Introduction

GABAergic synapses are the major sites of inhibitory transmission in the CNS. GABA (y-aminobutyric acid) is synthesized from glutamate by glutamic acid

'Brain Research Centre and Department of Psychiatry, University of British Columbia, Vancouver, BC, Canada V6T 2B5; [email protected], [email protected]

* Department of Anatomy and Division of Brain Korea 21 Biomedical Science, Korea University College of Medicine, Seoul, South Korea decarboxylase (GAD) and loaded into synaptic vesicles by vesicular GABA/ glycine transporter (VGAT). GABAergic terminals use molecular machinery in common with other terminal types to control calcium-dependent synaptic vesicle fusion and recycling. There are some important differences, for example, the absence of SNAP-25 at GABAergic terminals mediates differences in calcium dynamics and release rates in comparison with SNAP-25-dependent glutamatergic terminals1. But for the most part, the transmitter release apparatus is conserved between GABAergic and glutamatergic terminals. In contrast, the postsynaptic apparatus of GABAergic synapses is distinct from that of glutamatergic synapses. GABAergic postsynaptic elements lack the glutamatergic scaffolding and signaling proteins such as PSD-95 and CaMKII, but contain their own set of scaffolding and signaling proteins. Major components of GABAergic postsynaptic elements are GABAA receptor subunits, gephyrin, cadherins-catenins, the dystrophin glycoprotein complex (DGC), and neuroligin-2 (Figure 19.1). We review here these major elements of GABAergic postsynaptic sites, with emphasis on the primary functional element, GABAa receptors. Furthermore, as discussed below, none of these five major components have been found to directly interact, one of the indications that the study of GABAergic postsynaptic elements is still in its infancy, with much still to be discovered.

A particularly interesting aspect of GABAergic synapses is their distribution on postsynaptic neurons. Whereas glutamatergic synapses occur primarily on dendritic spines of mature pyramidal neurons, GABAergic synapses occur on dendrite shafts, soma, and the axon initial segment (Figure 19.2). Thus some inhibitory synapses are close to the site of action potential initiation and may have a strong influence on cell firing. In cortex in vivo, innervation of different postsynaptic domains is mediated by different classes of GABAergic neurons2. Double bouquet cells innervate distal dendrites, dendrite targeting cells innervate proximal dendrites, basket cells innervate somata and proximal dendrites, and axo-axonic cells selectively innervate the axon initial segment. This circuitry contributes to shape integrative properties at multiple levels, from locally shunting excitatory currents and regulating excitability within dendritic segments to defining oscillatory activity patterns of ensembles of cortical cells.

3. GABAa RECEPTORS: STRUCTURE AND FUNCTION

GABAA receptors mediate the majority of fast synaptic inhibition in the mammalian CNS. The larger family of GABA receptors is dividend into two classes, ionotropic (GABAA and GABAC) and metabotropic (GABAB). GABAB receptors are G-protein coupled receptors formed by heterodimerization of the 7-transmembrane proteins GABABi and GABAB23. Through G-protein signaling, GABAB receptors regulate voltage-gated K+ and Ca++ channels to mediate slow postsynaptic inhibition or to reduce transmitter release. GABAC receptors are structurally similar to GABAA receptors but are generally composed of distinct subunits p1-3. Expression of GAB Ac receptors is the highest in retinal bipolar terminals where they inhibit glutamate release4. Thus unlike GABAA receptors which function as the main signal transducers at GABAergic postsynaptic sites, GABAB and GABAC receptors primarily modulate transmission, often from the presynaptic side. A question still under investigation is whether in some instances GABAA and GABAC receptor subunits may co-assemble to mediate fast inhibition5.

GABAa receptors are members of the ligand-gated ion channel family which includes glycine receptors, nicotinic acetylcholine receptors, serotonin type 3 receptors, and the 5-hydroxytryptamine type 3 (5-HT3) receptors, and are composed of pentamers (Figure 19.1; ref. 6). By binding two molecules of GABA, a conforma-tional change occurs and an integral chloride channel is opened to flow Cl- ions down a concentration gradient. GABAA receptors are considered inhibitory because in mature systems they mediate Cl- influx and hyperpolarization. However, early in development, when the intracellular concentration of Cl- is higher than the extracellular concentration, GABAA receptor activation leads to neuronal excitation7. Benzodiazepines, appreciated clinically for their sedative, anticonvulsive, and anxiolytic effects, function by binding to GABAA receptors and allosterically potentiating the effects of GABA, effectively enhancing currents at submaximal GABA concentrations.

Gephyrin

Figure 19.1. Schematic Representation of the Major Postsynaptic Proteins at GABAergic Synapses. The subunit interfaces where GABA and benzodiazepines bind to GABAa receptors is indicated. Notably, no direct interactions among GABAA receptors, gephyrin, cadherins-catenin, the dystroglycan complex, and neuroligin-2 have been reported, indicating the presence of additional as yet unidentifed components.

