GABAARs [GABA (γ-aminobutyric acid) type-A receptors] are heteropentameric chloride-selective ligand-gated ion channels that mediate fast inhibition in the brain and are key therapeutic targets for benzodiazepines, barbiturates, neurosteroids and general anaesthetics. In the brain, most of the benzodiazepine-sensitive synaptic receptor subtypes are assembled from α1-3, β1-3 and γ2 subunits. Although it is evident that the pharmacological manipulation of GABAAR function can have profound effects on behaviour, the endogenous mechanisms that neurons use to promote sustained changes in the efficacy of neuronal inhibition remain to be documented. It is increasingly clear that GABAARs undergo significant rates of constitutive endocytosis and regulate recycling processes that can determine the efficacy of synaptic inhibition. Their endocytosis is regulated via the direct binding of specific endocytosis motifs within the intracellular domains of receptor β1-3 and γ2 subunits to the clathrin adaptor protein AP2 (adaptor protein 2). These binding motifs contain major sites of both serine and tyrosine phosphorylation within GABAARs. Their phosphorylation can have dramatic effects on binding to AP2. In the present review, we evaluate the role that these phospho-dependent interactions play in regulating the construction of inhibitory synapses, efficacy of neuronal inhibition and neuronal structure.
- adaptor protein (AP)
- γ-aminobutyric acid (GABA)
- γ-aminobutyric acid type-A receptor (GABAAR)
- synapse inhibition
Fast synaptic inhibition in the brain is mediated largely via GABAARs [GABA (γ-aminobutyric acid) type-A receptors], which are chloride-selective ligand-gated ion channels. These proteins are also clinically relevant drug targets for anxiolytic, sedative, anticonvulsant and hypnotic agents, including benzodiazepines, barbiturates and some general anaesthetics. Deficits in the functional expression of GABAARs are relevant in such neuropsychiatric disorders as autism, anxiety disorders, cognitive deficits, depression, epilepsy, schizophrenia and substance abuse. Given the roles that GABAARs play in neuronal inhibition, as drug targets and in pathology, there is significant interest in how neurons regulate both the number of these receptors that are expressed on the neuronal cell surface and their activity.
Molecular analysis has revealed that GABAARs belong to the superfamily of cysteine-loop ligand-gated ion channels that comprises nACh (nicotinic acetylcholine) receptors, strychnine-sensitive glycine receptors and 5-HT3 (5-hydroxytryptamine type-3) receptors . Members of this receptor family are heteropentameric glycoproteins composed of homologous subunits that specifically recognize one another and assemble around an intrinsic ion channel. Each subunit has a common structure consisting of a large extracellular ligand-binding N-terminal region and a short, barely extruding C-terminus separated by four highly conserved hydrophobic TM (transmembrane)1–4 domains. In addition, a major cytoplasmic loop lies between TM3 and TM4 .
To date, 19 GABAA receptor subunits have been identified in the mammalian brain. These can be divided into eight classes, namely α1-6, β1-3, γ1-3, δ, ε, π, θ and ρ1-3, and provide the basis for extensive heterogeneity of GABAAR structure . However, consensus opinion suggests that most of the synaptic GABAAR subtypes are composed of α1-3, β1-3 and γ2 subunits , whereas receptors containing α4/6, β1-3 and δ subunits form specialized populations of extrasynaptic receptors that mediate tonic inhibition [4,5]. Benzodiazepine-sensitive α1-5, β1-3 and γ2 subunit-containing receptors also mediate tonic inhibition .
GABAARs are dynamic entities on the plasma membrane that undergo constitutive endocytosis and recycling
After assembly within the endoplasmic reticulum, transport-competent GABAARs traffic through the secretory pathway and are then inserted into the plasma membrane primarily at extrasynaptic sites [6,7]. Extrasynaptic GABAARs exhibit high rates of lateral mobility, providing a mechanism for newly inserted receptors to access synaptic sites where they are stabilized via interaction with the inhibitory postsynaptic scaffold [7,8]. Of central importance in stabilizing GABAARs at these subcellular specializations is the inhibitory scaffold protein gephyrin. Gephyrin binds to specific amino acid motifs within the intracellular domain of the α2 subunit, linking GABAARs to the actin cytoskeleton and microtubules [9,10]. Extrasynaptic GABAARs are rapidly removed from the plasma membrane via clathrin-dependent endocytosis and, using biotinylation, it has been estimated that in excess of 20% of the total cell-surface population of GABAARs is internalized within 15 min [7,11]. Over short time courses, endocytosed receptors are rapidly recycled back to the plasma membrane for re-insertion, whereas over longer time periods they can be directed to lysozomes for degradation . This sorting decision is regulated via a direct interaction of GABAARs with HAP-1 (huntingtin-associated protein-1), which preferentially targets receptors for recycling . Consistent with this, mice devoid of HAP-1 have reduced numbers of cell-surface GABAARs and deficits in synaptic inhibition .
