Fig. 3: The β2 subunit main-chain Ile275:N-Pro273:O H-bond inhibits pore opening and is required to keep the unliganded channel closed.

a Current responses to 10–20 s pulses (arrows) of saturating GABA or 1 mM picrotoxin (PTX) for α1β2γ2 (WT) or α1(Leu9′Thr)β2γ2 (GoF) receptors after nonsense suppression incorporation of either the wild-type amino acid (Val, Ile) or its cognate α-hydroxy acid (Vah, Iah) at α1(Val279*) or β2(Ile275*). Vertical bars indicate the relative magnitude of PTX-sensitive (green) to total (gray) currents. b Unliganded open probability (P_o) per oocyte estimated as the fraction of PTX-sensitive to total current (left-to-right: n = 5, 7, 3; 8, 16, 17; 12, 9, 8). For example, the notation Ile275* denotes a TAG stop codon at position 275, and *Ile or *Iah imply nonsense suppression incorporation of Ile or Iah at the TAG site resulting in Ile275Ile or Ile275Iah, respectively. *Blank indicates an unchanged TAG stop codon. Box plots show median and interquartile intervals. P-values < 0.05 for Brown-Forsythe ANOVA with posthoc Dunnett’s T3 test shown. Values for GoF in the left or middle panels reflect oocytes from the same batches as the other recordings in each respective panel. c Free energy difference between closed and open states in the absence of ligand for each oocyte computed from the estimated open probabilities in panel b. Box plots and P-values as in panel (b). d Normalized concentration-response relations for GABA-elicited currents. Data are mean ± SEM across oocytes. Curves are the Hill equation fit to the means (Eq. 2). See Supplementary Tables 1,  2 for summary statistics and fit parameters [n per condition: left panel: GoF, 5; α1(Val279Vah), 7; α1(Val279Val), 6; middle panel: GoF, 5; β 2(Ile275Ile), 5; β 2(Ile275Iah), 8; right panel: WT, 8; β 2(Ile275Ile), 4; β 2(Ile275Iah), 6]. ^†For WT receptors, the reported unliganded open probabilities and free energies are upper and lower bounds, respectively, due to the limited ability to resolve small currents on the order of the noise.