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Figure 19.2. Cellular Distribution of GABAergic Synapses as Revealed by Immunofluorescence of Hippocampal Neurons in Dissociated Culture. Upper panels: GABAA receptors form postsynaptic clusters on the soma, dendrite shafts, and axon initial segment; the axon initial segment is identified by immunoreactivity for Na+ channels (arrow). Middle panels: GABAA receptors cluster selectively opposite GABAergic terminals immunoreactive for glutamic acid decarboxylase (GAD, arrows) and not opposite glutamatergic terminals immunoreactive for vesicular glutamate transporter 1 (VGlut1). Lower panels: GABAergic synapses identified by clusters of the scaffolding protein gephyrin form on dendrite shafts (arrows), whereas glutamatergic synapses identified by clusters of the scaffolding protein PSD-95 form mainly on dendritic spines (arrowhead) protruding from the shafts. The dendrite outline is roughly traced. Upper panel images were generated by Dr. Anuradha Rao.

Figure 19.2. Cellular Distribution of GABAergic Synapses as Revealed by Immunofluorescence of Hippocampal Neurons in Dissociated Culture. Upper panels: GABAA receptors form postsynaptic clusters on the soma, dendrite shafts, and axon initial segment; the axon initial segment is identified by immunoreactivity for Na+ channels (arrow). Middle panels: GABAA receptors cluster selectively opposite GABAergic terminals immunoreactive for glutamic acid decarboxylase (GAD, arrows) and not opposite glutamatergic terminals immunoreactive for vesicular glutamate transporter 1 (VGlut1). Lower panels: GABAergic synapses identified by clusters of the scaffolding protein gephyrin form on dendrite shafts (arrows), whereas glutamatergic synapses identified by clusters of the scaffolding protein PSD-95 form mainly on dendritic spines (arrowhead) protruding from the shafts. The dendrite outline is roughly traced. Upper panel images were generated by Dr. Anuradha Rao.

More generally, a molecular signal(s) from GABAergic axons, and not GABA itself, is the major cue for aligning postsynaptic receptors with the presynaptic release apparatus. Neuron culture studies revealed the surprising finding that, when GABAergic input is lacking or very limited, GABAA receptors and gephyrin do not remain diffusely localized but rather cluster opposite glutamatergic terminals45,46. The presence of such mismatched appositions was interpreted to indicate the existence of a shared family of proteins that participate in aligning pre-and post-synaptic elements at both GABA and glutamate synapses45. Indeed, neurexins and neuroligins were later found to fit this prediction. Neurexins alone presented to dendrites induce clustering of GABAA receptors and gephyrin via binding to neuroligin-2, and induce clustering of NMDA receptors and PSD-95 via binding to neuroligin-147. Further information about neuroligins and neurexins, as well as other components of GABAergic synapses and their potential roles in localizing GABAA receptors, is discussed in separate sections below on gephyrin, cadherins-catenins, the DGC, and neuroligin-2.

Excellent progress has been made in defining the role of the y 2 subunit in synaptic targeting of GABAa receptors, and mechanisms of y 2 localization. Deletion of y 2 in mice results in a profound decrease in postsynaptic clustering of a1 and a2 and a parallel reduction in mlPSC frequency32. Deletion of y2 also results in dispersal of the inhibitory postsynaptic scaffolding protein gephyrin, indicating that multiple postsynaptic components are dependent on y2 for synaptic localization. Deletion of y2 by a Cre-loxP strategy in hippocampus during the third postnatal week also results in loss of punctate immunoreactivity for a2 and gephyrin34. Thus y2 is required for development and ongoing maintenance of synaptic GABAA receptor localization. Loss of y2 did not affect presynaptic VGAT puncta, suggesting that postsynaptic receptor clustering is not important for maintaining contact by presynaptic terminals. Subunit y3 or the individual y2S or y2L splice variants could all partially compensate for y2 depletion and rescue the clustering of a1 and a2 subunit containing receptors48,49. To further dissect mechanisms of y2 synaptic targeting, Alldred et al.50 tested chimeric a2/y2 constructs for rescue of synaptic receptor localization and function in hippocampal neuron cultures from y2 knockout mice. The surprising finding is that TM4, not the major cytoplasmic loop, is necessary and sufficient for postsynaptic clustering of GABAA receptors, but both TM4 and the cytoplasmic loop are required for synaptic gephyrin clustering and restoration of mIPSCs. Thus transmembrane proteins as well as cytoplasmic components will likely feature in future studies of mechanisms underlying synaptic targeting of GABAA receptors.

