Figure 1.
Basal I− Permeability at Different I− Concentrations.
YFP-GABAA2 cells were exposed to different concentrations of I− in the absence or presence of GABA. Data at 1–10 s was adjusted to a baseline of 100%. A) Representative time-courses of fluorescent quench showing basal I− permeability and maximum GABA response (GABAmax) of cells exposed to 5 mM, 10 mM, 20 mM and 40 mM NaI buffer. 0 mM NaI buffer is included in each graph as a reference. B) Concentration-responses of GABA with 0 mM, 5 mM, 10 mM, 20 mM and 40 mM NaI buffer showing I− concentration-dependent baseline shift. The data represented are mean ± SD of quadruplicate wells. The experiment was repeated twice with similar results.
Figure 2.
Blocking of GABAA Ion Channel with Picrotoxin.
YFP-GABAA2 cells were exposed to 100 µM Picrotoxin for 15 min prior to addition of different concentrations of I−. The bars furthest to the right represent activation with EC80 of GABA in 10 mM NaI buffer and blocking with 1 mM Picrotoxin. The graph shows representative mean data ± SD from two independent experiments.
Figure 3.
The Influence of I− on GABA Potency.
GABA pEC50 values for 5, 10, 20 and 40 mM I− showing statistical significance at p<0.01 (**) and p<0.05 (*) when comparing pEC50 values at different I− concentrations. The graph shows mean ± SD of two experiments.
Figure 4.
Inhibition of GABAA with Bicuculline.
Concentration-response of Bicuculline was added to YFP-GABAA2 cells 15 min prior to addition of EC80 of GABA in 10 mM NaI buffer. Data are mean ± SD of triplicate wells. IC50 = 2.14 µM.
Figure 5.
Modulation of GABA with Diazepam.
YFP-GABAA2 cells were pre-incubated with DMSO or 1 µM Diazepam 15 minutes prior to addition of a concentration-responses of GABA at 10 mM I−. The graph shows representative mean data ± SD of quadruplicate wells. The experiment was repeated 3 times with similar results.
Figure 6.
Modulation of GABA signal with Diazepam (Dia) and TPA-023.
YFP-GABAA2 cells were exposed to EC20 of GABA alone or in the presence of 1 µM of modulator. Data at 1–10 s was adjusted to a baseline of 100%. Representative time-courses of fluorescent quench at basal (10 mM I−) and maximum GABA response (GABAmax). A) Modulation of GABA EC20 quench showing a significant increase in the presence of Diazepam. B) No significant change of GABA EC20 quench in the presence of TPA-023. The experiment was repeated three times with similar results.
Table 1.
Modulation of GABA Signal with Allosteric Modulators.