Molecular Organization of the Postsynaptic Membrane at Inhibitory Synapses

increasing number of neurological and neuropsychiatric diseases including: anxiety, depression, schizophrenia, epilepsy, stroke, substance abuse, neuropathic pain and hyperekplexia/startle disease. J.W. Hell, M.D. Ehlers (eds.), Structural and Functional Organization of the Synapse, Science+Business Media, LLC 2008 DO I : 10.1007/978-0-387-77232-5_ , © Springer 21 622 I.L. Arancibia-Carcamo et al. Fig. 1. Structure of inhibitory ligand gated channels. (a) Membrane topology of ligand gated ion channel subunits. Each subunit consists of a large extracellular N-terminus, four transmembrane domains (TM1-4) and a large intracellular loop between TM3 and TM4. (b) Proposed pentameric structures of GABAA, GABAC and glycine receptor subtypes. Most GABAA receptors are believed to be composed of 2α:2β:1γ subunits, whereas extrasynaptic receptors contain 2α:2β:1δ subunit combination. GABAC receptors are constructed from ρ1–3 subunits either as heteromeric or homomeric pentameric assemblies. Glycine receptors are pentamers constructed from α and β subunits in a ratio of 2:3. All these subunit combinations allow for the formation of a chloride permeable channel (c). (d) Site of action of pharmacological agents on GABAA receptors. The neurotransmitter GABA binds GABAA receptors on a site formed by the N-terminus of α and β subunits. GABAA receptors can be potentiated by binding of benzodiazepines to a site created at the interface of the α and γ subunit. In addition, GABAA receptors can also be modulated by barbiturates, ethanol and picrotoxin. The site of action for these drugs is believed to be inside the chloride permeable channel. dependent on the membrane trafficking of inhibitory receptors into and out of inhibitory postsynaptic domains, both by diffusion within the membrane and by membrane transport between surface and intracellular compartments. This process is regulated by interaction of receptors with proteins in the cytosol. The strength of inhibition can therefore be regulated by modulation of both the function and number of synaptic GABA and glycine receptors, controlled by receptor associated proteins that facilitate receptor activity, transport, and synaptic confinement. Molecular Organization of the Postsynaptic Membrane at Inhibitory Synapses 623 1 Pharmacology of GABAA and Glycine Receptors The three major GABA receptor types in the brain were initially identified based on their pharmacological and electrophysiological properties. GABA type A receptors (GABAA receptors) are ligand-gated ion channels, activated by muscimol and inhibited by bicuculline (141). A second GABA receptor (termed GABAB receptor) is insensitive to these agents, but is activated by baclofen, inhibited by 2hydroxysaclofen and produces a slower inhibitory response (20, 91). GABAB receptors are G protein-coupled receptors that mediate their inhibitory effects by coupling to ion channels (for a detailed review of GABAB receptors see (45)). In addition to GABAA and GABAB receptors, a third GABA receptor type (GABAC receptor), insensitive to both bicuculline and baclofen, but sensitive to the GABA analogue cis4-aminocrotonic acid and picrotoxin, has also been identified (56, 162, 180). GABAC receptors are ligand-gated ion channels, homologous in structure to the GABAA receptor but are primarily expressed in the retina (56, 162). Glycine receptors are primarily expressed in the brain stem and spinal cord where they play a crucial role in regulating inhibitory tone (49, 228). Glycine receptors are similar in structure to GABAA and GABAC receptors, are activated by glycine and also potentially taurine, but are insensitive to picrotoxin and instead are inhibited by the plant alkaloid strychnine (139). A range of compounds, several of which are clinically relevant therapeutic agents, can allosterically modulate GABAA receptor and glycine receptor function. GABAA receptor function is modulated by benzodiazepines, barbiturates, steroids, anesthetics and ethanol (141, 157, 181) (Fig.1). Benzodiazepines (such as diazepam) potentiate GABA responses by increasing GABAA channel opening frequency (141, 209). Diazepam has also been reported to increase channel conductance of some native receptors (58), which may be due to receptor interactions with GABAA receptor associated proteins (62). Barbiturates, such as pentobarbital, enhance GABA responses by increasing the open probability of GABA-activated channels and have also been reported to enhance single channel conductance of some native receptors, and directly activate GABAA receptors at high concentrations (11, 18, 59, 141, 144, 209). General anesthetics such as isoflurane and halothane increase GABA-induced currents by potentiating the action of GABA or by directly activating the channel (for reviews see (66, 81)). Ethanol has also been shown to have a potentiating effect on GABAA receptors (for reviews see (51, 141, 171, 189)). Recently, elegant work from several groups have reported that GABAA receptors containing the δ subunit are enhanced by low alcohol concentrations that are thought to mediate alcohol effects experienced during social drinking, although several controversies with respect to these findings remain (for detailed discussions see (158, 171, 189)). GABAA receptors are also subject to regulation by several endogenous modulators including steroids, protons and Zn (9, 93, 141). At low nanomolar concentrations which occur during stress, alcohol intoxication and pregnancy/oestrous, neurosteroids, such as allopregnanolone and tetrahydrodeoxycorticosteroneprogesterone, can potentiate GABA responses. At