergic synapses: a case study on neuroligin. Neuropharmacology 47(5), 724−733. Missler, M., Zhang, W., Rohlmann, A., Kattenstroth, G., Hammer, R. E., Gottmann, K., et al. (2003). Alpha-neurexins couple Ca2+ channels to synaptic vesicle exocytosis. Nature 423(6943), 939−948. Mizokami, A., Kanematsu, T., Ishibashi, H., Yamaguchi, T., Tanida, I., Takenaka, K., et al. (2007). Phospholipase C-related inactive protein is involved in trafficking of gamma2 subunit-containing GABA(A) receptors to the cell surface. J Neurosci 27(7), 1692−1701. Mody, I. (2008). Extrasynaptic GABAA receptors in the crosshairs of hormones and ethanol. Neurochem Int 52(1–2), 60−64. Mohrluder, J., Hoffmann, Y., Stangler, T., Hanel, K., & Willbold, D. (2007). Identification of clathrin heavy chain as a direct interaction partner for the gamma-aminobutyric acid type A receptor associated protein. Biochemistry 46(50), 14537−14543. Moss, S. J., & Smart, T. G. (2001). Constructing inhibitory synapses. Nat Rev Neurosci 2(4), 240−250. N'Diaye, E. N., Hanyaloglu, A. C., Kajihara, K. K., Puthenveedu, M. A., Wu, P., von Zastrow, M., et al. (2008). The ubiquitin-like protein PLIC-2 is a negative regulator of G protein-coupled receptor endocytosis. Mol Biol Cell 19(3), 1252−1260. Naylor, D. E., Liu, H., & Wasterlain, C. G. (2005). Trafficking of GABA(A) receptors, loss of inhibition, and a mechanism for pharmacoresistance in status epilepticus. J Neurosci 25(34), 7724−7733. Nishimune, A., Isaac, J. T., Molnar, E., Noel, J., Nash, S. R., Tagaya, M., et al. (1998). NSF binding to GluR2 regulates synaptic transmission. Neuron 21(1), 87−97. O'Sullivan, G. A., Kneussel, M., Elazar, Z., & Betz, H. (2005). GABARAP is not essential for GABA receptor targeting to the synapse. Eur J Neurosci 22(10), 2644−2648. Paarmann, I., Schmitt, B., Meyer, B., Karas, M., & Betz, H. (2006). Mass spectrometric analysis of glycine receptor-associated gephyrin splice variants. J Biol Chem 281 (46), 34918−34925. Papadopoulos, T., Korte, M., Eulenburg, V., Kubota, H., Retiounskaia, M., Harvey, R. J., et al. (2007). Impaired GABAergic transmission and altered hippocampal synaptic plasticity in collybistin-deficient mice. EMBO J 26(17), 3888−3899. Patrizi, A., Scelfo, B., Viltono, L., Briatore, F., Fukaya, M., Watanabe, M., et al. (2008). Synapse formation and clustering of neuroligin-2 in the absence of GABAA receptors. Proc Natl Acad Sci U S A 105(35), 13151−13156. Pfeiffer, F., Graham, D., & Betz, H. (1982). Purification by affinity chromatography of the glycine receptor of rat spinal cord. J Biol Chem 257(16), 9389−9393. Pirker, S., Schwarzer, C., Wieselthaler, A., Sieghart, W., & Sperk, G. (2000). GABA(A) receptors: Immunocytochemical distribution of 13 subunits in the adult rat brain. Neuroscience 101(4), 815−850. Pontier, S. M., Lahaie, N., Ginham, R., St-Gelais, F., Bonin, H., Bell, D. J., et al. (2006). Coordinated action of NSF and PKC regulates GABAB receptor signaling efficacy. EMBO J 25(12), 2698−2709. Prior, P., Schmitt, B., Grenningloh, G., Pribilla, I., Multhaup, G., Beyreuther, K., et al. (1992). Primary structure and alternative splice variants of gephyrin, a putative glycine receptor-tubulin linker protein. Neuron 8(6), 1161−1170. Rao, A., Cha, E. M., & Craig, A. M. (2000). Mismatched appositions of presynaptic and postsynaptic components in isolated hippocampal neurons. J Neurosci 20(22), 8344−8353. Rathenberg, J., Kittler, J. T., & Moss, S. J. (2004). Palmitoylation regulates the clustering and cell surface stability of GABAA receptors. Mol Cell Neurosci 26(2), 251−257. Rees, M. I., Harvey, K., Ward, H., White, J. H., Evans, L., Duguid, I. C., et al. (2003). Isoform heterogeneity of the human gephyrin gene (GPHN), binding domains to the glycine receptor, and mutation analysis in hyperekplexia. J Biol Chem 278(27), 24688−24696. Regan-Klapisz, E., Sorokina, I., Voortman, J., de Keizer, P., Roovers, R. C., Verheesen, P., et al. (2005). Ubiquilin recruits Eps15 into ubiquitin-rich cytoplasmic aggregates via a UIM-UBL interaction. J Cell Sci 118(Pt 19), 4437−4450. Renner, M., Specht, C. G., & Triller, A. (2008). Molecular dynamics of postsynaptic receptors and scaffold proteins. Curr Opin Neurobiol 18(5), 532−540.

