Neurotransmitter Receptor Trafficking and the Regulation of Synaptic Strength

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1 Traffic : Copyright C Munksgaard 2001 Munksgaard International Publishers ISSN Review Neurotransmitter Receptor Trafficking and the Regulation of Synaptic Strength Josef T. Kittler and Stephen J. Moss* Medical Research Council Laboratory of Molecular Cell Biology and UCL Department of Pharmacology, University College London, London WC1E 6BT, UK *Corresponding author: Stephen J. Moss, steve.moss@ucl.ac.uk. Modulation of the strength of synapses is thought to be one of the mechanisms that underlies learning and memory and is also likely to be important in processes of neuropathology and drug tolerance. This review focuses on the emerging role of postsynaptic neurotransmitter receptor trafficking as an essential mechanism underlying the dynamic regulation of synaptic strength. Key words: excitatory, inhibitory, synapse, AMPA, NMDA, GABA, glutamate, endocytosis, receptor sorting, dynamin, NSF, GABARAP, GRIP, PICK, LTP, LTD, adaptin, clathrin. Received and accepted for publication 4 May 2001 Communication between neurons occurs at highly specialized sites of cell cell contact formed between a presynaptic nerve terminal and a postsynaptic neuron, termed synapses. At synaptic sites, information is passed as a chemical messenger released from the presynaptic nerve terminal that activates specific neurotransmitter receptors in the postsynaptic domain. Synaptic transmission in the mammalian central nervous system is predominantly mediated by the neurotransmitters gamma aminobutyric acid (GABA) and glutamate. In the adult central nervous system (CNS), these neurotransmitters play essential but opposing roles by eliciting changes in the membrane potential of the postsynaptic neuron. Glutamate mediates the majority of excitatory neurotransmission in the brain, resulting in neuronal depolarization and ultimately the propagation of an action potential. In contrast, the majority of inhibitory neurotransmission is mediated by GABA, which acts to hyperpolarize nerve cells, reducing the probability of action potential firing. GABA and glutamate act as ionotropic receptors, which are ligand-gated ion channels, and at metabotropic receptors, which are G-protein-coupled receptors (1 4). It is the activation of ionotropic receptors resulting in the rapid flux of ions across the neuronal membrane that elicits the electrophysiological responses critical for fast synaptic transmission. For efficient synaptic transmission it is essential that ionotropic GABA and glutamate receptors are specifically segregated and concentrated opposite terminals for the corresponding neurotransmitter (see Figure 1) (5,6). A number of receptorassociated proteins have been identified that are important for the correct synaptic targeting and concentration of ionotropic receptors. Perhaps, and not surprisingly, different sets of proteins have been implicated in the clustering of inhibitory vs. excitatory receptors. Whereas glutamate receptors appear to be mainly associated with PDZ domain-containing proteins (7,8), GABA receptors are constructed around the core inhibitory synaptic scaffolding molecule gephyrin (9,10). A large amount of work has been dedicated to understanding the molecular mechanisms that regulate the formation and efficacy of synapses. The initial focus on the identification of proteins implicated in the targeting and anchoring of receptors at synaptic sites encouraged the belief that neurotransmitter receptors are held statically within the postsynaptic domain with long cell-surface half-lives. There has also been a significant focus on the modification of synapse strength and ionotropic receptor function by the regulation of presynaptic neurotransmitter release and the direct modulation of receptor channel properties by phosphorylation (11 18). Importantly, recent studies have revealed that some ionotropic receptors are dynamic molecules, which can rapidly translocate between synaptic sites and intracellular compartments. The ability of neurons to regulate the number of postsynaptic receptors is an additional attractive mechanism for modifying the efficacy of synaptic transmission which has important implications for synaptic plasticity. It is the trafficking of ionotropic receptors into and out of the postsynaptic domain and the implications of this receptor trafficking for the modulation of synaptic strength that will be discussed in this review. Ionotropic Glutamate Receptor Trafficking Structure of ionotropic glutamate receptors Ionotropic glutamate receptors comprise a diverse group of receptors encoded by distinct genes with additional subunit diversity generated by splice variation. Three classes of glutamate receptor have been identified from their pharmacological and molecular properties and have been named, based on the selective agonists that activate them: a-amino- 3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA) receptors (3,19). 437