higher concentrations (submicromolar to micromolar), which may occur during parturition, neurosteroids can directly activate the receptor (for detailed reviews see (9, 93, 124, 157)). GABAA receptors are also inhibited by a 624 I.L. Arancibia-Carcamo et al. number of pharmacological agents. Picrotoxin, a plant convulsant, is a noncompetitive GABAA receptor inhibitor that is thought to act by binding to a site within the channel (241) leading to stabilization of receptors in an agonist bound desensitized state (164). In addition, GABAA receptors lacking the γ subunit, and therefore likely extrasynaptic, are inhibited by Zn (201). Although the pharmacology of glycine receptors is not as rich as that of GABA receptors, glycine receptor function is enhanced by several of the same agents that potentiate GABA receptors, such as alcohols, anesthetics, and neurosteroids (30, 81, 87, 128, 153). Glycine receptors are also sensitive to Zn which potentiates receptor activation at submicromolar Zn concentrations, but causes inhibition at concentrations greater than 10 μM (13, 201). In a physiological context, low nanomolar basal Zn concentrations are sufficient to prolong the decay phase of glycinergic inhibitory postsynaptic currents (128). In contrast, an equivalent of benzodiazepines is lacking for glycine receptors. 2 Molecular Identification of GABAA Receptors and Glycine Receptors GABAA receptors and glycine receptors are members of the Cys-loop ligand gated ion channel family of receptors, which also include nicotinic acetylcholine receptors (nACh receptors), GABAC receptors and serotonin (5HT3) receptors (44, 192, 222). The glycine receptor was the first neurotransmitter receptor to be isolated from the mammalian brain using aminostrychnine-agarose affinity chromatography, taking advantage of the high affinity interaction (KD 1–10 nM) of the receptor with strychnine (175). The receptor appeared to be a complex of three proteins: a 48 kDa α subunit, 58 kDa β subunit, and an additional 93 kDa protein (80). Several biochemical studies of the purified form of glycine receptors have established that the integral membrane α and β subunit glycoproteins represent the constitutive subunits of the receptor (122). Cross-linking techniques showed that α and β subunits assemble to form the channel-containing transmembrane core of the glycine receptor. The size of the complex (250 kDa) suggested a pentameric assembly of the subunits giving a quaternary structure that is now well established for all members of the Cys-loop family of receptors (Fig. 1). In contrast to the receptor, the co-purifying 93-kDa protein, gephyrin, is a nonglycosylated polypeptide that can interact reversibly with the α/β pentamer. Gephyrin is a cytoplasmic extrinsic membrane protein that plays a key role in regulating glycine receptor localization as discussed below. 3 Molecular Diversity and Distribution Pattern of Glycine Receptor Subunit Isoforms Initial biochemical and immunochemical studies during spinal cord development established the notion of glycine receptor subtypes (128). A comparison of glycine receptor expression in neonatal and adult membranes revealed that a distinct form of glycine receptor predominated around birth (neonatal receptor, GlyRN). It is characMolecular Organization of the Postsynaptic Membrane at Inhibitory Synapses 625 terized pharmacologically by its relatively low strychnine-binding affinity. It is composed of pentamers of 49 kDa polypetides denominated α2 and it is found mainly during the fetal and neonatal stages. This supported the notion of a developmental switch from GlyRN to the adult form, GlyRA, corresponding to the formation of α1/β heteromers. This switch occurs in the spinal cord within 3 weeks after birth, and is not as complete in other regions of the nervous system. One current view is that postsynaptic glycine receptors correspond to a mixture of α2 pentamers and α2/β heteromers in immature neurons, while α1/β heteromers predominate in mature synapses. Currently, the greater diversity of glycine receptor subunits is established to include not only α1 and α2 but also α3 and α2* subunits (139) and in addition an α4 subunit has also been identified in mouse, which is absent in rat and human (145). These highly homologous isoforms can form glycine-gated chloride channels of comparable strychnine sensitivity, except α2*, which is 99% identical to human α2 shows a 500-fold lower sensitivity to strychnine (123). Further, alternative exon usage increases glycine receptor diversity. For the α1 subunit, eight additional amino acids can be inserted in the M3–M4 loop. This sequence contains a serine residue, which is a potential phosphorylation site allowing a functional modulation of the α1 subunit. Alternative splicing has also been identified for the α2 and α3 subunits (139). In situ hybridization (122, 182) revealed that the distribution of individual glycine receptor subunits was not identical for all variants. The mRNAs encoding the α1 and α3 subunits are mainly transcribed in spinal cord and brain stem at later postnatal stages. The α3 subunit mRNA is present in the infralimbic system, the hippocampal complex, and the cerebellar granular layer. Expression levels of the α2 subunit, by contrast, are high in the embryonic and perinatal stages, but barely detectable in the adult brain, with some expression in higher cortical regions. The α4 subunit, which is expressed at low levels in the adult brain and spinal cord, can form functional glycine receptors, which are restricted to the spinal cord and the sympathetic nervous system. Compared to the α subunit transcripts, the β subunit mRNA is more widely expressed throughout the embryonic and adult nervous system (103) and is found in brain loci devoid of strychnine-binding sites or glycine receptor immunoreactivity. The physiological significance of this widespread β subunit mRNA expression is not understood, and there is no evidence that glycine receptor β subunits assemble with subunits of other Cys-loop receptor family members. 