Sabatini, D. M., Barrow, R. K., Blackshaw, S., Burnett, P. E., Lai, M. M., Field, M. E., et al. (1999). Interaction of RAFT1 with gephyrin required for rapamycin-sensitive signaling. Science 284(5417), 1161−1164. Saiyed, T., Paarmann, I., Schmitt, B., Haeger, S., Sola, M., Schmalzing, G., et al. (2007). Molecular basis of gephyrin clustering at inhibitory synapses: role of G- and Edomain interactions. J Biol Chem 282(8), 5625−5632. Saliba, R. S., Michels, G., Jacob, T. C., Pangalos, M. N., & Moss, S. J. (2007). Activitydependent ubiquitination of GABA(A) receptors regulates their accumulation at synaptic sites. J Neurosci 27(48), 13341−13351. Saliba, R. S., Pangalos, M., & Moss, S. J. (2008). The ubiquitin-like protein Plic-1 enhances the membrane insertion of GABAA receptors by increasing their stability within the endoplasmic reticulum. J Biol Chem 283(27), 18538−18544. Schaerer, M. T., Kannenberg, K., Hunziker, P., Baumann, S. W., & Sigel, E. (2001). Interaction between GABA(A) receptor beta subunits and the multifunctional protein gC1q-R. J Biol Chem 276(28), 26597−26604. Scheiffele, P., Fan, J., Choih, J., Fetter, R., & Serafini, T. (2000). Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons. Cell 101(6), 657−669. Shen, X., Xu, K. F., Fan, Q., Pacheco-Rodriguez, G., Moss, J., & Vaughan, M. (2006). Association of brefeldin A-inhibited guanine nucleotide-exchange protein 2 (BIG2) with recycling endosomes during transferrin uptake. Proc Natl Acad Sci U S A 103(8), 2635−2640. Sheng, G., Chang, G. Q., Lin, J. Y., Yu, Z. X., Fang, Z. H., Rong, J., et al. (2006). Hypothalamic huntingtin-associated protein 1 as a mediator of feeding behavior. Nat Med 12(5), 526−533. Sieghart, W., & Sperk, G. (2002). Subunit composition, distribution and function of GABA(A) receptor subtypes. Curr Top Med Chem 2(8), 795−816. Smart, T. G., Hosie, A. M., & Miller, P. S. (2004). Zn2+ ions: modulators of excitatory and inhibitory synaptic activity. Neuroscientist 10(5), 432−442. Smit, A. B., Syed, N. I., Schaap, D., van Minnen, J., Klumperman, J., Kits, K. S., et al. (2001). A glia-derived acetylcholine-binding protein that modulates synaptic transmission. Nature 411(6835), 261−268. Smith, K. R., McAinsh, K., Chen, G., Arancibia-Carcamo, I. L., Haucke, V., Yan, Z., et al. (2008). Regulation of inhibitory synaptic transmission by a conserved atypical interaction of GABA(A) receptor beta- and gamma-subunits with the clathrin AP2 adaptor. Neuropharmacology 55(5), 844−850. Smotrys, J. E., & Linder, M. E. (2004). Palmitoylation of intracellular signaling proteins: regulation and function. Annu Rev Biochem 73, 559−587. Sola, M., Bavro, V. N., Timmins, J., Franz, T., Ricard-Blum, S., Schoehn, G., et al. (2004). Structural basis of dynamic glycine receptor clustering by gephyrin. Embo J 23(13), 2510−2519. Sola, M., Kneussel, M., Heck, I. S., Betz, H., & Weissenhorn, W. (2001). X-ray crystal structure of the trimeric N-terminal domain of gephyrin. J Biol Chem 276(27), 