Structure of GABAA Receptors
Regulation of GABAA Receptor Expression
The large number of GABAAR genes and the various types of neurons and glial cells in the brain with different patterns of subunit expression suggest a complex system regulating their transcription (Laurie et al. 1992a; Wisden et al. 1992; Olsen and Sieghart 2008). Major changes occur during development in the subunit expression patterns (Laurie et al. 1992b). Changes in receptor subunit expression also take place in adult brain. The changes are often suggested to reflect changes in neuronal activity. Activity-dependent signaling pathways modulate the function of both transcriptional activators and repressors, but the transcription factors responsible for the developmental and brain region/cell-specific expression of GABAAR subunits are presently unknown. Calcium is a crucial second messenger in the transduction of synaptic activity into gene expression, and it is involved in the mechanisms of GABAAR up- and downregulation (Gault and Siegel 1998; Lyons et al. 2001). Some mechanisms regulating α1 mRNA transcription have been revealed recently. The transcription factor cAMP Response Element Binding Protein (CREB) is induced in response to stimulation with neurotransmitters, neuromodulators, and neurotrophic factors. Activation of Protein Kinase C (PKC) in primary rat neocortical cultures increases transcription of α1 mRNA via phosphorylation of CREB that is bound to the GABRA1 promoter (Hu et al. 2008). In contrast, activation of Protein Kinase A (PKA) represses α1 mRNA transcription via Inducible cAMP Early Repressor (ICER) that forms inactive heterodimers with CREB (Hu et al. 2008). Brain-Derived Neurotrophic Factor (BDNF) decreases α1 transcription via activation of the Janus Kinase/ Signal Transducer and Activator of Transcription (STAT) pathway (Lund et al. 2008). BDNF-dependent phosphorylation of STAT3 induces the synthesis of ICER that binds with phosphorylated CREB at the GABRA1 promoter CRE site, thereby repressing transcription (Lund et al. 2008). BDNF has been shown to regulate transcription and cell surface expression of many GABAAR subunits, the effects being brain region- and subunit-specific (Uusi-Oukari and Korpi 2010).
Cell surface expression of GABAARs includes various interacting proteins affecting receptor cell surface expression and postsynaptic accumulation. Heterodimers of α and β subunits are initially formed in a process involving interaction with endoplasmic reticulum (ER) – associated chaperones calnexin and binding immunoglobulin protein (BiP). Heteropentameric GABAARs assembled in the ER are stabilized with ubiquitin-like protein Plic-1 that interacts with α and β subunits and facilitates the exit of assembled receptors from ER to the Golgi. GABAAR Eγ2 subunit is palmitoylated at cytoplasmic cysteine residues by the Golgi resident palmitoyltransferase GODZ, thus promoting translocation of receptors through the Golgi apparatus to the plasma membranes and to synapses. BIG2, a GTP exchange factor (GEF), is implicated in facilitating exit of GABAARs by interacting with β subunits. Translocation of GABAARs to the cell surface is further facilitated by several proteins including GABARAP (interacts with GABAAR γ subunits), N-ethylmaleimide-sensitive factor (NSF), and glutamate receptor interacting protein (GRIP). Postsynaptic clustering of GABAAR subtypes α1βγ2, α2βγ2, and α3βγ2 is facilitated by interaction of α1-α3 subunits with gephyrin, a multifunctional protein that serves as a subsynaptic scaffold organizing the spatial distribution of receptors and other proteins in inhibitory postsynaptic membranes. In addition, gephyrin-binding motifs have been identified in large cytoplasmic loops of β2 and β3. The interaction of α1-3βγ2 GABAARs with the postsynaptic cytoskeleton is regulated by the activity-dependent and calcineurin-regulated phosphorylation state of γ2 subunit. Transmembrane domain 4 and intracellular domain of the γ2 subunit have been shown to be necessary for recruiting gephyrin to the synapse. The role of gephyrin is to stabilize clustered GABAARs at the cell surface (Luscher et al. 2011; Vithlani et al. 2011; Mele et al. 2016).
