2.2. Expression of GABAA Receptors in HEK293 Cells

cDNAs of rat GABA_A receptor α1, α2, α3, α4, α5, β2 and β3 subunits were obtained from rat brain mRNA by reverse transcription PCR. cDNAs of α1, α2, α3, α4 and α5 subunits were cloned into vector pcDNA 3.1 (+) (Sigma, St. Louis, MO, USA) and that of β2 and β3 subunits into vector pIRES2 (EGFP) (Clontech, Mountain View, CA, USA), which allowed the expression of both the β2 or β3 subunit and green fluorescent protein from an internal ribosomal entry site (IRES). Plasmid Mini Kit (Omega, Doraville, GA, USA) was used to purify plasmids. HEK293 cells (ATCC, Manassas, VA, USA) were transfected with combinations of different α and β/EGFP subunits cDNAs using Lipofectamine 3000 transfection kit (Invitrogen, Carlsbad, CA, USA). The transfection mixture contained 1 μg α and 1 μg β/EGFP. The cells were propagated and plated on poly-l-lysine-coated glass coverslips in 35 mm dishes 10 h after transfection. EGFP-positive cells were used for electrophysiological recording 48 h after transfection.