Membrane potential dye

Morphine was a kind gift from the University of Sydney, Australia. Pregabalin and gabapentin were purchased from Tocris, Australia. Forskolin was provided by Ascent Scientific Ltd. Culture reagents and buffer salts were supplied by Sigma Aldrich (Castle Hill, Australia) or Thermo Fisher Scientific (Waltham, MA, USA). The CAMYEL plasmid encoding the cAMP sensor YFP-EPac-RLuc was originally from ATCC (MBA-277). Phospho-ERK1/2 antibody and anti-rabbit IgG HRP linked antibody were from Cell Signaling Technologies (Danvers, MA, USA). Antibiotics were from Invivogen (San Diego, CA, USA) and coelenterazine h was from Promega (Alexandria, Australia). The membrane potential dye was purchased from Molecular Devices, CA.

We used the FLIPR system (Molecular Devices) to quantify GABA-evoked activity of human GABAARs. We chose a membrane potential dye (Molecular Devices) to measure changes in membrane potentials and stably transfected HEK293 cells that expressed α₁, β₂ and γ_2. Since we observed an increase in GABA-evoked responses when transfected with γ₂ transiently, we describe the cells as having a low level of γ-subunit expression, indicating heterogeneity of GABA_AR compositions in the cell (α₁β₂ or α₁β₂γ₂). To assay for direct agonists, fluorescence was subtracted pre- and post compound addition. To assay for PAMs, cells were treated with compound at 20 uM and then with 5 uM GABA.