Like other neurotransmitter receptors51, GABAA receptors are presumably localized to synapses via dynamic protein-protein interactions, and thus in flux with receptor pools on the extrasynaptic plasma membrane and in intracellular compartments. Even for GABAA receptors that are concentrated at synaptic sites, there are detectable extrasynaptic levels. Quantitative immunogold labeling estimates the relative enrichment of GABAA receptors at postsynaptic sites to be ~200 fold, for a1 and P2/3 subunits in cerebellar granule cells52. Functional tagging of a1 and activity-dependent block was used to demonstrate mobility of functional GABAA receptors in cultured hippocampal neurons53. It was estimated that the entire cohort of synaptic receptors may turn over within ~14 min. Labeling GABAA receptors with antibodies against extracellular epitopes and monitoring redistribution in live cells also reveals considerable dynamics. Van Rijnsoever et al.54 report that surface-labeled receptors accumulate in an intracellular pool beneath the postsynaptic membrane, although perhaps limited accessibility of synaptic receptors to extracellular antibodies combined with lateral mobility may also contribute to the dynamics observed. Clearly, mechanisms that regulate trafficking of GABAA receptors, from synthesis in the endoplasmic reticulum through exocytotic transport and surface membrane insertion, lateral diffusion, endocytosis and recycling, and finally to degradation, will all be important in determining the steady-state numbers of synaptic GABAA receptors.

5. GABAa RECEPTORS: TRAFFICKING

Many studies suggest that receptor assembly occurs by defined pathways and there exist specific mechanisms that limit the combinations and arrangements of GABA receptor subunits contributing to functional surface GABAA receptors55. Most single GABAA receptor subunits expressed alone in HEK cells or Xenopus oocytes are retained in the endoplasmic reticulum. Exceptionally, while P3 subunits can form pentobarbitol-sensitive homomeric surface receptors56, P3 preferentially oligomerizes with other subunits, and P3 homomers are not thought to exist in neurons. When co-expressed, a1, P2, and Y2 form heteromeric receptors, with a1y2 and P2y2 combinations retained in the endoplasmic reticulum, and only a1P2 and a1P2y2 making functional surface receptor57. Additional studies have defined specific residues in the N-terminal regions that control subunit assembly55. Recombinant dimeric and trimeric concatenated subunits linking the C-terminus of one subunit to the N-terminus of the next subunit have been useful in defining the arrangement of subunits as yPaPa and in defining positional effects of different a subunits in the complex58.

Trafficking of GABAA receptors to and from the plasma membrane is regulated by extracellular signals and by intracellular interacting proteins (Figure 19.3). While yeast two-hybrid screens with the large intracellular loop of GABAA receptor subunits have not been so successful in identifying new synaptic interacting partners, many receptor binding proteins that appear to function in regulating receptor trafficking have been found. Perhaps the best known of these is GABAA receptor-associated protein (GABARAP). GABARAP can bind the y2 intracellular domain, gephyrin, tubulin, and NSF, and is homologous to GATE-16, an intra-Golgi transport factor59-61. GABARAP also binds GRIP1, a protein initially named as a glutamate receptor-interacting protein but later found to bind the kinesin molecular motor and steer this motor to dendrites62,63. GABARAP is enriched in the Golgi apparatus and intracellular membranous compartments61. These data all suggest that GABARAP may regulate trafficking of GABAA receptors to the cell surface. The homology of GABARAP and GATE-16 to a yeast protein involved in

Figure 19.3. Schematic Representation of the Trafficking Pathways of GABAa Receptors. Receptor subunits are synthesized and assembled in the endoplasmic reticulum and Golgi complex, sorted, and targeted to the extrasynaptic or synaptic plasma membrane opposite appropriate inputs. GABAa receptor-interacting proteins GABARAP, BIG2, and GODZ are thought to regulate the exocytotic pathway. Receptors undergo constitutive endocytosis and recycling or degradation, regulated by Plic-1 and HAP-1.