2 Kittler and Moss Figure 1: Subunit structure of the AMPA and GABA A/C receptors. (A) Structure of the AMPA receptor subunit with four membrane domains and extracellular N- and intracellular C-termini. (B) Structure of the GABA A and GABA C receptors with four transmembrane domains and extracellular N- and C-termini. (C) GABA A receptors and AMPA receptors are targeted to inhibitory and excitatory synapses respectively, as shown in dendrites from cultured hippocampal neurons stained with antibodies to corresponding receptors. Glutamate receptors are formed from the presumed tetrameric or pentameric assembly of homologous subunits around a central ion pore (3,19). The topology of the receptor subunits consists of a large extracellular N-terminus, four membrane-associated domains with the second transmembrane domain (TM II) forming a re-entrant loop which gives the receptor an intracellular carboxyl terminus (Figure 1A). NMDA receptors are formed from multimeric assemblies of NR1 subunits with NR2 (A-D) and NR3A subunits, whereas AMPA receptors are built from hetero-oligomeric assemblies of subunits GluR1-4 and kainate receptors are formed from subunits GluR5-7 and KA 1 and 2 (3,19,20). Studies of excitatory synaptic transmission have focused predominantly on AMPA and NMDA receptors. Glutamate directly activates AMPA receptors to mediate fast excitatory synaptic transmission. In contrast, NMDA receptors are inactive at resting membrane potentials because, in addition to glutamate binding, NMDA receptor activation also requires depolarization to relieve a voltage-dependent block of the channel pore by magnesium ions. Activation of NMDA receptors can be achieved by sustained activation of AMPA receptors at the same synapse, resulting in membrane depolarization and re- 438 Traffic 2001;2:

3 Ionotropic neurotransmitter receptor trafficking lease of the voltage-dependent block. Upon activation, NMDA receptors conduct sodium and calcium ions and mediate a slower phase of neurotransmission. AMPA and NMDA receptors are predominantly localized to excitatory postsynaptic domains (5). Studies have revealed that many of the proteins important for signaling, trafficking and synaptic targeting of glutamate receptors are members of a growing superfamily of proteins that contain PDZ domains (named after PSD-95, Discs-large and Z01 the first three proteins identified to contain this domain) that can bind with high affinity to a short PDZ-binding peptide motif found in the glutamate receptor intracellular domains (7,8,21). Recently, it has become clear that whereas both AMPA and NMDA receptors are anchored at synaptic sites (5), AMPA receptors also have the ability to be rapidly trafficked into and out of the postsynaptic domain to regulate synapse strength (8,22,23). AMPA receptor trafficking It has become evident that AMPA receptors appear to be dynamic in their association with the postsynaptic domain and can translocate into and out of this location (24 30). In addition, AMPA receptors associate with a number of other proteins, including the PDZ-containing proteins GRIP1 (31), GRIP2/ABP (32,33), SAP-97 (34) and the PKC-interacting protein PICK1 (35,36). Interestingly, an association was also found between AMPA receptor GluR2 subunits and N-ethylmaleimide-sensitive factor (NSF) (37 39), a protein critical for intracellular membrane trafficking events (40). Disrupting the association between AMPA receptors and NSF causes a Figure 2: GABA A receptors are localized to synaptic sites in complexes with gephyrin, and modulators of GABA A receptor function protein kinase C bii and RACK 1. Receptors are endocytosed via a dynamin-dependent clathrin-mediated pathway in a complex with the AP2-adaptor, and are sorted to endosomes where they may be recycled or targeted for degradation. The GABA A receptor-associated protein GABARAP recruits N-ethylmaleimide-sensitive factor (NSF) to intracellular pools of receptors and may modulate receptor trafficking. Traffic 2001;2:

4 Kittler and Moss rapid decrease in AMPA receptor currents, suggesting that NSF is important for the cell-surface trafficking and cycling of these receptors (41,42). These results provided evidence to support the suggestion that AMPA receptors may be cycling between cell surface and intracellular pools (Figure 2). Further studies showed that perfusion of neurons with compounds that disrupt exocytosis results in a decrease in AMPA receptor currents, whereas inhibitors of endocytosis cause an increase in AMPA-mediated currents, presumably by blocking the constitutive removal of these receptors by dynamin-dependent endocytosis (42). In addition, using immunofluorescence techniques, it has also been demonstrated in cultured hippocampal neurons that treatment with glutamate can induce AMPA receptor internalization that is blocked by dominant negative dynamin (43). These results provide compelling evidence that receptors are cycling between cell surface and intracellular compartments by a process dependent on NSF and SNARE function for receptor membrane insertion and dynamin-dependent endocytosis for receptor removal (42). The above results have stimulated a significant amount of research directed at identifying the signaling pathways important for regulating glutamate receptor exocytosis and endocytosis (Figure 3). A number of recent studies have gone a long way to understanding the mechanisms that regulate AMPA receptor endocytosis (44 46) and the processes important for both activity-dependent and -independent re-insertion of receptors (8,23,46 48). AMPA receptors can be stimulated to endocytose by a number of processes including ligand binding, NMDA receptor activation, neuronal activity and treatment with the growth factor insulin (43 46,49). Selective AMPA receptor agonists stimulate AMPA receptor internalization and, in addition, the AMPA receptor blocker CNQX can also stimulate endocytosis of AMPA receptors, suggesting that ligand binding itself can induce a conformational change which promotes receptor internalization (45). In the case of AMPA stimulation a component of this receptor endocytosis is reduced by blocking voltage-gated L-type calcium channels (which will open upon AMPA receptor-mediated membrane depolarization) and calcium influx (44,45). NMDA receptor channel activation can also stimulate AMPA receptor endocytosis, in this case via calcium influx through the NMDA receptor itself by a mechanism that is dependent on the activity of the calcium/calmodulin-dependent protein phosphatase calcineurin (45,46) and protein kinase A (46). Interestingly, NMDA appears to be more effective at promoting AMPA receptor internalization in distal dendrites of the neuron, whereas AMPA promotes internalization of AMPA receptors at the soma and proximal dendrites. This suggests there may be a spatial regulation of receptor endocytosis that allows for a location-dependent control of the internalization process. Although in broad agreement, differences in some of the results from the above studies suggest that the pathways important for AMPA receptor endocytosis are complex (22,44 46,50). One study found that AMPA-mediated endocytosis is independent of phosphatase activity, in agreement with another study (46) which provides evidence that only NMDA-stimulated and not AMPA-stimulated AMPA receptor endocytosis is dependent on calcium and calcineurin. However, a third study (44) found that NMDA-dependent endocytosis is calcium and calcineurin dependent, but in contrast to the other two studies found that AMPA-mediated endocytosis is also dependent on calcium and calcineurin. The reason for these differences is unclear but may be due to differing experimental protocols and preparations or due to an underlying heterogeneity of AMPA receptor structure (50). Insulin can also stimulate AMPA receptor internalization (49); however, this appears to be via a distinct dynamin-dependent pathway that is independent of calcium influx but may be dependent on both kinase and phosphatase activity. Interestingly, by immunofluorescence, insulin-dependent AMPA receptor internalization occurs mainly in the cell soma and produces a different (possibly late endosomal) staining pattern to AMPA- and NMDA-stimulated internalization (45). Importantly, in one study the fate of internalized receptors was followed (46). This revealed that different protocols that stimulate receptor endocytosis appear to determine the final destination of internalized receptors (46). Whereas NMDA stimulates AMPA receptors to endocytose and then recycle back to the cell surface, AMPA-stimulated internalized receptors are not rapidly re-inserted into the postsynaptic domain but instead may be sorted for degradation (46). This may allow for a regulatory mechanism of activity-dependent sorting, providing a switch to maintain AMPA receptors only at synapses with sufficient NMDA receptor activity (46,48). Interestingly, the rapid endocytosis and re-insertion of AMPA receptors upon NMDA treatment results in little change in the total number of postsynaptic receptors. This fast cycling may allow for a very rapid and sensitive control of glutamatergic synapse strength simply by altering the degree of either receptor internalization or re-insertion (46,48). In addition, in the case of AMPA-stimulated receptor endocytosis, targeting endocytosed receptors for lysosomal degradation provides a further mechanism for modifying synapse strength over longer time scales, which could be important for synapse development