4 Molecular Heterogeneity of GABAA Receptors A total of 16 genes encoding GABAA receptor subunits have been identified in the mammalian nervous system. The GABAA receptor was initially purified using GABA/benzodiazepine affinity chromatography. SDS-PAGE analysis revealed two receptor subunits, α and β, with molecular weights of 53 kDa and 58 kDa respectively (199, 200). Oligonucleotides, designed based on peptide microsequencing results, were used for screening bovine cDNA libraries and allowed for the cloning of both α and β subunits. Analysis of the amino acid sequences of α and β subunits revealed that these receptor subunits showed significant homology with each other and with other members of the Cys-loop receptors such as the nAChRs. Based on 626 I.L. Arancibia-Carcamo et al. amino acid sequence homology six α subunits as well as three β subunits have been identified (96, 98, 133, 134, 137, 142, 238). Co-expression of α and β subunits in heterologous systems, however, formed receptors that lacked benzodiazepine sensitivity, a pharmacological trait observed in neuronal GABAA receptors. A novel GABAA receptor subunit that when co-expressed with α and β subunits inferred benzodiazepine sensitivity on the assembled receptors was then identified (179). This subunit, termed γ2, shows 42% and 35% identity to the α1 and β1 subunits respectively. Three γ subunits have been identified in total (179, 197, 231, 239). In addition to α, β and γ subunits other receptor subunits have been identified: δ (197), ε (50), π (90) and θ (17). In addition some of these genes, including the α6 (121), β2 (146), β3 (101), ε (230) and γ2 subunits (229), undergo alternative splicing resulting in a long (L) and short (S) version of the gene product. However, a complete understanding of the physiological relevance of these splice variants remains to be addressed. In situ hybridization and immunohistochemical studies have provided a detailed picture of the regional and cellular expression patterns of GABAA receptor subunit mRNA and corresponding receptor subunit protein, in the nervous system (67, 82, 126, 127, 159, 174, 176, 198, 232, 240). The α1, β2/3, and γ2 subunits are the most abundant and broadly expressed GABAA receptor subunits in the brain and spinal cord (126, 174, 176, 232). In agreement with this, gene deletion studies have revealed that α1 and β2 subunits account for up to half of all GABAA receptors in the CNS (212). The α1 subunit is found to be present, at high levels, in most regions of the adult brain (88, 176) but not in embryonic or neonatal brain, where it is expressed at low levels (127). The α2 subunit is found in most brain regions, except the thalamus and globus pallidum, although at lower levels than the α1 subunit (176). The α3 and α4 are the least abundant α subunits in the adult brain. The α3 subunit is highly expressed in early developmental stages, but at low levels in the adult brain (176, 240) where it is concentrated in the cerebral cortex, mainly on monoaminergic neurons (72), the basal forebrain, on cholinergic neurons (73), and the thalamic reticular nucleus (69). In contrast the α4 subunit is restricted to the thalamus, the striatum and the molecular layer of the dentate gyrus (176, 232). The α5 subunit is evenly expressed throughout development and enriched in the CA1 region of the hippocampus, although expression levels show a continuous decrease with aging (240). In contrast the α6 subunit is found almost exclusively, postnatally in the granular layer of the cerebellum (126, 127, 176). Despite a wide distribution of all three β subunits, β1 is expressed at much lower levels compared to β2 and β3 subunits (176). Although there is considerable overlap in the expression of β2 and β3 subunits, there are some regions where higher expression of one β subunit in certain brain regions comes at the expense of another. For example, β2 subunits are highly expressed in the adult thalamus in comparison to β1 and β3 subunits (176, 194, 232), although the β3 subunit is expressed at higher levels than the β2 in the fetal and neo-natal thalamus (127). The β3 subunit is expressed at high levels in the striatum where very low levels of the β2 subunit are found. In addition, β1 and β3 subunits are found at higher concentrations than the β2 subunit in the hippocampus (232). Furthermore, in the hippocampus, the β2 subunit is found concentrated in non-pyramidal neurons, whereas β1 and β3 subunits are found in primary pyramidal neurons (155, 161, 176, 232). Molecular Organization of the Postsynaptic Membrane at Inhibitory Synapses 627 Of the γ subunit isoforms, the γ2 subunit is, by far, the most abundant. However, all three γ subunits are found widely distributed, with the γ1 subunit expressed at higher concentrations in the pallidum and substantia nigra whereas the γ3 subunit is slightly more concentrated in the cerebral cortex (126, 127, 176, 232). Similar to other GABAA receptor subunits, the expression of γ1 and γ3 is altered during development, with these two subunits being expressed at higher levels pre-natally (127). Furthermore, the γ2 subunit also shows a small but significant reduction in expression levels with aging (240). Importantly, the γ1 and γ3 subunits are unable to fully substitute for the essential γ2 subunit in γ2 knockout mice, which show a lethal phenotype (4, 61). In contrast to the γ subunits, the δ subunit of GABAA receptors is most highly expressed in the thalamus and in the granular layer of the cerebellum where it is thought to assemble into receptors which contain either α4 or α6 subunits, respectively (176). The ε, π and θ subunits are the least abundant GABAA receptor subunits expressed in the CNS. The ε subunit has been observed in the hypothalamus, amygdala and brainstem (160). Interestingly, the θ subunit has also been observed in all these regions as well as the substantia nigra and hippocampus (160). The π subunit appears to be expressed mainly in the uterus, although low expression levels have been observed in the hippocampus (90). 