To examine the consequences of this rapid exchange of GABAARs between the cell surface and intracellular compartments, the effects of global inhibitors of endocytosis on the efficacy of synaptic inhibition have been examined. Blockade of dynamin activity with P4 peptide, an accepted inhibitor of endocytosis, results in a significant increase in the amplitude of mIPSCs (miniature inhibitory postsynaptic currents; approx. 100%) over a time course of 30 min in hippocampal neurons [13–15]. This finding suggests that blockade of endocytosis results in an increase in the number of functional GABAARs at synaptic sites. Consistent with this, imaging studies have revealed that P4 peptide also dramatically increases the residence time of GABAARs on the plasma membrane .
Phospho-dependent binding of the clathrin adaptor AP2 to GABAAR β subunit isoforms
A critical determinant of membrane protein endocytosis is recruitment into clathrin-coated pits prior to the formation of endocytotic vesicles. This process is facilitated by the clathrin adaptor protein AP2, which forms a link between cargo and clathrin. AP2 is a heterotetrameric complex composed of two large (~100 kDa) α and β2 subunits, a medium (50 kDa) μ2 subunit, and a small (19 kDa) σ2 subunit. These subunits are, in this context, commonly referred to as adaptins. The α-adaptin is responsible for targeting the protein to the plasma membrane, where the β2-adaptin interacts with clathrin to trigger clathrin assembly, forming coated pits. This in turn leads to the activation of μ2-adaptin phosphorylation, inducing a conformational change in the subunit that allows the complex to directly bind to endocytic motifs in cell-surface receptors, clustering the protein cargo into the assembling coated pit [16,17].
Consistent with their high rates of clathrin-dependent endocytosis, GABAARs are found in clathrin-coated pits and are also intimately associated with AP2 as measured via co-immunoprecipitation [13,18–20]. In vitro binding assays were used to further assess which components of AP2 bind to GABAAR subunits. These assays revealed that the μ2–AP2, but not the α, β2 or σ2 subunit, is capable of binding to the intracellular domains of the GABAAR β1-3 and γ1-3 subunits, but not to the corresponding regions of the α1, α3 or α6 subunits [13,21].
Molecular analysis was utilized to delineate the amino acids responsible for μ2–AP2 binding in the receptor β subunit isoforms. This revealed a conserved amino acid motif between residues 400 and 412 of these subunits that is sufficient to mediate μ2–AP2 binding, namely KTHLRRRSSQLK in the case of β3 . Similar atypical ‘basic patch’ binding motifs for μ2–AP2 have been identified in AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptors and the vesicle-associated protein Stg1 [22,23]. Intriguingly, this motif also contains the principal phosphorylation sites for both cAMP-dependent PKA (protein kinase A) and PKC (protein kinase C) within the β3 subunit Ser408/Ser409 [24,25]. Thus phosphorylation of the β3 subunit on Ser408/Ser409 may be of significance in regulating GABAAR binding to μ2–AP2 and thus their endocytosis. To study this, the effects of phosphorylating Ser408/Ser409 on the binding of μ2–AP2 to the β3 subunit were analysed. As measured by in vitro binding, phosphorylation of Ser408/Ser409 by either PKA or PKC drastically reduced the binding of μ2–AP2 to the β3 subunit. Likewise, phosphorylation of the β1 subunit on Ser409 abolished μ2–AP2 binding to the intracellular domain of this receptor subunit. Using surface plasmon resonance, high-affinity binding of μ2–AP2 to a peptide corresponding to residues 400–412 of the β3 subunit was evident (Kd=300 nM), which was reduced to 1900 nM for peptides chemically phosphorylated on Ser408/Ser409 .
To explore the significance of this phospho-dependent interaction for synaptic inhibition, the effects of a peptide corresponding to residues 400–412 of the β3 subunit on the properties of mIPSCs (miniature excitatory postsynaptic currents) have been measured. This peptide produced a time-dependent increase in the amplitudes of mIPSCs in cultured neurons in a manner that was occluded by inhibitors of dynamin without modifying the properties of mIPSCs . In contrast, a peptide corresponding to residues 400–412 chemically phosphorylated on Ser408/Ser409 did not modify the properties of mIPSCs .
Consistent with these electrophysiological studies, enhancing the phosphorylation of Ser408/Ser409 by the pharmacological activation of PKC increased the cell-surface expression levels of GABAARs containing β3 subunits. In parallel with this enhanced cell-surface expression and phosphorylation, reduced binding of the β3 subunit to the AP2 adaptin was evident as measured by immunoprecipitation [25a].