25294−25301. Song, M., & Messing, R. O. (2005). Protein kinase C regulation of GABAA receptors. Cell Mol Life Sci 62(2), 119−127. Stegmuller, J., Werner, H., Nave, K. A., & Trotter, J. (2003). The proteoglycan NG2 is complexed with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by the PDZ glutamate receptor interaction protein (GRIP) in glial progenitor cells. Implications for glial-neuronal signaling. J Biol Chem 278(6), 3590−3598. Studer, R., von Boehmer, L., Haenggi, T., Schweizer, C., Benke, D., Rudolph, U., et al. (2006). Alteration of GABAergic synapses and gephyrin clusters in the thalamic reticular nucleus of GABAA receptor alpha3 subunit-null mice. Eur J Neurosci 24(5), 1307−1315. Studler, B., Fritschy, J. M., & Brunig, I. (2002). GABAergic and glutamatergic terminals differentially influence the organization of GABAergic synapses in rat cerebellar granule cells in vitro. Neuroscience 114(1), 123−133. Sugita, S., Saito, F., Tang, J., Satz, J., Campbell, K., & Sudhof, T. C. (2001). A stoichiometric complex of neurexins and dystroglycan in brain. J Cell Biol 154(2), 435−445. Sumita, K., Sato, Y., Iida, J., Kawata, A., Hamano, M., Hirabayashi, S., et al. (2007). Synaptic scaffolding molecule (S-SCAM) membrane-associated guanylate kinase with inverted organization (MAGI)-2 is associated with cell adhesion molecules at inhibitory synapses in rat hippocampal neurons. J Neurochem 100(1), 154−166. Swant, J., Stramiello, M., & Wagner, J. J. (2008). Postsynaptic dopamine D3 receptor modulation of evoked IPSCs via GABA(A) receptor endocytosis in rat hippocampus. Hippocampus 18(5), 492−502. Tabuchi, K., Blundell, J., Etherton, M. R., Hammer, R. E., Liu, X., Powell, C. M., et al. (2007). A neuroligin-3 mutation implicated in autism increases inhibitory synaptic transmission in mice. Science 318(5847), 71−76. Takamiya, K., Kostourou, V., Adams, S., Jadeja, S., Chalepakis, G., Scambler, P. J., et al. (2004). A direct functional link between the multi-PDZ domain protein GRIP1 and the Fraser syndrome protein Fras1. Nat Genet 36(2), 172−177. Taniguchi, H., Gollan, L., Scholl, F. G., Mahadomrongkul, V., Dobler, E., Limthong, N., et al. (2007). Silencing of neuroligin function by postsynaptic neurexins. J Neurosci 27 (11), 2815−2824. Terunuma, M., Jang, I. S., Ha, S. H., Kittler, J. T., Kanematsu, T., Jovanovic, J. N., et al. (2004). GABAA receptor phospho-dependent modulation is regulated by phospholipase C-related inactive protein type 1, a novel protein phosphatase 1 anchoring protein. J Neurosci 24(32), 7074−7084. Terunuma, M., Xu, J., Vithlani, M., Sieghart, W., Kittler, J., Pangalos, M., et al. (2008). Deficits in phosphorylation of GABA(A) receptors by intimately associated protein kinase C activity underlie compromised synaptic inhibition during status epilepticus. J Neurosci 28(2), 376−384. Thomas, P., Mortensen, M., Hosie, A. M., & Smart, T. G. (2005). Dynamic mobility of functional GABAA receptors at inhibitory synapses. Nat Neurosci 8(7), 889.

