Fig. 1. Identification of h5-HT3A residues as voltage-clamp fluorometry reporter positions. (A) Surface contoured cartoon representation of a homology model structure of the pentameric h5-HT3A receptor (left) from Ladefoged et al. (2018). The principal and complementary subunits are highlighted in cyan and green, respectively, with indications of the ECD and TMD. The cross-sectional view (right) illustrates the location of the five equivalent 5-HT binding pockets (dashed circles) formed at subunit interfaces in the ECD, with 5-HT indicated in yellow surface contour. The chemical structure of the endogenous agonist 5-HT is shown below. (B) Magnified view of the ECD interface structure in cartoon representation with the orthosteric binding site and the loop A to G regions highlighted in different colors and bound 5-HT in yellow surface contour. Positions where Cys residues were introduced by mutagenesis, and the endogenous Cys residues 157 and 171 are indicated as orange and yellow spheres, respectively. (C) Representative recording traces of parallel fluorescence and current measurements from uninjected oocytes and oocytes expressing WT h5-HT3A, Y89C, Q146C, F221C, and M223C mutants during application of 100 µM 5-HT (black line). Upper traces (red) show the membrane fluorescence signal (F), and lower traces (black) show the corresponding membrane currents (I). (D-E) Summary of fluorescence changes (ΔF) (D) and currents (E) evoked by application of 100 µM 5-HT. Shown are scatter plots of ΔF from individual uninjected oocytes and oocytes injected with WT h5-HT3A or Cys-mutant h5-HT3A. The ΔF value is calculated as the percentage difference between baseline fluorescence and steady-state fluorescence during 5-HT application (see Materials and Methods). Bars represent mean ΔF (±S.E.M): uninjected; −0.71 ± 0.28 (13 oocytes), WT; −0.81% ± 0.23% (22 oocytes), D74C; 1.1% ± 0.3% (nine oocytes), Y89C; 30% ± 2% (77 oocytes), Q146C; 11% ± 1% (80 oocytes), V196C; −1.2% ± 0.4% (nine oocytes), F221C; 4.6% ± 0.7% (48 oocytes), M223C; 14% ± 1% (83 oocytes), S225C; 1.6% ± 0.6% (seven oocytes), I238C; 0.63% ± 0.15% (eight oocytes), R239C; 1.8% ± 0.4% (eight oocytes), R241C; 0.39% ± 0.11% (six oocytes), L243C; −1.8% ± 0.3% (13 oocytes), V246C; 1.1% ± 0.2% (four oocytes), V247C; 0.66 ± 0.10 (five oocytes), L249C; and 0.56 ± 0.21 (five oocytes). * Denotes mean response significantly different from WT; P < 0.05 (ANOVA with Dunnet’s correction for multiple comparisons).