and elimination (46). AMPA receptor trafficking and synaptic plasticity Modulation of AMPA receptor-mediated synaptic transmission underlies a number of forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD), that have been suggested as cellular models of learning and memory (18,51 54). A model has been put forward which provides a mechanism for modulating synaptic strength based on activity-dependent regulation of AMPA receptor trafficking between synaptic and nonsynaptic sites (8,22,23,47,55,56) (Figure 3). A cornerstone of this model is the existence of synapses that contain only NMDA receptors but lack AMPA receptors. These synapses, are termed silent synapses, because at normal resting membrane potentials and in the absence of functional AMPA receptors, NMDA receptors are unable to respond to synaptically released 440 Traffic 2001;2:

5 Ionotropic neurotransmitter receptor trafficking Figure 3: AMPA receptors are endocytosed via a dynamin-dependent clathrin-mediated pathway in a complex with the AP2- adaptor. Glutamate stimulates internalization of AMPA receptors. AMPA receptors internalized upon AMPA activation are targeted for degradation, whereas AMPA receptors internalized upon NMDA receptor activation are rapidly recycled and re-inserted. NMDA receptormediated AMPA receptor internalization is dependent on calcium (Ca 2π ) and calcineurin activation. In addition, the trafficking and endocytosis of AMPA receptors is modulated by direct interactions of AMPA receptor subunits with NSF and the PDZ containing proteins GRIP/ABP, PICK and SAP97. glutamate due to a voltage-dependent magnesium block. In this model, AMPA receptors can translocate into previously silent synapses to increase their strength, resulting in LTP, or can be removed from AMPA-containing synapses, weakening synapse strength, resulting in LTD. A large amount of evidence has been put forward in support of this model. The presence of silent synapses containing only NMDA receptors is supported by both electrophysiological and morphological studies (24,29,57 60). Furthermore, protocols that induce LTP and LTD also appear to regulate receptor trafficking (23). Induction of NMDA receptor-dependent LTP Traffic 2001;2: results in the rapid appearance of AMPA receptor-mediated responses at these silent synapses (23,48). Using GFP-tagged glutamate receptors it has also been possible to show that LTPinducing stimuli or calcium/calmodulin-dependent kinase II (CamKII) activity results in the rapid translocation of GluR1 into dendritic spines and clusters in live neurons (25,26). The same model of AMPA receptor redistribution may also be important for LTD. Studies have provided evidence that the rapid removal of synaptic AMPA receptors by clathrin-mediated endocytosis may be critical for LTD (22,42,49,61). In cultured hippocampal neurons the expression of LTD results in a decrease in the number of cell-surface AMPA receptors without affecting levels of 441

6 Kittler and Moss NMDA receptors. The expression of hippocampal and cerebellar LTD is also blocked by perfusion of peptides or fusion proteins that inhibit dynamin-dependent endocytosis and removal of surface AMPA receptors (61). Furthermore, inhibiting endocytosis also blocks the effect of insulin, which causes a depression of AMPA receptor currents that occludes LTD (49,61). In addition, the signaling pathways that can mediate LTD appear to regulate AMPA receptor transport. Hippocampal NMDA receptor-dependent LTD is dependent on a rise in intracellular calcium with the subsequent activation of the phosphatases calcineurin and protein-phosphatase 1 (16). Consistent with this, as described in the studies above, NMDA receptor activation causes a calcium- and calcineurin-dependent endocytosis of AMPA receptors in cultured hippocampal neurons. The association of AMPA receptors with interacting proteins is also important for AMPA receptor trafficking-dependent forms of synaptic plasticity (8,22,62,63). Disruption of the association of AMPA receptors with NSF occludes hippocampal NMDA receptor-dependent LTD (64), whereas disrupting NSF function with N-ethylmaleimide reduces hippocampal LTP (42). In addition, recent studies provide evidence that the association of AMPA receptors with PDZ-containing proteins is important for synaptic plasticity (26,62,63,65). In the cerebellum blocking the association of AMPA receptors with GRIP/ ABP and PICK1 using peptides blocks LTD, and in the hippocampus blocking the association of AMPA receptors with GRIP/ABP also blocks LTD. The ability of different AMPA receptor subunits to associate with different associated proteins may explain the recent report of subunit specific rules governing AMPA receptor trafficking to synapses determined by subunit composition (47). Whereas GluR1/GluR2-containing hetero-oligomers require activity and