GABAAergic Signaling Is Developmentally Shifted from Depolarizing (Excitatory) to Hyperpolarizing (Inhibitory)
During brain development, GABA signaling is established before glutamatergic transmission, suggesting that GABA is the principal excitatory transmitter during early development. GABAARs are expressed well before synapses are formed. GABA is released at an early developmental stage and acts as a trophic factor to modulate several essential developmental processes including neuronal proliferation, migration, differentiation, synapse formation, neuronal growth, and network construction. This early intercellular communication is based on diffusion and distal paracrinic actions that contrasts with the local fast communication provided by synaptic currents. GABA tonically reduces the speed of cell migration via GABAAR activation. Astrocytes may generate a microenvironment that controls the degree of GABAAR activation and the migration of neuronal precursors (Ben-Ari et al. 2007; Oh et al. 2016).
Proliferation of neocortical progenitors in ventricular and subventricular zones of the developing cortex is downregulated by GABAAR activation that leads to depolarization of plasma membrane and increase in intracellular Ca2+. The interneuronal precursors synthesize and release GABA, and express GABAARs to respond to the secreted GABA. Cortical entry of tangentially migrating interneuronal precursors arriving from the medial ganglionic eminence is enhanced by GABA and GABAARs. This enhanced motility of interneurons is dependent on GABAAR-mediated depolarization and downstream activation of L-type calcium channels. However, soon after interneurons enter the cortex, their spontaneous calcium oscillations and their migration terminate. This is due to an increase in the expression of KCC2 transporter which reduces the [Cl−]i and terminates depolarizing activity of GABAARs (Jovanovic and Thomson 2011).
Developmental changes in GABA signaling are determined by the progressive negative shift in EGABA that in turn reflects the developmental reduction of intracellular [Cl−]i. KCC2 is the principal transporter for Cl− extrusion from neurons. KCC2 extrudes K+ and Cl− using the electrochemical gradient for K+. Cl− extrusion is weak in immature neurons and increases with neuronal maturation. The KCC2 is strongly expressed in mature neurons (while the expression of NKCC1 is strongly downregulated), thus underlying the developmental changes in Cl− extrusion. K+-Cl− cotransport also contributes to the low [Cl−]i in mature neurons. Developmental expression of KCC2 is pivotal for development of hyperpolarizing GABAAR-mediated inhibition (Ben-Ari et al. 2007).
Synaptic (Phasic) and Extrasynaptic (Tonic) GABAA Receptor-Mediated Inhibition
The receptor subtypes α1-3βγ2 form clusters in synapses. They are activated with a very high local concentration of presynaptically released GABA. A large number of clustered synaptic receptors are activated very quickly and their desensitization is also fast. In contrast to α1-3βγ2 combinations that are clustered in synapses, α5βγ2 receptor subtype is predominantly clustered extrasynaptically by interaction with phospho-activated radixin, which links these receptors to submembrane microfilaments (Luscher et al. 2011). Part of αβγ2 receptors are freely moving in extrasynaptic portion of plasma membrane. In addition to α4βδ and α6βδ receptors that are localized exclusively extrasynaptically, part of each αβγ2 receptor subtypes contribute, in addition to αβδ receptors, to the production of the continuous tonic inhibition (Fig. 4). α4/6βδ receptors are particularly suited for tonic activity, because they possess high affinity for GABA and are thus activated by the ambient GABA leaking out from synapses or released extrasynaptically. In addition, α4/6βδ receptors desensitize very slowly, therefore being tonically active (Luscher et al. 2011; Belelli et al. 2009).
In addition to production of receptor subtype-selective drugs with minimal adverse effects, one of the major challenges in GABAAR research is to resolve the signaling molecules and pathways responsible for developmental and brain region/cell-specific regulation of GABAAR subunit and receptor subtype expression. The work is already in progress and new challenges are arising from the progression.
- Hu Y, Lund IV, Gravielle MC, Farb DH, Brooks-Kayal AR, Russek SJ. Surface expression of GABAA receptors is transcriptionally controlled by the interplay of cAMP-response element-binding protein and its binding partner inducible cAMP early repressor. J Biol Chem. 2008;283:9328–40.PubMedCrossRefPubMedCentralGoogle Scholar