Screening of residues located along the extracellular domain (ECD) of m5-HT3AR as potential anchors for fluorescent probes and establishment of four reporter sensors.

A. Left panel: visualization of the residues mutated, TAMRA-labelled and tested for variation of current and fluorescence on the Apo Cryo-EM structure of m5-HT3A (from Basak et al., 2018), in lateral (zoom on the ECD) and top view with two subunits in cartoon representation and the three others represented in surface mode (PDB: 6BE1). Middle panel: representation of the two isomers of the labeling fluorescent probe mixture used in this study, MTS-TAMRA (5(6)-carboxytetramethylrhodamine methanethiosulfonate). Right panel: representation of currents evoked by 50-100 µM of 5-HT on the mutants and of the absolute values of variation of fluorescence (difference between the baseline fluorescence and steady-state fluorescence upon 5-HT perfusion) recorded simultaneously. Note the representation of the four selected sensors: in dark green, I160C/Y207W; in magenta, S204C; in purple, V106C/L131W and in cyan, R219C/Y140W. B. The sensor S204C, shown on 5-HT bound conformation (5-HT represented in red; PDB: 6DG8), is located on loop C. Note that the fluorescence variation is significatively increased by the addition of the tryptophan in the sensors I160C/Y207W and V106C/L131W. (unpaired t-tests, I160C/Y207W versus I160C P value = 0,013 (*); V106C/L131W versus V106C P value = 0,0081 (**)) C. The sensor I160C/Y207W where I160C is the point of labelling with MTS-TAMRA and is represented in ball representation in fluorescent green, Y207 is mutated into tryptophan and colored in dark green. D. The sensor V106C/L131W where V106C is represented in ball representation in purple and L131 is mutated into tryptophan and colored in purple. Note the vestibular positioning of this sensor. E. The sensor R219C/Y140W, where R219C, located in pre-M1 loop, is represented in ball representation in cyan and Y140, located in the Cys-loop, is mutated into tryptophan, and colored in dark cyan. F. Molecular structures of the ligands used in this study: agonists (in red, 5-HT; in blue, mCPBG; in salmon, varenicline) and antagonists (all represented in orange, A: alosetron, G: granisetron, O: ondansetron, M: metoclopramide). G. Effect of desensitization on the dynamic of the fluorescence recordings. Examples of desensitizing currents promoted by prolonged perfusion of strong agonists (to elicit robust desensitization): mCPBG perfused on the sensor I160C/Y207W or 5-HT perfused on the sensors S204C, V106C/L131W and R219C/Y140W. Traces show that the fluorescent signal remains stable during desensitization for all the sensors. Note that the differences in the desensitization kinetics of the displayed traces are due to the variability of different oocyte batches, since the four conditions do not show significant differences in desensitization kinetics (Fig S2).

Electrophysiological and fluorescence characterization of S204C and I160C/Y207W, two sensors located in proximity to the ligand-binding site.

A. Exploration and characterization of the sensor S204C. Left upper panel: macroscopic ligand-gated currents (in black) and fluorescence (in magenta) recorded at −60mV on S204C labelled with MTS-TAMRA evoked by saturating concentrations of agonists (in red, 5-HT, 200µM ; in blue, mCPBG, 200µM; in salmon, varenicline, 400µM) and antagonists (all represented in orange, A: alosetron, G: granisetron, O: ondansetron, M: metoclopramide, all at 3µM). Left bottom panel: graphical representation of ligand-induced relative changes of current and fluorescence compared to 5-HT. The normalized values for all the ligands are compared with the mean values obtained for 5-HT. Middle left top panel: representative recording of current and fluorescence variations of S204C labelled with MTS-TAMRA upon different concentrations of perfused 5-HT. Middle left bottom panel: dose-response curves for ΔI (black) and ΔF (magenta) with mean and SEM (normalized by the maximum current of each oocytes) for application of 5-HT. Middle right top panel: representative recording of current and fluorescence variations of S204C labelled with MTS-TAMRA upon different concentrations of perfused mCPBG. Middle right bottom panel: dose-response curves for ΔI (black) and ΔF (magenta) for application of mCPBG. Right top panel: representative recording of current and fluorescence variations of S204C labelled with MTS-TAMRA upon different concentrations of perfused varenicline. Right bottom panel: dose-response curves for ΔI (black) and ΔF (magenta) for application of varenicline. B. Effect of loss-of-function mutation N101K on the sensor S204C. Same experiments and legends as for panel A. but the construct here is the sensor S204C with the additional loss-of-function mutation N101K. C. Exploration and characterization of the sensor I160C/Y207W. Same experiments and legends as for panel A, but here currents are represented in black and fluorescence in green. Note also that for the left lower panel, currents and fluorescence have been compared to mCPBG instead of 5-HT.