the GluR1 carboxyl tail (which can bind SAP-97) for synaptic delivery, GluR2/GluR3- containing hetero-oligomers continuously replace synaptic receptors dependent on the GluR2 carboxyl tail (which binds NSF, GRIP/ABP and PICK1) but independent of activity (47). These results suggest the presence of both activity-dependent and constitutive cycling of receptors providing pathways which can be used to regulate synaptic function. Ionotropic GABA Receptor Trafficking The structure of GABA A and GABA C receptors Ionotropic GABA receptors are chloride-selective ligand-gated ion channels which can be differentiated into GABA A and GABA C receptors based on their distinct pharmacology (66,67). GABA A receptors are the major sites of fast synaptic inhibition in the brain, while GABA C receptor expression is largely restricted to the retina. GABA A receptors are also the sites of action for anxiolytic and sedative agents, including both the benzodiazepines and barbiturates (4,68). Ionotropic GABA receptors are members of the ligand-gated ion channel superfamily that includes muscle and neuronal nicotinic acetylcholine receptors, glycine receptors and serotonin type 3 receptors (66,69). Members of this superfamily are hetero- pentamers with each receptor subunit having a similar membrane topology consisting of extracellular amino and carboxy termini, four transmembrane (TM) domains and a large intracellular loop between TM domains III and IV (69) (Figure 1B). GABA A receptors can be assembled from 7 subunit classes with multiple members: a(1 6), b(1 3), g(1 3), d, e, q and p, generating the potential for extensive heterogeneity of receptor structure in the CNS (4,68). However the consensus opinion suggests that in vivo most GABA A receptors are composed of a, b and g subunits in a stoichiometry of 2:2:1 (4,68,70). GABA C receptors are simpler in structure compared to GABA A receptors and are believed to be homomeric or heteromeric assemblies of (1 3) subunits (67). Trafficking of ionotropic GABA receptor In common with glutamate receptors, there has been increasing evidence for the cycling of GABA A receptors between synaptic sites and intracellular compartments (Figure 4). In stellate neurons of the cerebellum, the number of postsynaptic GABA A receptors has been found to determine inhibitory synaptic strength (10,71). This provides good evidence that regulating the number of postsynaptic GABA A receptors can have a significant effect on inhibitory synaptic transmission. Furthermore, recent studies have shown that the redistribution of GABA A receptors between intracellular compartments and cell-surface domains can occur on relatively rapid (10 s of minutes) time scales. For instance, insulin treatment of neurons in culture (72) causes the redistribution of GABA A receptors from intracellular compartments to the cell surface. Recent evidence also suggests a role for brainderived neurotrophic factor (BDNF) in regulating the cell-surface expression of GABA A receptors containing the a2 subunit in hippocampal pyramidal neurons (73). The mechanisms underlying the modulation of GABA A receptor cell-surface expression by these differing growth factors have, however, not been reported. In addition, the number of postsynaptic GABA A receptors can be modulated by activity (74) and GABA A receptors can be downregulated by an agonistdependent mechanism (75,76). Specifically, treatment of cultured neurons with either GABA or benzodiazepine agonists results in an increase in the number of GABA A receptors in clathrin-coated vesicles (77 79). This loss of cell-surface GABA A receptors by endocytosis may be of clinical significance and may partially explain the development of tolerance to benzodiazepines (75,76,79,80). To further elucidate the mechanisms underlying GABA A receptor trafficking, the membrane stability of GABA A receptors has been studied in heterologous expression systems. In HEK 293 cells, GABA A receptors have been found to constitutively endocytose to endosomal compartments through a clathrin-dependent mechanism (81 83). Interestingly GABA A receptor subunit composition appears to play a role in the intracellular fate of internalized GABA A receptors. Specifically a/b-containing receptors appear to be targeted to peripheral endosomes, while a/b/g-containing receptors are targeted to late perinuclear endosomes (81). It is of central importance to understand the signaling mechanisms that 442 Traffic 2001;2:

