Figure 8. Both LGS-associated GABRB3(E180G) and IS-associated GABRB1(F246S) de novo mutations produced spontaneously gated GABA_A receptors.

(A) Representative traces from whole cell recordings showing a shift in the baseline current (seen as an outward current) with 10 μM Zn²⁺ application from cells expressing GABA_A receptors with β3(E180G) and β1(F246S) subunits (grey traces), but minimal or absent in cells expressing wt receptors (black traces). (B) Bar graphs are presented showing average outward current responses to 10 μM Zn²⁺ application. Representative spontaneous single-channel current traces recorded from cells expressing wt β3 or β3(E180G) (C), or wt β1 or β1(F246S) (E) subunit-containing GABA_A receptors in absence (upper panels) and presence (bottom panels) of 100 μM Zn²⁺. (D, F) Bar graphs show single-channel amplitude and Po of wt (black bars) and spontaneously activated mutant (gray bars) receptors. For wt β3 subunit-containing receptors the low and high conductance openings were 12.5 pS (1.1 ± 0.07 pA, n = 7) and 21 pS (1.8 ± 0.10 pA, n = 4) with Po of 0.13 ± 0.02 (n=7) and 0.06 ± 0.01 (n = 4), respectively. The β3(E180G) subunit mutation significantly increased the Po of low conductance openings (0.34 ± 0.05, 1.1 ± 0.08 pA, n = 10), without altering high conductance openings (0.10 ± 0.03, 1.7 ± 0.03 pA, n = 4, p > 0.05). The wt β1 subunit-containing receptors had conductance level 0.99 ± 0.06 pA, (n = 5) with Po of 0.03 ± 0.01 (n = 5). The β1(F246S) subunit mutation increased Po to 0.13 ± 0.02 (n = 8) but did not alter single channel conductance (1.1 ± 0.04 pA n = 8, p > 0.05). Values represent mean ± S.E.M. Two-way ANOVA with Tukey’s multiple comparisons (D) test or unpaired t-test (F) was used to determine statistical significance. ** indicate p < 0.01.