Gephyrin

Figure 19.1. Schematic Representation of the Major Postsynaptic Proteins at GABAergic Synapses. The subunit interfaces where GABA and benzodiazepines bind to GABAa receptors is indicated. Notably, no direct interactions among GABAA receptors, gephyrin, cadherins-catenin, the dystroglycan complex, and neuroligin-2 have been reported, indicating the presence of additional as yet unidentifed components.

Electron microscopic imaging of GABA receptors revealed a channel with a diameter of about 8 nm in the plane of the membrane, around which five subunits are arranged pseudosymmetrically8. The subunits constituting a pentameric receptor have a common structure made of a large extracellular amino terminus which contains a signature cysteine-cysteine loop prior to the first of four transmembrane domains (TM1-4) and a very short extracellular carboxyterminus.

TM2 is thought to form a major lining of the ion channel. A large intracellular loop between TM3 and TM4 is the most divergent and includes multiple binding sites for trafficking and postsynaptic scaffolding proteins and phosphorylation sites for diverse serine/threonine and tyrosine kinases. We further review here GABAa receptor distributions and trafficking; for reviews of functional modulation by phosphorylation see refs. 9 and 10.

A huge diversity of mammalian GABAa receptors is generated by combinations of subunits a1-6, P1-3, y1-3, 5, s, %, and 011. Spatiotemporal patterns of expression and certain rules for generating a functional receptor limit the number of possible combinations. Nonetheless, heterogeneity of GABAA receptor subtypes confers differential localization and functional and pharmacological properties, making possible fine tuning of GABAergic synaptic transmission. It is generally believed that most receptors consist of two a subunits, two P subunits, and one y subunit. Almost 60% of all mammalian GABAA receptors are thought to be a1p2y2 type, followed in abundance by a2p3y2 (15-20%) and a3pxy2 (10-15%)12. Receptors containing a4-6 or P1 form a minor population, and y1, y3, 5, s, n, or 0 are thought to replace the y2 subunit in less abundant GABAA receptor subtypes. In the rare receptors containing nonidentical a subunits, their arrangement in the pentamer confers distinct properties. Finally, additional diversity is generated by alternative splicing of the y2 subunit. The short (y2S) and long (y2L) forms are distinguished by the absence or presence of eight amino acids within the cytoplasmic loop between TM3 and TM413.

The physiological significance of individual GABAA receptor subunits has been studied intensively with genetically targeted mice (Table 1; reviewed in ref. 14). The Y 2 subunit is essential for mouse survival, presumably due to its presence in the majority of GABAA receptors. Heterozygous Y 2 +/- mice exhibit increased reactivity toward aversive stimuli and enhanced responsiveness in trace fear conditioning, and thus may provide an animal model of anxiety disorders33. Deletion of the P3 subunit also results in a severe phenotype, poor coordination, hyperactivity, hyper-responsiveness, increased seizures, and cleft palate, the latter due to non-neuronal expression of P329,38. In contrast, deletion of P2 results in little phenotype; P3 may be more essential due to its expression earlier in development. A series of mice with point mutations (H101R) rendering individual a subunits insensitive to benzodiazepine site ligands revealed that a1 is largely responsible for the sedative and amnesic effects of benzodiazepines, whereas a2 is largely responsible for their anxiolytic actions12. Furthermore, in visual cortex, GABAergic circuitry involving a1 but not a2 subunit mediates critical period ocular dominance plasticity, as also demonstrated using the H101R mutant mice39.

4. GABAa RECEPTORS: SYNAPTIC AND EXTRASYNAPTIC DISTRIBUTIONS

The remarkable heterogeneity of GABAA receptor subunits appears in part related to different functions mediated by specific subunit combinations at different subcellular locations10,40. GABAA receptors a1,2,3,6PxY 2 are concentrated at postsynaptic sites in hippocampus, cerebral cortex, and cerebellum, where they mediate phasic inhibition initiated by discrete vesicular GABA release. Receptor combinations a5PxY 2 and a4,6Px8 are predominantly or exclusively

Table 1. Summary of GABAa Receptor Functional Analysis by Targeted Mutagenesis in Mice.

Subtype (Reference)

Animal phenotype

Cellular phenotype

al KO (15-17)

Viable, fertile, body weight reduction, tremor on handling

No spontaneous seizures, but increased bicuculine-induced seizures

Loss of more than 50% of all GABAA receptors Reduced a6,P2/3 and 72 Increased a2, a3

al H101R* (18,19)

Normal appearance

Decreased DZ-induced sedation and amnesia and anticonvulsant actions

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