Figure 19.3. Schematic Representation of the Trafficking Pathways of GABAa Receptors. Receptor subunits are synthesized and assembled in the endoplasmic reticulum and Golgi complex, sorted, and targeted to the extrasynaptic or synaptic plasma membrane opposite appropriate inputs. GABAa receptor-interacting proteins GABARAP, BIG2, and GODZ are thought to regulate the exocytotic pathway. Receptors undergo constitutive endocytosis and recycling or degradation, regulated by Plic-1 and HAP-1.

ubiquitination-like modifications during autophagy, and participation of recombinant GABARAP in this pathway, suggest another potential role in post-translational modification and protein degradation64. Another Golgi-associated protein that may regulate trafficking of GABA receptors to the surface is brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2), via direct binding to P subunits65.

A particularly interesting finding to come from the yeast two-hybrid screens is the association and direct palmitoylation of GABAa receptor y2 by the Golgi-specific DHHC zinc finger palmitoyltransferase GODZ66. Palmitoylation, or thioacylation, of cysteine residues is a reversible post-translational modification appreciated as a key regulator of plasma membrane association of cytosolic proteins. A number of neuronal transmembrane proteins are also palmitoylated, including synaptotagmin I, Kv1.1 voltage-gated channel, nicotinic a7 acetylcholine receptor, AMPA receptor subunits GluR1-4, and GABAa receptor67'68. Palmitolyation can regulate trafficking of membrane proteins and modulate function independent of trafficking. Initial experiments suggest that palmitoylation is necessary to attain normal surface levels and synaptic clustering of GABAa receptors in hippocampal culture69. Regulation by palmitoylation can be quite complex; palmitoylation of AMPA receptors at one site by GODZ reduces surface levels while palmitoylation at another site regulates agonist-induced internalization68.

GABAA receptors are subject to constitutive clathrin-mediated endocytosis and recycling, in a regulated manner (reviewed in refs. 9,10). For example, in cultured cortical neurons, within 30 min about one-quarter of surface P3-contain-ing receptors are internalized, and about half of the internalized pool is recycled back to the plasma membrane over the same time period70. Efficient endocytosis occurs by binding of the clathrin adaptor AP2 to the intracellular loop of GABAA receptor P subunits71. GABAA receptor degradation is inhibited and thus surface levels increased by binding of P subunits to Huntingtin associated protein 1 (HAP1) and by binding of a or P subunits to the ubiquitin-like protein Plic-170,72. It was suggested that Plic-1 reduces GABAA receptor polyubiquitination, thus reducing receptor targeting to the proteasome, as suggested for other ubiquitin-like proteins. For HAP1, the mechanisms of regulation of GABAA receptor might involve the interaction of HAP1 with Hrs and its involvement in receptor sorting or with the motor associated protein dynactin p150Glued. In addition to direct binding to these regulatory proteins, GABAA receptor trafficking is also extensively regulated by kinase and phosphatase pathways, providing mechanisms for modulation by multiple extracellular signals.

Three major extracellular signals have been found to regulate GABAA receptor trafficking and inhibitory synaptic signaling: insulin, epileptic activity, and brain-derived neurotrophic factor (BDNF). Insulin induces a rapid increase in surface levels of GABAA receptor and synaptic currents by a mechanism requiring phosphorylation of the P subunit by the downstream serine/threonine kinase Akt73,74. Perhaps of greatest physiological relevance is the regulation of GABAA receptors by epileptic activity. Kindling-induced epilepsy increases the synaptic content of GABAA receptors by ~75%, resulting in a corresponding increase in synaptic currents in hippocampal dentate granule neurons75. Increases in expression of multiple GABA receptor subunits have been found in dentate neurons in several experimental models of epilepsy40. Remarkably, these neurons also upregulate GAD67 and VGAT and are thought to co-release GABA with glutamate under epileptic conditions76. BDNF, released by pyramidal neurons in an activity-regulated manner, promotes the development of GABAergic interneurons. Several studies also suggest that BDNF regulates GABAergic signaling postsynaptically, by regulating cell surface levels of GABAA receptors, but the precise findings vary greatly77-80. Perhaps the magnitude and even direction of regulation of GABAA receptor trafficking by BDNF may depend on the cell type, stage of development, and duration of treatment.

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