GABAA receptors are ligand-gated ion channels found in the synapses of neurons that conduct chloride ions across the neuronal cell membrane. The receptor contains two binding sites for γ-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the central nervous system. Upon activation, the GABAA receptor selectively conducts Cl- through its pore. This results in a hyperpolarization of the neuron which has an in- hibitory effect on neurotransmission by diminishing the chance of a successful action potential occurring and thereby decreasing the excitability of the neuron. There are at least nineteen different individual GABAA receptor subunits that assemble the pen- tameric structure in different individual combinations to form the native receptor (α1-6, β1-3, γ1-3, δ, ρ1-3, and minor subunits) . Of these potential combinations, the recep- tors containing two of the α1-6 subunits, two of any βsubunits and one of the γ2 subunit are the most prevalent in the brain. These receptors subtypes mediate the diverse ef- fects of benzodiazepine modulation.
Here we present the establishment of an automated patch-clamp assay using tran- siently transfected mammalian cells (HEK293T) with the IonFlux instrument (Fluxion Biosciences). The assay was validated by measuring the electrophysiological response of benzodiazepine-based compound HZ166. The use of transiently transfected cells allows for maximum flexibility for the assembly of different GABAA receptor types for screening purposes and the robustness of the developed assay makes it well suited for the high-throughput screening using the IonFlux HT system. Transient transfection ap- proaches will also enable the mutant screening for other ion channel proteins.