The F-loop of the γ2 subunit contributes to diazepam efficacy in the GABAA receptor

CL Padgett and SCR Lummis. Dept of Biochemistry, University of Cambridge, Cambridge CB2 1QW

The GABAA receptor is a member of the Cys-loop ligand-gated ion channel family. GABA binding to the receptor opens an anion-selective pore and an influx of Cl- ions hyperpolarises the post-synaptic membrane. The GABAA receptor has a pentameric structure; 2α , 2β and a δ or γ subunit with ligands binding at subunit interfaces constructed of binding loops (A-F). The receptor is targeted by a number of clinically and pharmacologically interesting drugs including benzodiazepines (BZDs). BZDs bind at the a /γ subunit interface and allosterically modulate GABA efficacy. This study investigates the role of the F-loop residues R194 and W196 of the γ2 subunit in BZD binding and action.

We used the Kunkel method (1985) to mutate γ2R194 to an Asn, Gln and Ala and γ2W196 to a Tyr and Ala. HEK293 cells were co-transfected with α1 , β2 and mutant γ2 subunits at a ratio of 1:1:5 by calcium phosphate precipitation. After three days cells were harvested and 3Hflumazenil binding studies were performed with 100 μM diazepam to define non-specific binding, using methods previously described (Lummis SCR et al., 1993). RNA was prepared and injected into Stage V-VI oocytes for two-electrode voltage clamp recording 48h later in ND96 buffer (Reeves DC et al., 2001). Diazepam was always co-applied with GABA with a 5min washout period between applications.

Mutations at either γ2R194 or γ2W196 did not significantly alter the dissociation constants of 3Hflumazenil binding (Table 1). Diazepam (1 μM) potentiated an EC10 GABA in all receptors, however mutations at either γ2R194 or γ2W196 significantly altered this potentiation (Table 1).

Table 1. Dissociation constants of 3Hflumazenil and percentage potentiation of EC10 GABA response by 1 μM diazepam given as mean ± s.e.m., n = 3-9, statistical significance calculated by one-way ANOVA; * = P<0.01.

We conclude that γ2R194 and γ2W196 do not contribute to BZD binding but do contribute to allosteric modulation of the receptor. This suggests the F-loop has an important role in inter-subunit communication.

Kunkel TA (1985) Proc Natl Acad Sci USA 82(2):488-92.
Lummis SCR et al. (1993) Eur J Pharmaco 243(1):7-11.
Reeves DC et al. (2001) J Biol Chem 276(45):42035-42.