Extended Data Figure 1: Limited proteolysis preserves the 5-HT₃ receptor structural and functional integrity.

All data shown were obtained with purified 5-HT₃ receptor submitted to limited digestion in the presence of trypsin. a, Size-exclusion chromatography profile on a Superose 6 10/300 column with a major peak of the pentameric protein–detergent complex. b, SDS–PAGE of the peak pooled fractions presents two bands corresponding to the N-terminal and C-terminal fragments of the receptor. c, Single-particle analysis using negative-stained transmission electron microscopy of the same fraction depicts the structural envelope of an intact split receptor (left, electron micrograph scale bar: 50 nm; right, selection of class averages, box sizes: 38.5 nm). d, After reconstitution in GUVs (middle panel), the split receptor exhibits single-channel activities provoked by serotonin present in the patch pipette (left panel) with a single-channel conductance of ∼30 pS (right panel). e, The split receptor in detergent (blue) or reconstituted in liposomes (black) presents equivalent [³H]GR65630 binding profiles (K_d = 0.3 nM in both cases) compared to the wild-type receptor in cell membranes²⁰ (red). VHH15 was found to be a potent inhibitor of the 5-HT₃ receptor. f, Schematic of the protocol used to quantify the inhibition of 5-HT₃ receptor by the nanobody using automated two-electrode voltage-clamp recording on Xenopus oocytes. g, Representative recordings of serotonin-evoked activation after VHH incubation (red traces) or after buffer incubation (grey traces, negative control). The time of incubation with VHH or the buffer is not scaled as only 20 s of recording is shown. h, Inhibition of the serotonin signal upon different nanobody concentrations. The Hill equation was used to fit the data (red line) leading to a half maximal inhibitory concentration of 29 nM. 11 oocytes have been tested. i, Validation of the VHH15 inhibitory effect on the HEK 293 cells expressing the 5-HT₃ receptor used for overexpression. The pulse train contains serotonin applications (orange: 5 μM, 30 s) and VHH15 application (red: 100 nM, 3 min). j, Analysis of the interaction of VHH15 with the 5-HT₃ receptor by surface plasmon resonance. Experimental sensorgram (dashed curve) was recorded in single-cycle mode⁵⁶ with sequential injection of increasing concentrations of 5HT-3 receptors (3, 9, 27, 81 and 243 nM) on the VHH15-functionalized surface. The sensorgram was fitted with a 1:1 binding model including mass transfer and drift correction (solid line) yielding an association rate constant k_on = 6.2 × 10⁴ M⁻¹ s⁻¹ and a dissociation rate constant k_off = 4.7 × 10⁻⁶ s⁻¹ with a derived equilibrium dissociation constant . k, Ligand binding for VHH15 competing against [³H]GR65630. Samples were incubated with the indicated concentrations of VHH15 before measuring the specific [³H]GR65630 binding. Normalized VHH15 competition binding isotherms lead to IC₅₀ value of ∼2 nM. Competition binding affinity of VHH15 calculated according to the Cheng–Prusoff equation is in the range of hundreds of pM.