It is important to note that phosphorylation of Ser408/Ser409 in the β3 subunit was subject to dynamic modulation by both neurotransmitter and growth factor receptors that activate both PKA and PKC signalling cascades. These modulatory receptors included D1 (dopamine type-1) and D3 and TrkB (tropomyosin receptor kinase B) receptors [24,26,27]. This functional cross-talk may provide input-specific control of GABAAR phosphorylation and thus affect the efficacy of synaptic inhibition by modulating receptor endocytosis and hence accumulation on the plasma membrane.
Phospho-dependent binding of the clathrin adaptor AP2 to GABAAR γ subunit isoforms
Approaches similar to those outlined above were used to determine the amino acid residues within the γ2 subunit that mediate binding to μ2–AP2. This resulted in the identification of a classical tyrosine-based binding motif (YXXφ, where φ is a hydrophobic amino acid) centred on Tyr367 in the (YGY367ECL) in the γ2 subunit [17,28]. Significantly, both Tyr367 and the adjacent tyrosine residue Tyr365 are the principal sites of phosphorylation for Src family members in GABAARs [29,30]. Using surface plasmon resonance coupled with crystallography, it was evident that this motif bound μ2–AP2 with an affinity of 40 nM, an interaction critically dependent on Tyr367 . Phosphorylation of either Tyr365 or Tyr367 also ablated μ2–AP2 to the γ2 subunit. Introduction of a peptide containing Tyr365/Tyr367 into neurons produced a large increase in mIPSC amplitude that was accompanied by an increase in the number of receptors on the cell surface , an effect not replicated by a peptide in which Tyr365/Tyr367 had been mutated to alanine residues.
In addition to this tyrosine-based motif, an additional μ2–AP2 motif was identified between residues 324 and 335 of the γ2 subunit, namely RKPSKDKDKKK. This motif is enriched in basic amino acids, similar to that identified in receptor β subunit isoforms . It is notable that this sequence contained Ser327, a site of phosphorylation for both PKC and CaMKII (Ca2+/calmodulin-dependent protein kinase II) with GABAARs [32,33]. It will be interesting to examine the role that phosphorylation of Ser327 plays in regulating the binding of GABAARs to μ2–AP2.
GABAAR endocytosis regulates the number and size of inhibitory synapses and the maturity of dendritic spines
To examine the significance of GABAAR endocytosis in the construction of inhibitory synapses, imaging studies were performed in neurons expressing fluorescent β3 subunits in which Ser408/Ser409 had been mutated to alanine residues (β3S408A/S409A). This mutation mimics the effects of phosphorylation by significantly reducing the binding of the β3 subunit to μ2–AP2 [25a]. As measured by live imaging, GABAARs containing β3S408A/S409A exhibited enhanced levels of cell-surface expression compared with those containing wild-type β3 subunits, a phenomenon that was mediated by reduced endocytosis [25a]. In keeping with this, the size and number of inhibitory synapses was enhanced in neurons expressing β3S408A/S409A, which was paralleled by a significant increase in both the amplitude and frequency of mIPSCs. Neurons expressing β3S408A/S409A exhibited marked deficits in the number of mature spines together with a reduction in the expression levels of PSD-95 (postsynaptic density 95) at excitatory synapses [34,35]. This deficit in maturity was clearly due to enhanced GABAergic inhibition, as it was reversed by pharmacological blockade of GABAARs. Given the critical role that spines play in excitatory transmission and in information storage, this observation suggests a critical role for GABAAR membrane trafficking in regulating spinogenesis and has profound implications for cognition.
Fast synaptic inhibition mediated by GABAARs plays a critical role in neuronal function. Deficits in this process are central to neuropsychiatric disorders ranging from autism to epilepsy. It is increasingly clear that GABAARs undergo significant rates of phospho-dependent endocytosis, a process that can shape the size and number of inhibitory synapses and neuronal excitation. Thus long-term changes in the strength of inhibitory connections by the phospho-dependent modulation of GABAAR endocytosis may contribute to synaptic plasticity and ultimately to behaviour.
S.J.M. is supported by the National Institute of Neurological Disorders and Stroke [grant numbers NS047478, NS048045, NS051195, NS056359 and NS054900].
S.J.M. serves as a consultant for Wyeth Pharmaceuticals, a relationship that is regulated by Tufts University and does not have any impact on this work.
Neuronal Glutamate and GABAA Receptor Function in Health and Disease: Biochemical Society Focused Meeting held at University of St Andrews, St Andrews, U.K., 21–24 July 2009. Organized and Edited by Chris Connolly and Jenni Harvey (Dundee, U.K.).
Abbreviations: AP2, adaptor protein 2; D1, dopamine type-1; GABA, γ-aminobutyric acid; GABAAR, GABA type-A receptor; HAP-1, huntingtin-associated protein-1; mIPSC, miniature inhibitory postsynaptic current; PKA, protein kinase A; PKC, protein kinase C; TM, transmembrane
- © The Authors Journal compilation © 2009 Biochemical Society