I.L. Arancibia-Cárcamo, J.T. Kittler / Pharmacology & Therapeutics 123 (2009) 17–31 Tretter, V., Jacob, T. C., Mukherjee, J., Fritschy, J. M., Pangalos, M. N., & Moss, S. J. (2008). The clustering of GABA(A) receptor subtypes at inhibitory synapses is facilitated via the direct binding of receptor alpha 2 subunits to gephyrin. J Neurosci 28(6), 1356−1365. Triller, A., & Choquet, D. (2008). New concepts in synaptic biology derived from singlemolecule imaging. Neuron 59(3), 359−374. Triller, A., Cluzeaud, F., & Korn, H. (1987). gamma-Aminobutyric acid-containing terminals can be apposed to glycine receptors at central synapses. J Cell Biol 104(4), 947−956. Unwin, N. (1993). Nicotinic acetylcholine receptor at 9 A resolution. J Mol Biol 229(4), 1101−1124. Ushkaryov, Y. A., Petrenko, A. G., Geppert, M., & Sudhof, T. C. (1992). Neurexins: synaptic cell surface proteins related to the alpha-latrotoxin receptor and laminin. Science 257(5066), 50−56. Varoqueaux, F., Aramuni, G., Rawson, R. L., Mohrmann, R., Missler, M., Gottmann, K., et al. (2006). Neuroligins determine synapse maturation and function. Neuron 51(6), 741−754. Varoqueaux, F., Jamain, S., & Brose, N. (2004). Neuroligin 2 is exclusively localized to inhibitory synapses. Eur J Cell Biol 83(9), 449−456. Wang, H., Bedford, F. K., Brandon, N. J., Moss, S. J., & Olsen, R. W. (1999). GABA(A)receptor-associated protein links GABA(A) receptors and the cytoskeleton. Nature 397(6714), 69−72. Wang, H., & Olsen, R. W. (2000). Binding of the GABA(A) receptor-associated protein (GABARAP) to microtubules and microfilaments suggests involvement of the cytoskeleton in GABARAPGABA(A) receptor interaction. J Neurochem 75(2), 644−655. Wang, J., Liu, S., Haditsch, U., Tu, W., Cochrane, K., Ahmadian, G., et al. (2003). Interaction of calcineurin and type-A GABA receptor gamma 2 subunits produces longterm depression at CA1 inhibitory synapses. J Neurosci 23(3), 826−836. Wang, Q., Liu, L., Pei, L., Ju, W., Ahmadian, G., Lu, J., et al. (2003). Control of synaptic strength, a novel function of Akt. Neuron 38(6), 915−928.

31

Wyszynski, M., Kim, E., Dunah, A. W., Passafaro, M., Valtschanoff, J. G., Serra-Pages, C., et al. (2002). Interaction between GRIP and liprin-alpha/SYD2 is required for AMPA receptor targeting. Neuron 34(1), 39−52. Wyszynski, M., Valtschanoff, J. G., Naisbitt, S., Dunah, A. W., Kim, E., Standaert, D. G., et al. (1999). Association of AMPA receptors with a subset of glutamate receptorinteracting protein in vivo. J Neurosci 19(15), 6528−6537. Xiang, S., Kim, E. Y., Connelly, J. J., Nassar, N., Kirsch, J., Winking, J., et al. (2006). The crystal structure of Cdc42 in complex with collybistin II, a gephyrin-interacting guanine nucleotide exchange factor. J Mol Biol 359(1), 35−46. Xiang, S., Nichols, J., Rajagopalan, K. V., & Schindelin, H. (2001). The crystal structure of Escherichia coli MoeA and its relationship to the multifunctional protein gephyrin. Structure 9(4), 299−310. Ye, B., Liao, D., Zhang, X., Zhang, P., Dong, H., & Huganir, R. L. (2000). GRASP-1: a neuronal RasGEF associated with the AMPA receptor/GRIP complex. Neuron 26(3), 603−617. Yu, W., Charych, E. I., Serwanski, D. R., Li, R. W., Ali, R., Bahr, B. A., et al. (2008). Gephyrin interacts with the glutamate receptor interacting protein 1 isoforms at GABAergic synapses. J Neurochem 105(6), 2300−2314. Yu, W., Jiang, M., Miralles, C. P., Li, R. W., Chen, G., & de Blas, A. L. (2007). Gephyrin clustering is required for the stability of GABAergic synapses. Mol Cell Neurosci 36 (4), 484−500. Yuan, X., Yao, J., Norris, D., Tran, D. D., Bram, R. J., Chen, G., et al. (2008). Calciummodulating cyclophilin ligand regulates membrane trafficking of postsynaptic GABA(A) receptors. Mol Cell Neurosci 38(2), 277−289. Zita, M. M., Marchionni, I., Bottos, E., Righi, M., Del Sal, G., Cherubini, E., et al. (2007). Post-phosphorylation prolyl isomerisation of gephyrin represents a mechanism to modulate glycine receptors function. EMBO J 26(7), 1761−1771.