The epilepsy mutation, ;2(R43Q) disrupts a highly conserved inter-subunit contact site, perturbing the biogenesis of GABA A receptors Tim G. Hales, a Haiyan Tang, b Karen A. Bollan, b Sara J. Johnson, b Dale P. King, b Neil A. McDonald, b Aixin Cheng, b and Christopher N. Connolly b, T a Department of Pharmacology and Physiology, The George Washington University, 2300 Eye Street NW, Washington, DC 20037, USA b Department of Pharmacology and Neuroscience, Ninewells Medical School, University of Dundee, Dundee DD1 9SY, UK Received 2 November 2004; accepted 5 January 2005 Available online 8 March 2005 Abstract Given the association of a ;2 mutation (R43Q) with epilepsy and the reduced cell surface expression of mutant receptors, we investigated a role for this residue in A1B2;2 receptor assembly when present in each subunit. Regardless of which subunit contained the mutation, mutant GABA A receptors assembled poorly into functional cell surface receptors. The low level of functional expression gives rise to reduced GABA EC 50 s(A1(R43Q)B2;2 and A1B2(R43Q);2) or reduced benzo- diazepine potentiation of GABA-evoked currents (A1B2;2(R43Q)). We determined that a 15-residue peptide surrounding R43 is capable of subunit binding, with a profile that reflected the orientation of subunits in the pentameric receptor. Subunit binding is perturbed when the R43Q mutation is present suggesting that this residue is critical for the formation of inter-subunit contacts at (+) interfaces of GABA A subunits. Rather than being excluded from receptors, ;2(R43Q) may form non- productive subunit interactions leading to a dominant negative effect on other receptor subtypes. D 2005 Elsevier Inc. All rights reserved. Introduction g-Aminobutyric acid type A (GABA A ) 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: a(1–6), h(1–3), g(1–3), y and q. The most prevalent subunit combination consists of a1h2g2(McKernan and Whiting (1996) with a stoichiometry of two a1, two h2 and a single g2(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, GABA A receptor antagonists promote epileptic seizures, whereas agonists are anticonvulsants (Morimoto et al., 2004). In keeping with this view of decreased GABA A receptor activity as a contributing factor to epileptogenesis, over the past 3 years, a number of GABA A 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 g2(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 y (Dibbens et al., 2004), the TMII–III second extracellular domain; K289M in g2(Bianchi et al., 2002; Baulac et al., 2001; Macdonald et al., 2003; Ramakrishnan and Hess, 2004), the TMIII domain; A322D/A294D in a1(Cossette et al., 2002; Fisher, 2004; Gallagher et al., 2004) and the large intracellular loop between TMIII–IV; premature stop codon in g2 (Harkin et al., 2002). The functional effects of these mutations may provide insights into GABA A 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 GABA A 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 g2(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 desensiti- zation or sensitivity to benzodiazepine modulation but a decrease in current amplitude that might be attributed to a decreased surface expression of a1h2g2(R43Q) receptors. Recent analyses of the g2(R43Q) mutation have revealed a reduction in the number of benzodiazepine-binding sites (Sancar and Czajkowski, 2004), 1044-7431/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2005.01.002 T Corresponding author. Fax: +44 1382 667120. E-mail address: c.n.connolly@dundee.ac.uk (C.N. Connolly). Available online on ScienceDirect (www.sciencedirect.com). www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 29 (2005) 120 – 127