Thompson AJ , Lummis SC .

081925 Wellcome Trust

	Figure 1. Structures of the ligands used in this study (A) and an alignment (B) of the channel-lining residues of the human 5-HT3A and 5-HT3B subunits. Accession numbers for the alignment are 5-HT3A = P46098; 5-HT3B = O95264; α7 nACh = P36544.
	Figure 2. Concentration-responses curves for 5-HT, dopamine and mCPBG at wild type homomeric and heteromeric 5-HT3 receptors. Dopamine was a partial agonist at both 5-HT3A receptors (A) and 5-HT3AB receptors (B). mCPBG-evoked responses were similar to 5-HT (C), but were greater at 5-HT3AB receptors (D). Example traces are shown besides each curve. Data are normalized to the maximum 5-HT response in each oocyte and plotted as the mean ± SEM for a series of oocytes. Parameters derived from these curves are in Table 1.
	Figure 3. Concentration-response curves and the long-lived inhibition of subsequent 5-HT3 receptor agonist responses by quipazine. Partial agonist responses are seen with quipazine at both 5-HT3A and 5-HT3AB receptors (A & B). Example traces are shown besides each curve. Application of quipazine causes inhibition of subsequent 5-HT responses, and recovery at both 5-HT3A and 5-HT3AB receptors is slow (C & D). Data are normalized to the maximum 5-HT response in each oocyte and plotted as the mean ± SEM for a series of oocytes. The small size of the quipazine responses meant it was not possible to make accurate measurements of the EC50 values.
	Figure 4. Concentration-response curves and long-lived inhibition of 5-HT3 receptor responses by VUF10166. (A) Concentration-response curves showing the agonist responses to 5-HT and VUF10166 at 5-HT3A receptors. Examples VUF10166 agonist responses are compared with a maximal (30 μM) 5-HT response at 5-HT3A receptors. (B) VUF10166 activation of 5-HT3AB receptors was negligible (Rmax = 0.03 ± 0.02, n = 3). (C) Recovery from VUF10166 inhibition was slower at 5-HT3A than at 5-HT3AB receptors; at these concentrations 5-HT3AB receptors recovered from inhibition within ∼8 min, whereas recovery for 5-HT3A receptor responses was ∼25 min. Parameters from these curves are in Table 1. Data in this figure are duplicated from Thompson et al. (2012b) with kind permission.
	Figure 5. Effects of a 5-HT3AT6'S mutation on the responses of homomeric and heteromeric 5-HT3 receptors. (A) Relative to wild type responses, a decrease in 5-HT EC50 was seen at 5-HT3AT6'S receptors. (B) Quipazine, dopamine and mCPBG also have decreased EC50 values, and Rmax was increased for dopamine and quipazine. (C) Incorporation of the 5-HT3B subunit also resulted in decreased EC50 values and Rmax of dopamine and quipazine compared to wild-type receptors. mCPBG showed an apparent decrease in maximal current, but it is possible that this is due to the relative increase in 5-HT efficacy. (D) At 5-HT3AT6'SB receptors VUF10166 was a full and potent agonist. (E) The slow recovery from VUF10166 inhibition was not altered at 5-HT3AT6'S mutant receptors, but was no longer seen at 5-HT3AT6'SB receptors. Data are normalized to the maximum 5-HT response in each oocyte and plotted as the mean ± SEM for a series of oocytes. Parameters from these curves are in Table 1.
	Figure 6. Radioligand binding and 5-HT3B subunit expression in HEK293 cells. (A) Example data for competition of [3H]granisetron binding with quipazine at wild type 5-HT3AB receptors transiently expressed in HEK293 cells. The curve is representative of four similar experiments and is typical of the curves for all of the ligands studied here. Parameters derived from curves like these were averaged and the mean ± SEM values are shown in Table 2. (B) Typical immunofluorescent labelling (pAb77; Reeves and Lummis, 2006) of 5-HT3B subunits expressed with either wild type or mutant 5-HT3A subunits in HEK293 cells.

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