The epilepsy mutation, γ2(R43Q) disrupts a highly conserved inter-subunit contact site, perturbing the biogenesis of GABAA receptors
Introduction
γ-Aminobutyric acid type A (GABAA)a receptors are the major sites of fast synaptic inhibition in the brain. In mammals, they are constructed as pentameric ion channels from multiple subunits selected predominantly from the following distinct classes: α(1–6), β(1–3), γ(1–3), δ and ε. The most prevalent subunit combination consists of α1β2γ2 (McKernan and Whiting (1996) with a stoichiometry of two α1, two β2 and a single γ2 (Tretter et al., 1997).
A reduction in GABAergic inhibition has been associated with anxiety (Crestani et al., 1999) and a number of models of epilepsy and is consistently expressed as a component of the epileptic phenotype (Morimoto et al., 2004). Moreover, in general, GABAA receptor antagonists promote epileptic seizures, whereas agonists are anticonvulsants (Morimoto et al., 2004). In keeping with this view of decreased GABAA receptor activity as a contributing factor to epileptogenesis, over the past 3 years, a number of GABAA receptor mutations associated with epilepsy have been discovered, each responsible for a reduction in GABAergic inhibition (Baulac et al., 2001, Bianchi et al., 2002, Bowser et al., 2002, Dibbens et al., 2004, Fisher, 2004, Gallagher et al., 2004, Harkin et al., 2002, Macdonald et al., 2003, Marini et al., 2003, Wallace et al., 2001). These mutations lie within the amino-terminal extracellular domain; R43Q in γ2 (Bianchi et al., 2002, Bowser et al., 2002, Kang and Macdonald, 2004, Macdonald et al., 2003, Marini et al., 2003, Wallace et al., 2001), and at E177A and R220C in δ (Dibbens et al., 2004), the TMII–III second extracellular domain; K289M in γ2 (Bianchi et al., 2002, Baulac et al., 2001, Macdonald et al., 2003, Ramakrishnan and Hess, 2004), the TMIII domain; A322D/A294D in α1 (Cossette et al., 2002, Fisher, 2004, Gallagher et al., 2004) and the large intracellular loop between TMIII–IV; premature stop codon in γ2 (Harkin et al., 2002). The functional effects of these mutations may provide insights into GABAA receptor structure and how this impacts on function (Connolly and Wafford, 2004). In addition to a possible primary role in a genetic predisposition to epilepsy, a reduction in GABAA receptor function is also induced by epileptic activity (Wu et al., 2004) as a result of receptor internalization by endocytosis via clathrin-coated vesicles (Blair et al., 2004).
Analysis of the functional implications of the γ2(R43Q) mutation revealed an increase in the rate of receptor desensitization coupled with a decrease in the rate of deactivation (Bowser et al., 2002). In contrast, Bianchi et al. (2002) reported that there were no changes in either the rates of receptor deactivation and desensitization or sensitivity to benzodiazepine modulation but a decrease in current amplitude that might be attributed to a decreased surface expression of α1β2γ2(R43Q) receptors. Recent analyses of the γ2(R43Q) mutation have revealed a reduction in the number of benzodiazepine-binding sites (Sancar and Czajkowski, 2004), decreased surface expression (Sancar and Czajkowski, 2004, Kang and Macdonald, 2004) and localization within the endoplasmic reticulum (Kang and Macdonald, 2004), raising the possibility that the fault lies in receptor assembly and/or exit from the endoplasmic reticulum.
A high degree of structural homology is thought to exist between distinct members of the ligand-gated ion channel superfamily (Brejc et al., 2001, Ernst et al., 2003, Hosie et al., 2003, Sigel, 2002, Trudell, 2002). As such, the function of these receptors is now being investigated in the light of their predicted structures, as determined by a comparison to the crystal structure of the acetylcholine binding protein (Brejc et al., 2001). Given this proposed high degree of structural similarity and the complete conservation of R43 in all members of the cys-loop superfamily, we investigated the role of this mutation on the assembly, surface transport and function of GABAA receptors when introduced into either the α1, β2 or γ2 subunits. In support of previous findings (Kang and Macdonald, 2004, Sancar and Czajkowski, 2004), we observed an inability of the γ2(R43Q) mutant to access the cell surface.
Furthermore, this mutation prevents the binding of this region to β2, suggesting a failure of β2–γ2 interface formation during receptor assembly. Similar results were observed when the mutation was present in either the α1 or β2, implying a common role for this region in receptor assembly.
Section snippets
Results
A comparison (T-Coffee; http://www.ch.embnet.org/software/TCoffee.html, Notredame et al., 2000) of the primary sequence surrounding the R43Q site in γ2 (residues 1–68) to the homologous regions in α1 and β2 reveal low (17.6%) sequence homology, with 30.9% (1) and 25% (2) when compared individually to γ2 and 27.9% between α1 and β2. However, a region of conservation is apparent (72.7%) when the proximal region (LLEGYDNKLRP) is compared (Fig. 1A). Upon closer examination, this arginine is part of
Discussion
Epilepsy is a common neurological disorder affecting about 1% of the population and is characterized by the presence of sporadic spontaneous seizures. Epileptic seizures are thought to result from an imbalance in excitation (glutamatergic) and inhibition (GABAergic). Indeed, seizure activity can be induced by electrical stimulation, the over-excitation of glutamate receptors or the inhibition of GABAA receptors (Morimoto et al., 2004).
A decrease in GABAA receptor function is thought to
Cell culture and transfection
COS 7 cells (ATCC CRL 1651) and HEK293 cells were maintained in DMEM (Life Technologies Ltd, UK) supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 100 mg/ml streptomycin and 100 U/ml penicillin in an atmosphere of 5% CO2. Exponentially growing cells were transfected by electroporation (400 V, infinity resistance, 125 mF, Biorad Gene Electropulser II), in the case of COS cells, and calcium phosphate precipitation, in the case of HEK cells (Bollan et al., 2003). 10
Acknowledgments
This work has been supported by the BBSRC (grant 94/C17336 awarded to CNC), Tenovus Scotland (CNC), Anonymous Trust (CNC) and the NIH (grant GM058037 awarded to TGH).
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