5 GABAA and Glycine Receptor Structure GABAA receptors, glycine receptors and the other members of the Cys-loop receptor family are polytopic type I membrane proteins that share a common subunit structure (44). Each subunit encompasses a large extended N-terminal domain, bearing potential glycosylation sites, four highly conserved transmembrane domains (deduced by hydropathy analysis), and a large intracellular loop of significantly lower homology between TM3 and TM4 which protrudes into the cytoplasm (TM3–TM4 loop; Fig. 1). The four membrane-spanning domains form α helices (TM2) and/or β strands (TM1, TM3, TM4). Based on extensive biochemical and electron microscopy analysis of the nicotinic acetylcholine receptor, members of this family are assumed to be pentameric in structure where subunits are arranged around a central aqueous pore (222). The tertiary and quaternary structure of the soluble pentameric acetylcholine-binding protein (AChBP), which shares approximately 20% sequence homology with nAChR, GABA and glycine receptors, has been useful in further elucidating structure–function parameters for receptors of the family (21, 202). In addition to improving our understanding of the binding of agonists, antagonists and modulators, (1, 35, 92, 93, 201), the AChBP crystal structure provides a powerful model of the Nterminal domain of the Cys-loop receptor family. AChBP is mainly a sandwich of antiparallel β sheets positioning conserved residues in order to stabilize the protomers, whereas variable residues are at their interface (21, 202). The topology of the transmembrane domains (Fig. 1) delineates the ion permeation pathway away from the hydrophobic core of the phospholipid bilayer. 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 the lipids and isolate TM2 from a hydrophobic environment (44). Recent studies on the GlyR α1 subunit have challenged the 628 I.L. Arancibia-Carcamo et al. four-helix model and provide evidence that whereas TM2 and TM4 are entirely helical, TM1 and TM3 also contain β strands. 6 Glycine Receptor Assembly The α1 and β subunits assemble as a pentameric complex of 2α:3β stoichiometry (84, 102). Short amino acid sequences, named assembly boxes, all located in the Nterminal domain of the β subunit and corresponding to three diverging motifs in α and β subunits, have been identified (83, 102, 154). They have a role in determining receptor assembly. Replacement of these motifs in the β subunit by the corresponding α1 motifs results in the loss of the subunit ratio in α/β oligomers, suggesting that different amino acid positions are determinants in the early step of subunit–subunit interaction. These residues impose a mutually exclusive mode of assembly, either in complexes of invariant α/β stoichiometry or in homo-oligomers. A 2α−3β stoichiometry has recently been established following affinity purification of expressed engineered tandem subunits (84). These experiments indicated that the β subunit contributes to the agonist binding properties of hetero-oligomeric α1/β glycine receptors. In fact, ionic interactions at the α/β interface are required to stabilize glycine in its binding pocket. This feature, with an agonist binding shared between two adjacent subunits is characteristic of the Cys-loop receptor family and also holds true for GABAA receptors (102, 162). 7 GABAA Receptor Assembly and Composition of Plasma Membrane Receptors The large diversity of GABAA receptor subunits generates the potential for a bewildering heterogeneity of receptor structure. However, a number of functional, biochemical and immunocytochemical studies have revealed that only a limited number of receptor subunit combinations are likely to exist on the neuronal cell surface (162, 198). Restriction on GABAA receptor molecular heterogeneity in the brain is due to a number of factors. GABAA receptor subunit composition is in part restricted by the regional and temporal selectivity in subunit expression (69, 176, 198). In addition, a number of assembly rules further restrict the heterogeneity of native GABAA receptors (44). Expression studies indicated that individual GABAA receptor subunits do not result in the formation of GABA gated channels. Expression of β1 and β3 subunits alone results in the formation of chloride permeable channels that are sensitive to picrotoxin and barbiturates, however, these channels are insensitive to application of GABA. In contrast, co-expression of α and β subunits results in the production of functional GABA gated chloride channels sensitive to picrotoxin, bicuculline, barbiturates and Zn (55, 133, 192). This is in agreement with the binding site for GABA lying at the interface of α and β subunits (14, 15, 141). However, the co-expression of α and β subunits does not form channels that are sensitive to benzodiazepine modulation, which represents the pharmacology of most native receptors. It is only Molecular Organization of the