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.