7 Ionotropic neurotransmitter receptor trafficking Figure 4: AMPA receptor trafficking is important for neuronal plasticity. Long-term potentiation (LTP) of synapses results in increased insertion of AMPA receptors (and possibly decreased removal), whereas in long-term depression (LTD) the removal of AMPA receptors is necessary. In addition, protein kinase activity and interactions of AMPA receptors with NSF, and PDZ domain containing proteins (SAP97, GRIP/ABP and PICK1) have been implicated in the trafficking of receptors during LTP and LTD. In hippocampus and cerebellum, LTD is blocked by blocking dynamin function and clathrin-mediated endocytosis. In cerebellum blocking, the association of AMPA receptors with GRIP/ABP and PICK1 using peptides blocks LTD and, in hippocampus blocking, the association of AMPA receptors with GRIP/ABP and NSF also blocks LTD. control GABA A receptor trafficking and it is apparent that PKC activity plays a central role in controlling GABA A receptor cellsurface levels in a number of heterologous systems (81,83,84). Interestingly, PKC activity appears to predominantly act to inhibit receptor recycling, at least in HEK 293 cells (81). Clathrin-dependent endocytosis of GABA A receptors has also been demonstrated to occur from the soma and dendrites of cultured cortical and hippocampal neurons (81,85). However, receptor endocytosis appears to be more prominent for receptors on cell bodies compared to dendrites (81,85). A mechanism for the recruitment of GABA A receptors into clathrin-coated pits is suggested by the observation that receptors can associate, via their intracellular loops, with the adaptor complex AP2 (85). Interestingly, there is a strong subunit dependence for the interaction of GABA A receptors with the AP2 adaptor complex, which is restricted to the Traffic 2001;2: b1 3 and g2 subunits. In contrast, a subunit isoforms appear to be incapable of interacting with the AP2 complex (85). The functional significance of GABA A receptor endocytosis has been examined using peptide inhibitors of dynamin-dependent endocytosis (85). Blocking endocytosis in cultured hippocampal neurons using intracellular dialysis of a peptide that disrupts the association of dynamin with amphiphysin (86,87) results in a 100% increase in the amplitude of inhibitory synaptic currents over a period of 40 min (85). The rapid increase in the strength of inhibitory synaptic transmission upon blocking endocytosis suggests that GABA A receptors are being continually inserted and removed at synaptic sites, and provides strong evidence for the dynamic nature of synaptic GABA A receptors. The signaling pathways that govern GABA A receptor trafficking remain unknown; however, receptor activity, phosphorylation and rearrangements of the actin cytoskeleton mediated by the small GTPase Rac-1 have all been implicated (10,88,89). 443

8 Kittler and Moss Molecular and cellular studies have been utilized to identify molecules that facilitate the membrane trafficking and synaptic clustering of GABA A receptors (Figure 4). One protein that has been strongly implicated in GABA A receptor synaptic targeting and clustering is the microtubule binding protein gephyrin (90). This multifunctional protein is important for the synthesis of the molybdenum cofactor Moco and is also critical for the synaptic clustering of glycine receptors via a direct interaction with the glycine receptor b subunits (91). Gephyrin also binds tubulin (92) and thereby links glycine receptors to the cytoskeleton. A number of other gephyrin-binding partners have been isolated, including collybistin, an exchange factor for Cdc-42 (93), a small GTPase that regulates the actin cytoskeleton and that is implicated in polarized membrane trafficking (94,95). Gephyrin also interacts with Raft1 which mediates protein translation. Therefore gephyrin acts as a key component of the inhibitory synaptic scaffold at glycinergic synapses, recruiting proteins important for the control of actin dynamics and protein translation (9,10). Gephyrin is also found at GABAergic synapses throughout the CNS, suggesting that it may play a role in GABA A receptor clustering (96,97). Furthermore, abolishing gephyrin expression results in a drastic loss of GABA A receptor synaptic clusters in gephyrin-deficient mice (98). There is no apparent loss of GABA A receptor number in these animals, but the remaining receptors are diffusely distributed on the plasma membrane, with increased levels of intracellular GABA A receptor staining (98). The mechanisms underlying gephyrininduced GABA A receptor clustering remain unknown, but the receptor g2 subunit has been implicated, as deletion of this subunit in mice abolished receptor clustering and reduced levels of gephyrin expression (99). These experiments strongly suggest that gephyrin plays a central role in the clustering and stabilization of GABA A receptors at inhibitory synapses in the CNS. GABARAP is another microtubule-binding GABA A receptorassociated protein isolated using a yeast 2-hybrid screen that may be important for the membrane trafficking of GABA A receptors. GABARAP is a 17-kDa polypeptide that binds to the GABA A receptors via the intracellular domain of the g2 subunit and is