Postsynaptic Membrane at Inhibitory Synapses 629 when γ subunits are additionally co-expressed with α and β subunits that GABA gated, benzodiazepine sensitive channels are formed (239) (179), which are in addition insensitive to Zn modulation (201). As shown with fluorescence, only certain GABAA receptor subunit combinations can access the cell surface whereas other subunit combinations are retained within the endoplasmic reticulum (ER) (15, 43, 44, 162). This suggested the existence of molecular mechanisms to restrict the surface expression of receptors with given compositions. With exception of the β1, β3 and γ2S subunits, individual GABAA receptor subunits are mostly retained in the ER (43). Moreover, pulse chase experiments demonstrated that α1 and β2 subunits are rapidly degraded when expressed alone (77), suggesting that these proteins are targeted for degradation from the ER. Importantly, GABAA receptor subunits have been shown to associate with BiP and calnexin (43, 77), two chaperone molecules which assist in protein quality control in the ER (114). Initial studies suggested that assembly was dependent on the N-terminus of receptor subunits. Using a chimeric approach, four amino acids in the N-terminal domain of the β3 subunit have been shown to mediate functional cell surface expression of this subunit compared to β2 (215, 216). Introduction of these four amino acids into the Nterminus of the β2 subunit is sufficient to enable β2 homomerization and ER exit. These four amino acids are also important for the oligomerization of β subunits with γ subunits but not with α subunits. Interestingly, mutation of these four amino acids within the β3 subunit abolished its ability to form homomers but not its ability to oligomerize with γ2L suggesting that at least one alternative signal for the assembly of β and γ subunits must exist (215, 216). A conserved domain in the N-terminus of α subunits has also been identified to play a role in the oligomerization of α with β but not γ subunits (15, 16, 215, 216). Furthermore, conserved glutamine and arginine residues have been shown to independently play a key role in determining the assembly of α with β subunits (15, 16, 215, 216), whereas a conserved arginine in α, β and γ subunits has been shown to be essential for subunit oligomerization (85). Finally, two regions within the N-terminus of the γ2 subunit have been shown to mediate assembly of this subunit with α and β subunits (111, 112). 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 (16, 68, 138, 141, 162). 8 Role of GABAA Receptor Subunit Composition in Determining Subcellular Localization Immunofluorescence and EM immunogold studies have revealed that α1, α2, α3, α6, β2/3 and γ2 subunits are enriched at postsynaptic domains of inhibitory synapses in many brain regions including cortex, hippocampus, globus pallidus and cerebellum (61, 64, 67–69, 166, 167, 198). These synaptically targeted receptor subunits can also be found extrasynaptically (64). Immunocytochemical and immunogold electron microscopy studies have revealed that GABAA receptor subunit combinations can be targeted to different subcellular domains (67, 68, 166–168). For example, although the α1–3 and α5 subunits are all expressed in hippocampal and cerebellar cells, the 630 I.L. Arancibia-Carcamo et al. α2 subunit is found concentrated in the axon initial segment (AIS) of the majority of cells where it colocalizes with the inhibitory synaptic marker gephyrin (25, 168). In contrast, the α1 subunit is found throughout the cell and shows both diffuse and clustered staining suggesting both a synaptic and extrasynaptic localization (25). Interestingly, the α1 subunit cannot be found in the AIS on its own and is always colocalized there with the α2 subunit suggesting that subunit composition of GABAA receptors in the AIS consists of one copy of α1 and one of α2 (25). Recent functional experiments in hippocampal pyramidal neurons have revealed that fast phasic responses are mediated by synaptic α2 subunit containing GABAA receptors on the cell soma but synaptic α1 containing GABAA receptors on dendrites (177). Receptors containing α3 are differentially targeted depending on the cell type where they are expressed. In pyramidal cells, the α3 subunit is found in clusters at postsynaptic sites, whereas in a subset of hippocampal cells characterized by a round cell body and numerous short dendrites, α3 containing receptors show a diffuse expression pattern across the membrane and are not found at synaptic sites (25). α5 subunits have been found highly expressed in extrasynaptic locations on hippocampal dendrites where it has been proposed they may contribute to tonic inhibition (25), but have also been localized to synapses both by immunofluorescence and EM postembedding immunogold (40, 195). In addition to the α subunit, receptor subcellular localization is also determined by the γ and δ subunits. In the cerebellum, immunocytochemical studies on electron microscopic sections demonstrated that synaptic and extrasynaptic GABAA receptors differed in their subunit composition. Synapses in cerebellar granule neurons are positive for the GABAA receptor γ2 subunit, whereas the δ subunit was found exclusively in extrasynaptic membranes (165). In the hippocampus δ subunits are localized perisynaptically and not at synapses (227). Interestingly, in the cerebellum phasic and tonic GABAA receptor mediated inhibition has been observed and this has been attributed to synaptic and extrasynaptic GABAA receptors respectively (22, 23). Furthermore, in addition to immunocytochemical studies that show γ2 and δ subunits being respectively targeted to synaptic and