Electrophysiological and fluorescence characterization of V106C/L131W, located in the extracellular vestibular area and R219C/Y140W positioned at the interface area between ECD and TMD.

A. Exploration and characterization of the sensor V106C/L131W. Left top panel: macroscopic ligand-gated currents (in black) and fluorescence (in purple) recorded at −60mV on the construct with the sensor V106C/L131W labelled with MTS-TAMRA evoked by saturating concentrations of agonists (in red, 5-HT; in blue, mCPBG; in salmon, varenicline) and antagonists (all represented in orange, A: alosetron, G: granisetron, O: ondansetron, M: metoclopramide). Left bottom panel: graphical representation of ligand-induced relative changes of current and fluorescence compared to 5-HT. The normalized values for all the ligands are compared with the mean values obtained for 5-HT. Middle top panel: representative recording of current and fluorescence variations of V106C/L131W labelled with MTS-TAMRA upon different concentrations of perfused 5-HT. Middle bottom panel: dose-response curves for ΔI (black) and ΔF (purple) with mean and SEM (normalized by the maximum current of each oocyte) for application of 5-HT. Right top panel: representative recording of current and fluorescence variations of V106C/L131W labelled with MTS-TAMRA upon different concentrations of perfused varenicline. Right bottom panel: dose-response curves for ΔI (black) and ΔF (magenta) with mean and SEM (normalized by the maximum current of each oocyte) for application of varenicline. Note the significative difference between dose response of current and fluorescence at 0.5, 1 and 5 µM of perfused varenicline (unpaired t-test). B. Exploration and characterization of the sensor R219C/Y140W. Same experiments and legends as for panel A. but the construct here is the sensor R219C/Y140W, current is represented in black and fluorescence in cyan (in trace recordings and dose-response representations). C. Effect of loss-of-function mutation N101K on the sensor R219C/Y140W. Same experiments and legends as for the panel A. but the construct here is the sensor R219C/Y140W with the additional loss-of-function mutation N101K, current is represented in black and fluorescence in cyan (in trace recordings and dose-response representations).

Current and fluorescence maximum evoked by ligands (agonist and antagonists) on m5-HT3A mutants.

EC50 values for current (EC50c) and fluorescence (EC50f) responses to agonists (5-HT, mCPBG, varenicline) at labeled and unlabeled m5-HT3A mutants. The top part of the table represents the characterization of the sensors with different ligands and the associated controls (rows 1 to 13) and the second part the additional allosteric mutations added (rows 14 to 16).

Hypothetical integrative model of VCF data.

Schematic representation of the 5-HT3R representing two subunits in side-view with the orthosteric site and ion channel M2 helices highlighted. The four fluorescent sensors are represented as hexagons following the color code of figure 1 (S204C in magenta, I160C/Y207W in green, V106C/L131W in purple, R219C/Y140W in cyan). Ligand-elicited fluorescence changes are represented as a light halo. VCF data identifies four different conformations whose fluorescence patterns match known high-resolution structures. These conformations are called resting (matching apo structures, PDB 4PIR, 6BE1, 6H5B, 6Y59), inhibited (matching setron-bound structures, PDB 6HIS, 6W1J, 6W1M, 6W1Y, 6Y1Z), intermediate (matching I1 and state1, PDB 6HIO, 6DG7) and active (matching F, state2, and open, PDB 6HIN, 6DG8, 6Y5A).