Figure 2. α5-SOP002 selectively targets α5 subunits of
GABAARs. Whole-cell current clamp recordings in
α5β2γ2-, α1β2γ2-, and α2β2γ2-HEK293 cells. HEK293 cells stably
expressing α5β2γ2- (A), α1β2γ2- (B), or
α2β2γ2-GABAARs (C). Immunofluorescent imaging
with 40x oil immersion objective lens shows cell surface expression of
α5, α1 or α2- (cyan), β2- (red), and γ2-GABAAR subunits
(green). (A-C) also show all the three channels merged showing α-, β2-,
and γ2-GABAAR subunit co-localisation at the cell
surface (white) along with the DIC image of the cells. Scale bar
represents 10 µm. All three stable cell lines responded to 10 µM
puff-applied GABA (D-F) in control extracellular solution
(black traces), extracellular solution containing 1 µM α5-SOP002 (red
traces), and following puff-application of 1 µM diazepam (blue traces)
at a holding membrane potential of -60 mV. The corresponding plots for
α5β2γ2-HEK293 cells (G-I) show the changes in membrane
potential in response to 10 µM GABA puffed locally, in the presence of
bath-applied α5-SOP002, and in the presence of diazepam. Only the
α5β2γ2-HEK293 cells showed an inverse agonist effect (response to GABA)
of α5-SOP002. All three cell lines however, showed an enhancement of
response to GABA in the presence of diazepam. Statistically significant
data are shown with * for P <0.05 and ** for P<0.01.