extrasynaptic sites, electrophysiological studies have confirmed that the γ2 subunit mediates phasic inhibition whereas the δ subunit plays a role in mediating tonic inhibition (23, 24, 206). In agreement with this γ2 subunit GABAA receptor knockout (KO) experiments have demonstrated the critical role of the γ2 subunit in mediating the synaptic targeting of GABAA receptors which is critical for correct animal behavior (2, 47, 61). Overall, it is proposed based on work from immunofluorescent, EM, functional and gene deletion studies, that α1, α2 or α3 subunits, co assembled with the γ2 subunit and β subunit variants, are the major receptor subtypes localized to inhibitory synapses and contributing to phasic inhibition (64). In contrast, α5 subunit containing receptors assembled with the γ2 subunit and β subunit variants, and α4 or α6 subunits co assembled with the δ subunit and β subunit variants are primarily localized extrasynaptically and mediate tonic inhibition (29, 64). Recently it has been demonstrated that in some cell types such as hippocampal interneurons, the α1 subunit may also assemble with the δ subunit and β subunit variants to form extrasynaptic receptors mediating tonic inhibition suggesting that novel GABAA receptor subunit partnerships may yet be identified (76). Molecular Organization of the Postsynaptic Membrane at Inhibitory Synapses 631 9 Components of the Inhibitory Postsynaptic Domain and Their Role in the Formation and Maintenance of Inhibitory Synapses Fig. 2. Schematic representation of a central inhibitory terminal. The specification and validation of inhibitory synapses and the correct apposition of inhibitory presynaptic terminals with inhibitory postsynaptic domains is thought to be determined in part by the co-ordinated action of adhesion molecules including cadherins, the neurexin-neuroligin 2 complex and the dystroglycan complex. Inhibitory neurotransmitter receptors (GABAA and glycine) are confined to these inhibitory specializations by scaffold molecules, most notably gephyrin, which can form a sub-synaptic clustered lattice important for synaptic receptor retention. Either directly or via intermediate binding partners, gephyrin can interact with cytoskeletal elements including tubulin and actin. The dystroglycan complex can interact with cytoplasmic binding partners including dystrobrevin and S-SCAM, which may also play a role in regulating receptor clustering and trafficking and furthermore, may also facilitate the co-clustering of both neurexin-neuroligin 2 and neurexindystrolgycan complexes. Specific mechanisms must exist to specify the formation and maturation of synapses and the correct apposition of presynaptic terminals with postsynaptic domains containing the correct cognate neurotransmitter receptors. In addition mechanisms must exist to direct and retain inhibitory receptors from intracellular or extrasynaptic compartments within the inhibitory postsynaptic apparatus. Several components of the inhibitory postsynaptic domains are proposed to play a key role in these processes, either as components of the inhibitory synaptic scaffold or by regulating transport of receptors within the cell (Figs. 2 and 4). 632 I.L. Arancibia-Carcamo et al. 9.1 A Critical Role for Gephyrin in the Molecular Organization of Inhibitory Synaptic Domains One of the first proteins to be found enriched at inhibitory synapses is the ubiquitously expressed 93 kDa protein gephyrin (221). Initially isolated as a protein copurifying with glycine receptors from rat spinal cord (175), gephyrin was found to be present in front of GABAergic terminal boutons (220) and was then demonstrated to play a central role in the organization of GABAergic synapses. Immunofluorescence and EM studies have conclusively shown that gephyrin is enriched at inhibitory GABAergic and glycinergic synapses (reviewed in (162)). However, several key differences remain between gephyrin regulation of glycinergic and GABAergic synapses. Firstly, whereas a direct interaction between gephyrin and glycine receptors has been demonstrated between a 14 residue stretch in the glycine receptor β-subunit loop and gephyrin (151), the exact molecular mechanism whereby gephyrin is recruited to GABAA receptors and whether or not gephyrin can interact either directly or indirectly via a bridging molecule with GABAA receptors remains unclear. Furthermore, in contrast to glycine receptor clustering, not all GABAA receptor subtypes are clustered by gephyrin dependent mechanisms. However, gephyrin and γ2 subunit GABAA receptor mouse knockout experiments have emphasized the relationship between GABA receptor clustering and gephyrin (see below) (2, 61). 9.2 Gephyrin Structure and Lattice Formation Several recent biochemical and structural studies have led to a model for the ability of gephyrin to forms clusters at inhibitory postsynaptic domains (7, 125). Gephyrin has a modular structure consisting of an N-terminal G-domain linked by a 170 residue central region to a C-terminal E-domain (99, 204). This structure originates from the fusion of two genes of bacterial origin MogA and Moe, important for the biosynthesis of molybdenum co-factor and homologous to gephyrin Gand E-domains respectively (3, 204, 236). Structural and biochemical data have revealed that the gephyrin N-terminal G-domain and the C-terminal E-domain can form trimers and dimers respectively (99, 203), suggesting a mechanism for the formation of a hexagonal submembrane lattice onto which inhibitory receptors can be sequestered (Fig. 3). In agreement with this, experiments using Blue Native-PAGE of affinity purified gephyrin expressed in Xenopus oocytes found gephyrin to