GABA_Creceptors are members of the Cys-loop family of ligand gated ion channels (LGICs), which also includes the GABA_A, 5-HT₃, glycine, and nicotinic acetylcholine receptors. These receptors are pentameric in nature, with 5 subunits surrounding a central pore and the agonist binding site located at the interface between 2 adjacent subunits in the extracellular domain. The GABA 1 receptor is homo-pentameric, and its pharmacology suggests that it is a subclass of the GABA_C receptor type. The function of the GABA 1 receptor has been assessed using whole cell voltage clamp of HEK293 and COS cells, and also two-electrode voltage clamp in Xenopus oocytes (Kusama et al., 1993). However, its existence as an ionotropic protein permits its investigation by the FLEXstation, using a membrane potential dye. Here, we present data supporting the validity of the FLEXstation in assessing GABA 1 receptor function, and also reveal the importance of tyrosine (Y) 102 to this receptor's action.

Human GABA 1 subunit (Torres et al., 2002) was subcloned into pcDNA3 (Invitrogen) and Y102 mutated to W, S, and A using the Kunkel Method (Kunkel, 1985). Plasmids were transfected into HEK293 cells using electroporation, and subsequently grown on 96 well plates. FLEXstation assays were performed 2-3 days after electroporation using the Membrane Potential Assay Kit (Molecular Devices) according to recommendations, with the following modifications. Cells were washed twice with 1x Reagent Buffer, then 50µl of this buffer plus 50µl Membrane Potential Assay Reagent was incubated with cells at room temperature for 1h before starting the assay.

Robust responses were observed from the GABA 1 receptor in response to GABA and also the partial agonist muscimol. Analysis of these data yielded pEC₅₀ values of 6.23 ± 0.08 (n=3) for GABA and 5.98 ± 0.08 (n=3) for muscimol. These results are in agreement with previously published data (e.g. Chang et al., 2000). Studies with the competitive antagonist 3-aminopropylphosphonic acid showed that GABA responses were abolished by 300µM of this compound.

Responses of the Y102 mutants were also robust, though all of the mutants yielded pEC₅₀ values significantly different to wildtype (P<0.05, Students t-test). The observed pEC₅₀ values using GABA were: Y102S = 3.83 ± 0.09 (n=4), Y102W = 3.50 ± 0.10 (n=3), Y102A = 2.77 ± 0.21 (n=3). The Y102S and Y102W mutations have been tested in oocytes (Torres et al., 2002), and also show large shifts in EC₅₀s. A model of the GABA 1 receptor extracellular domain has been created (Harrison, unpublished data) and shows Y102 to be at the interface between subunits, suggesting this residue may be involved in ligand binding.

We conclude that the FLEXstation is a suitable instrument for characterising GABA 1 receptor responses, and that Y102 is a critical residue for correct receptor function.

Chang, Y. et al. (2000) Mol Pharmacol 58, 1375-80.
Kunkel, T.A. (1985) Proc Natl Acad Sci USA 82, 488-92.
Kusama, T. et al. (1993) Br J Pharmacol 109, 200-06.
Torres, V.I. et al. (2002) J Biol Chem 277, 43741-8.