Pharmacology of recombinant human GABAA receptor subtypes measured using a novel pH-based high-throughput functional efficacy assay

To facilitate the discovery of novel compounds that modulate human GABAA receptor function, we have developed a high throughput functional assay using a fluorescence imaging system. L(tk-) cells expressing combinations of human GABAA receptor subunits were incubated with the pH-sensitive dye 2′,7′bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein, then washed and placed in a 96-well real-time fluorescence plate reader. In buffer adjusted to pH 6.9 there was a robust and persisting acidification response to addition of GABA, which was antagonised by the GABAA receptor antagonist bicuculline. The concentration–response relationship for GABA was modulated by allosteric ligands, including benzodiazepine (BZ) site agonists and inverse agonists. The effects of BZ site ligands on the pH response to GABA for receptors containing α1β3γ2, α3β3γ2 or α5β3γ2 subunits were well correlated with results from electrophysiological studies on the same receptor subunit combinations expressed in Xenopus oocytes. Most modulatory compounds tested were found to be relatively unselective across the three subunit combinations tested; however, some showed subtype-dependent efficacy, such as diazepam, which had highest agonist effects on the α3β3γ2 subtype, substantial but lesser agonism on α1β3γ2 and still substantial but the least agonism on α5β3γ2. This indicates that the α subunit within the recombinant receptor expressed in L(tk-) cells can affect the efficacy of the response to some BZ compounds. Inhibitors of Na+/Cl− cotransport, anion/anion exchange and the gastric type of H+/K+ ATPase potently inhibited GABA-evoked acidification, indicating that multiple transporters are involved in the GABA-evoked pH change. This novel fluorescence-based high throughput functional assay allows the rapid characterization of allosteric ligands acting on human GABAA receptors.