run as hexamers, possibly dimers of trimers, in addition to some higher order complexes (188). In contrast oligomerization mutants no longer formed hexamers in this expression system. The gephyrin hexamers may be a natural intermediate of gephyrin lattice formation. Initial structural and mapping studies have located the GlyR β-loop binding to the C-terminal E-domain of gephyrin (89, 193). More recently atomic resolution structural data define the GlyR β-loop as binding to each E-domain monomer in a pocket adjacent to the dimer interface. Complementary mutagenesis experiments reveal that β-loop binding is mediated by a hydrophobic interaction between phenylalanine 330 of gephyrin and two residues of the glycine receptor β-loop (phenylalanine 398, isoleucine 400) that are essential for this interaction. In contrast, the mechanisms of gephyrin recruitment to GABAA receptors or GABAA receptor recruitment to gephyrin, remain unknown. Molecular Organization of the Postsynaptic Membrane at Inhibitory Synapses 633 Fig. 3. Gephyrin structure and binding partners. (a) Domain and splicing map of gephyrin. The 93kDa protein is divided into three domains: An N-terminal G-domain (homologous to the bacterial gene product MogA), a linker region (Ldomain) and a C-terminal E-domain (homologous to the bacterial gene product MoeA). Gephyrin can exist in several splice variants termed C1-C7 and C4’-C6’, the sites where cassettes are subject to alternative splicing are shown. (b) Gephyrin may assemble as a hexagonal lattice by the formation of dimers of trimers. G-domains can come together to form a trimeric structure, which in turn can form dimers via their E-domains. (c) Molecular partners of the glycine receptor/gephyrin complex. Gephyrin interacts with the intracellular loop of glycine β-subunits via its E-domain. This domain is also responsible for the interaction of gephyrin with other binding partners such as collybistin, profilin and VASP. RAFT-1 has been shown to interact with the Eand L-domains of gephyrin. Gephyrin has also been shown to bind microtubules via its Gdomain as well as the light chain of the microtubule motor dynein. Interestingly, gephyrin has several splice variants (147–149, 172, 173, 186) which may generate functional diversity and direct specific roles in regulating GABAAR function (Fig. 3). Gephyrin splicing appears to have no effect on high affinity interaction with the GlyR β-subunit but whether splicing may underlie gephyrin’s interaction with GABAA receptor clustering remains less clear (147–149, 172, 173). On the other hand, gephyrin splicing within its central domain affects its ability to interact with various gephyrin associated proteins (102). Of greatest interest, splicing also regulates the ability of gephyrin to form oligomers. Two groups 634 I.L. Arancibia-Carcamo et al. have recently demonstrated that insertion of a gephyrin splice cassette C5’ into the gephyrin N-terminal G-domain interferes with N-terminal trimerization (7, 188). Several studies (e.g. (7)) have now demonstrated that interfering with gephyrin dimerization and trimerization inhibits gephyrin’s ability to form clusters in neurons. Gephyrin expression constructs containing substitutions at oligomerization interfaces have confirmed the essential role of Gand E-domains for clustering gephyrin at synapses. Furthermore, expression of recombinant gephyrin Gor Edomains, or dimerization or trimerization defective gephyrin mutants, disrupts gephyrin clusters in cultured neurons. Furthermore this causes disruption of both GABAA receptor and glycine receptor clusters at inhibitory postsynaptic sites supporting a critical role of gephyrin lattice formation in the maintenance of inhibitory postsynaptic domains. 9.3 Gephyrin Associated Proteins Several gephyrin interacting partners have been identified that tie gephyrin functionally to cytoskeletal transport and anchoring processes (Fig. 3). Key gephyrin associated proteins identified so far include: the cdc42 guanylate exchange factor (GEF) collybistin (100), tubulin (178), the motor protein component dynein light chain (Dlc) (71), Mena/VASP (75), profilin (143) and RAFT (187). Gephyrin binding to tubulin provides a physical linkage between gephyrin and microtubules. Gephyrin also interacts directly with the actin monomer binding proteins profilin I and profilin II, and the actin microfilament adaptors Mena and VASP, providing a link to the actin cytoskeleton (5, 75, 143). Gephyrin can form complexes with profilin and Mena dependent on the gephyrin E-domain and competes with G-actin and phospholipids for the same binding region on profilin (75). In cell lines or cultured hippocampal neurons, profilin and Mena/VASP partially colocalize with inhibitory synaptic markers and gephyrin clusters and may contribute to a link between gephyrin dependent receptor clustering mechanisms and the microfilament system to regulate the dynamics of receptor localization at inhibitory synapses (163). Of particular interest was the identification by yeast two-hybrid screening of a direct interaction of gephyrin with guanine nucleotide exchange factor (GEF) collybistin (hPEM-2 in humans), which can accelerate the GDP-GTP exchange on small GTPases (100). Collybistin is a member of the Dbl family of GEFs which are composed of tandem Dbl-homology (DH) and pleckstrin-homology (PH) domains and are specific for the Rho GTPases (Cdc42, Rac and Rho and their isoforms) which can regulate, among other processes, the reorganization of the actin cytoskeleton. Collybistin exists in a number of splice variants with three alternatively spliced Cterminal isoforms combined with presence or absence of an N-terminal