also capable of interacting with microtubules (100,101). GABARAP has sequence similarity to two other small microtubule-binding proteins GATE-16 and light chain 3 of the microtubule-associated proteins (MAPs) 1A and 1B ( ). These three proteins share homology with the yeast protein Aut7 which facilitates vesicular transport. GATE 16 has also been shown to play a role in Golgi transport via its ability to interact with NSF (103). To examine the role of GABARAP in GABA A receptor trafficking, the subcellular localization of these two proteins has been examined. These studies reveal that GABARAP is preferentially localized to the Golgi apparatus and postsynaptic cisternae in neurons and is not a major component of the inhibitory synaptic scaffold (104,105). Recent studies have demonstrated that GABARAP, like its homolog GATE-16, is also capable of directly interacting with NSF. Furthermore, complexes of GABARAP and NSF can be found on neuronal intracellular membranes, strongly supporting a role for this protein in GABA A receptor transport (105). Interestingly, overexpressing GABARAP in mammalian cells with GABA A receptors results in an increase in cell-surface receptor clustering and also increased receptor agonist affinity (106). This intriguing observation suggests that at least in recombinant systems GABARAP can influence the level and/or functional properties of GABA A receptors expressed at the cell surface. Clearly, further studies are required to determine the precise role that GABARAP and NSF complexes play in mediating the intracellular transport of GABA A receptors. Additional GABA A receptor-associated proteins include the receptor for activated C-kinase and the bii isoform of PKC which bind directly to GABA A receptor b(1 3) subunits and are responsible for basal phosphorylation of these proteins (107,108). This intimate association of GABA A receptors with these signaling molecules may be highly significant, given that receptor phosphorylation has been suggested to play a role in controlling both GABA A receptor function and cell-surface stability (10,13,76). Finally, GABA C receptors are predominantly expressed in retinal bipolar neurons and have distinct subcellular distributions compared to GABA A receptors, suggesting differing trafficking itineraries. Interestingly, GABA C receptors selectively interact with MAP-1B (109), inferring that this protein may play a role in controlling the subcellular distribution of these receptors. Furthermore, disrupting this interaction modifies GABA C receptor agonist affinity in retinal bipolar cells (110). The precise mechanism underlying this modulation remain unresolved, but evidence from MAP-1B knockout mice suggests MAP-1B does not affect the size of GABA C receptor clusters (111). However, this result should be treated with caution as it is widely accepted that phenotypes from knockout mice can often be masked by undefined compensatory mechanisms Concluding Remarks and Future Directions It is evident that changes in the membrane trafficking of ionotropic receptors play a key role in regulating synaptic strength (8,10,22,23). Furthermore, in the case of excitatory synaptic transmission, the exocytosis and endocytosis of glutamate receptors has been directly implicated in the processes of LTP and LTD, proposed cellular models for learning and memory. Significant progress has been made in understanding the signaling pathways important for regulating the translocation of glutamate receptors into and out of the synapse (8,22,23). LTP and LTD have also been observed at GABAergic synapses in a number of brain regions (88, ). It will be important for future research to determine if processes important for GABA A receptor trafficking can underlie these forms of GABAergic LTP and LTD. These changes in the strength of GABAergic synaptic transmission could be important in modulating neuronal plasticity since this will have 444 Traffic 2001;2:

9 Ionotropic neurotransmitter receptor trafficking an important effect on the integrative properties and input output relationship of a neuron (10,115). For example, LTD of GABAergic inhibition has recently been shown to underlie the increased excitability of CA1 neurons associated with LTP (88). Finally, there is also growing evidence that the mechanisms important for the trafficking of receptors are likely to be dependent on specific protein protein interactions, some of which are regulated by phosphorylation (8,21,26,37, ). The recent observation that specific protein associations may play a role in the regulation of GABA A and glutamate receptor trafficking is important, since this may provide a mechanism for the specificity necessary for the independent regulation of receptor translocation into and out of excitatory and inhibitory synapses. 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