The human GABAA receptors α2, α6, β3, and γ2L cloned into pcDNA3.1 expression vectors were a generous gift from Dr. Robert L. Macdonald, Vanderbilt University, Nashville, TN. L929 cells, a mouse fibroblast cell line (CCL-1), were obtained from American Type Culture Collection (Manassas, VA). L929 cells were cultured in Dulbecco’s modified Eagle’s medium (Lonza, Basel, Switzerland) supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen; ThermoFisher, Grand Island, NY) and maintained in humidified 95% air and 5% CO2 at 37°C. L929 cells were transfected using FuGENE 6 (ThermoFisher) transfection reagent in Opti-MEM reduced serum medium (Life Technologies, Benicia, CA) with an equal amount of each of the subunits (1:1:1) in combination with GFP expressed from the pEGFP-C1 vector (Invitrogen). The ratio of total cDNA to transfection reagent was 2:1. Cells were detached by trypsinization 48 hours post-transfection, washed, and plated onto poly-L-lysine–coated glass coverslips. Transfected cells were identified as GFP-expressing cells using an epifluorescence microscope for electrophysiological whole-cell voltage-clamp studies. Correct incorporation of the γ subunit was tested by determining sensitivity to diazepam, a GABAA receptor positive allosteric modulator that binds at the α/γ interface as previously described (Pressly et al., 2018).

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