SH3 domain. Collybistin 2 (the shortest version) recruits gephyrin to submembranous clusters which recruit glycine receptors (89, 100). Collybistin is a selective GEF for the GTPase Cdc42, which can regulate the reorganization of actin filaments. Since gephyrin can interact with components of the cytoskeleton, collybistin may regulate gephyrin function and receptor clustering by controlling local actin dynamics around the gephyrin lattice. Recent structural and biochemical results indicate that gephyrin binding has an inhibitory function on collybistin GEF activity (235). Collybistin may therefore play a role in terminating Cdc42 signaling during the initial stages of inMolecular Organization of the Postsynaptic Membrane at Inhibitory Synapses 635 hibitory synapse formation. In addition to control of cytoskeletal dynamics, the ability of gephyrin to associate with mTOR/RAFT1 (rapamycin and FKB12 target protein) suggests that gephyrin may also be able to participate in translational control at the synapse (187). This is in line with the demonstration that glycine receptor alpha subunit mRNAs can be found localized in the vicinity of the postsynaptic membrane (183) where a protein synthesis machinery is also present (74). Gephyrin also interacts with dynein light chain 1 (also known as dynein light chain 8) and its homologue dynein light chain 2 (71). Dynein light chain interacts with a 63 amino acid binding domain within the central linker region of gephyrin and this interaction allows the recruitment of gephyrin and glycine receptors to dynein motor complexes for retrograde transport in neurons (71, 140). Whether gephyrin can similarly recruit GABAA receptor complexes to dynein motors remains unknown. 9.4 Functional Role of Gephyrin in Regulating the Formation and Maintenance of Inhibitory Glycinergic and GABAergic Synapses Evidence supports a role for gephyrin in regulating the formation of inhibitory synapses and the recruitment of receptors to these postsynaptic specializations. Loss of function studies using antisense or gephyrin KO mice (104) (65, 104, 132) demonstrated that this protein is essential for the synaptic recruitment and/or clustering of all synaptic glycine receptors, both in the brain and spinal cord. Furthermore, gephyrin knockout mice die soon after birth, exhibiting a rigid hyperextended posture similar to animals treated with strychnine. In contrast the consequences of gephyrin depletion on the function of GABAergic synapses have remained more controversial. Antisense treatment against gephyrin in cultured hippocampal neurons causes a dramatic reduction in the density of clusters for GABAA receptor α2 and γ2 subunits (61). Similarly RNAi mediated gephyrin knockdown resulted in a significant reduction but did not completely abolish the clustering of α2 and γ2 containing GABAA receptors (94). A complete absence of α2 and γ2 containing clusters in hippocampal cultures from gephyrin knockout mice was reported in one study (117). In contrast other studies have shown that, whereas surface and synaptic clusters of α2 and γ2 subunit containing GABAA receptors are significantly reduced, many clusters could still be detected, and furthermore α1 subunit clusters remain unaffected in gephyrin knockout neurons (116, 131, 132). Therefore in most neuronal types, gephyrin is not essential for GABAA receptor clustering but does appear to contribute to the aggregation of α2, α3 and γ2 but not α1 or α5 subunits. In agreement with this, GABAergic miniature inhibitory postsynaptic currents (mIPSCs), although reduced in amplitude, are present in hippocampal neurons lacking gephyrin, suggesting that gephyrin independent mechanisms of GABAA receptor clustering exist and may compensate during inhibitory synapse development in the absence of gephyrin (132). Intriguingly, the clustering of gephyrin at GABAergic synapses is dependent on GABAA receptors. Gephyrin does not form clusters in the absence of GABAA receptors in cultured neurons and gephyrin clustering is disrupted in vivo GABAA receptor α1, α3 and γ2 subunit knockout mice (2, 61, 125, 207). For example, in α3 knockout neurons of the reticular nucleus of the thalamus, the absence of in neurons from 636 I.L. Arancibia-Carcamo et al. GABAA receptors results in gephyrin forming large intracellular aggregates rather than synaptic clusters (207). Similarly in α1 knockout mice gephyrin clustering at postsynaptic sites is disrupted, leading to intracellular aggregates (125). In agreement with these results, artificial aggregation of GABAA receptors induces co-clustering of gephyrin (132). These results suggest that clustered synaptic GABAA receptors may recruit gephyrin to synapses. Interestingly, the glycinergic or GABAergic mixed phenotype of the presynaptic element determines the postsynaptic accumulation of specific cognate receptors but not of gephyrin and the postsynaptic accumulation of gephyrin alone cannot account for the formation of glycine receptor rich microdomains (130). A developmental analysis revealed that, at mixed glycine-GABAergic synapses, GABAA receptors formed clusters first, followed by gephyrin and then by glycine receptors (57). Gephyrin may then form a submembranous lattice that could serve to further stabilize newly recruited GABAA receptors at these sites. In agreement with this notion, RNAi mediated gephyrin knockdown combined with fluorescence imaging of GFP-labeled GABAA receptors found that gephyrin regulates GABAA receptor cell surface dynamics by reducing the mobility of GABAA receptor clusters (94). This would provide a feedback loop to promote and validate the formation of inhibitory postsynaptic specializations.