Figure 2.

Competitive inhibition of neurosteroid analogue photolabeling. (A) Structures of the neurosteroid analogue photolabeling reagents KK200 and KK123. (B) Structure of the α₁β₃ GABA_A receptor transmembrane domains highlighting residue Y442 on β₃-TM4 (purple) which is labeled by KK123 and residue G308 on β₃-TM3 (red) which is labeled by KK200. The α₁ subunit is shown in grey and the β₃ subunit in turquoise. (C) photolabeling efficiency of β₃ subunit TM3 by 3 μM KK200 in α₁β₃ GABA_A receptors in the absence (control) or presence of either 10 or 30 μM allopregnanolone (ALLO), ent-allopregnanolone (ent-ALLO), pregnanolone (PREG) or ent-pregnanolone (ent-PREG). There was a statistically significant difference between groups as determined by one-way ANOVA (F(6, 14) = 9.393, p < 0.001). Bonferroni’s post-hoc multiple comparison test of the means showed that the effects of ALLO, PREG, and ent-PREG were all significantly different than control (p < 0.001 vs. control for each comparison) whereas the effects of ent-ALLO were not significantly different than control (p = 0.85 for control vs. 10 µM ent-ALLO and p = 0.06 for control vs. 30 µM ent-ALLO). (D) Photolabeling efficiency of β₃ subunit TM4 by 3 μM KK123 in α₁β₃ GABA_A receptors in the absence (control) or presence of 30 μM competitive steroid. There was a statistically significant difference between groups as determined by one-way ANOVA F(4, 10) = 237.0, p < 0.0001). Bonferroni’s post-hoc multiple comparison test of the means showed that the effects of ALLO, PREG, ent-ALLO and ent-PREG were all significantly different than control (p < 0.0001 vs. control for each comparison) In Panels C and D data are shown as mean ± range with n =3 for each point. Statistical differences: *** = p < 0.001; **** = p < 0.0001; ns = not significant.