Whole-cell patch clamp recordings of HEK293 cells
Electrophysiological recordings of HEK293 cells stably expressing GABAARs were performed in a whole-cell, current clamp mode. The chamber containing coverslips with the cell line was continuously superfused at a flow rate of 1.8 mL/min with the extracellular medium composed of 130 mM NaCl, 4 mM KCl, 10 mM Hepes, 20 mM NaHCO3, 10 mM glucose, 1 mM MgCl2, and 2 mM CaCl2, and was equilibrated with 5% CO2/95% O2 and maintained at room temperature (~ 21-25 ̵̊C). The electrodes were filled with an intracellular solution containing (in mM), 130 KCl, 3 NaCl, 4.5 phosphocreatine, 10 Hepes, 1 EGTA, 3.5 Na-ATP, 0.45 Na-GTP, and 2 MgCl2 (adjusted to pH 7.2 with KOH, 290–300 mosmol/L), and had a final resistance of 3-8 MΩ. To test the target selectivity of α5-SOP002, the responsiveness to applied GABA was investigated and measured in HEK293 cells stably expressing either, α5β2γ2, α1β2γ2 or α2β2γ2 subunits of GABAARs. The pharmacological properties of the expressed receptors were investigated by puffer-application of GABA (1 µM; Tocris Bioscience, UK) and subsequent bath-application of α5-SOP002 (0.5-1 µM), followed by diazepam (1 µM, Tocris Bioscience, UK). The change in membrane potential after GABA puff application response was recorded. The statistical test used was one-way ANOVA with a 95% confidence interval.