Fig. 2: Nonsense suppression incorporation of α-hydroxy acids to eliminate specific main-chain H-bonds.
From: A single main-chain hydrogen bond required to keep GABAA receptors closed
a Depiction of a main-chain H-bond with the amide NH of an isoleucine (Ile) amino acid residue and its ablation upon substitution with its cognate α-hydroxy acid (Iah). b Oocytes are coinjected with cRNA for GABAAR subunits with a TAG stop codon at the site of interest along with tRNA ligated to either the wild-type amino acid (AA), its cognate α-hydroxy acid (ncAA), or nothing (blank). The result is expression of wild-type channels, channels incorporating the α-hydroxy acid at the TAG site, or no channels due to truncation at the TAG site, respectively. c Current trace for α1(Leu9′Thr)β2γ2 gain-of-function (GoF) receptors. Arrows indicate approximate onset of pulses of either 1 mM picrotoxin (PTX) or increasing concentrations of GABA. Open probability (Po) estimated by normalizing from the zero-current baseline in PTX to the maximal current elicited with GABA. d Total current per oocyte as illustrated in panel (c) (left to right: n = 5, 7, 3, 8; 8, 16, 17, 15; 12, 9, 8, 8) suggests reliable nonsense suppression incorporation of AA and ncAA with little to no read-through (i.e., relative lack of current for blank) in both GoF and wild type (WT) backgrounds. For example, the notation Val279* denotes a TAG stop codon at position 279, and *Val or *Vah imply nonsense suppression incorporation of Val or Vah at the TAG site resulting in Val279Val or Val279Vah, 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.
