α5-SOP002 selectively targets α5 subunits of
GABAARs
An α5β2γ2-HEK293 cell line was developed to investigate the selectively
of α5-SOP002 towards the α5-containing GABAARs. The cell
surface expression of all three GABAAR subunits in this
cell line was characterised using immunocytochemistry (Figure 2 (A))
with subunit-specific antibodies. The responsiveness of the
α5β2γ2-HEK293 stable cell line to GABA and diazepam demonstrated the
presence of functional α5β2γ2-GABAARs at the cell
surface (Figure 2 (D)), while application of α5-SOP002, immediately
following GABA, confirmed its activity as a negative allosteric
modulator (i.e. inverse agonist) of these receptors. These
experiments were repeated using the α1β2γ2-HEK293 and α2β2γ2-HEK293
stable cell lines in order to test the specificity of α5-SOP002. The
cell surface expression of α1β2γ2-and α2β2γ2-GABAARs was
also demonstrated using immunocytochemistry with subunit-specific
antibodies (Figure 2 (B-C)), as shown previously (Brown et al., 2016;
Fuchs et al., 2013).
HEK293 cells expressing α5β2γ2-GABAARs responded to GABA
(10 μM), puff-applied (5 s) in close proximity, with a large
hyperpolarisation, recorded at a membrane holding potential of -60 mV.
This was also recorded in the α1β2γ2-HEK293 and α2β2-HEK293 stable cell
lines (Figure 2 (E-F)).
The response of the three cell lines to GABA was measured and the
changes of the response after bath-application of 1 μM α5-SOP002
followed by puff-application of the broad spectrum
GABAAR modulator, diazepam (1 μM) was also analysed
(Figure 2 (D-F)).
Bath-application of α5-SOP002 (1 μM) significantly reduced the
hyperpolarising GABA inhibitory response in cells expressing α5β2γ2-
GABAARs (mean ± SEM: control GABA: 10.0 ± 5.0 mV;
α5-SOP002: 5.12 ± 2.2 mV; P <0.05, n = 8 ), while
puff-application of diazepam had an opposite effect leading to a
significant enhancement of GABA response (12.26 ± 6.94, P<0.05, n= 8, one-way ANOVA, Figure 2 (G)). In contrast,
there were no significant changes in the GABA response in the presence
of α5-SOP002 in cells expressing α1β2γ2-GABAARs (control
GABA: 18.0 ± 5.0 mV; α5-SOP002: 18.0 ± 4.5, n =6; Figure 2 (E)) or
α2β2γ2-HEK293 (control GABA: 13.5 ± 11.5 mV; α5-SOP002: 13.0 ± 10.5 mV,n =6; Figure 2 (F)). Puff-application of diazepam nevertheless
significantly enhanced the hyperpolarising inhibitory GABA response in
both, α1β2γ2-HEK293 (24.0 ± 7.6 mV, P <0.01, n=6) and α2β2γ2-HEK293 cells (17.0 ± 12.0, P <0.05,n= 6) (Figure 2 (H-I)). This confirmed the selectivity of
α5-SOP002 towards GABAARs containing the α5 subunit.