Extended Data Fig. 7: TMPPAA binds to an allosteric site in the TMD.
From: Conformational transitions of the serotonin 5-HT3 receptor
a, Cavities in surface mode for the T (blue), I1 (yellow), F (purple) and I2 (green) states. Note the re-arrangement of the cavities between states. The protein is depicted in cartoon, with sticks in the right panels for residues in which mutations impair the effect of TMPPAA. Noisy densities are found in the I2 reconstruction, in or close to the cavities, but they do not permit ligand modelling and could correspond to parts of a lipid or detergent molecule. b, Side view of the transmembrane domain of I2 with mutated positions depicted as green spheres (for residues for which mutations had negligible effect on TMPPAA potency), orange spheres (for residues for which mutations reduced TMPPAA potency substantially) or red spheres (for mutations that completely or almost completely eliminated TMPPAA activity at concentrations up to 100 µM). Yellow circles indicate residues for which different mutations produced different effects. c, Functional properties exhibited by serotonin and TMPPAA as agonists at 5-HT3A receptors expressed in tsA201 cells in an FMP assay. The human 5-HT3A receptor was used for these experiments, since TMPPAA evokes a more robust agonist response through this receptor than through mouse 5-HT3A in this assay17. The colour code is similar to that in b. n.d., not determinable, w.a., weak activity. A complete table of data for all tested mutant receptors is shown in Supplementary Table 1. d, Concentration-response curves for serotonin (closed symbols) and TMPPAA (open symbols) tested as agonists at wild-type, W472A and L243W human 5-HT3A receptors expressed in tsA201 cells, using the FMP assay. Data are from a single representative experiment determined in the same 96-well plate and are given as mean based on duplicate determinations. The experiment was repeated independently with similar results 3 times for the W472A and L243W receptors and 66 times for the wild-type receptor (n for wild-type and all mutant receptors are given in c). e, Loss of TMPPAA potency at the mouse W456A mutant receptor. Currents evoked by 10 µM serotonin (blue curve) or 10 µM serotonin plus 100 µM TMPPAA (red curve) are equivalent. The grey trace, obtained in an oocyte expressing wild-type receptors (response to 10 µM serotonin, normalized for the peak amplitude), shows that desensitization in wild-type and W456A mutant receptors is similar. Current traces are representative of 3 independently repeated experiments.
