(a) Representative traces obtained from outside-out membrane patches as in Fig. 2. Currents are from WT rTRPV1, rTRPV1-Y511A-T550A, rTRPV1-Y511A-S512A-T550A and CkTRPV1-A558T channels. Black traces were obtained in response to pH 5.5, and turquoise, gold, blue and pink traces were obtained in the presence of pH 5.5 after 3 min of 5 μM OA. (b) Fraction of remaining currents after OA as normalized to the initial current in the presence of pH 5.5; rTRPV1 (n=22); rTRPV1-Y511A-S512A (n=10); rTRPV1-T550A (n=14); rTRPV1-Y511A-T550A (n=8); rTRPV1-Y511A-S512A-T550A (n=9), rTRPV1-Y511A-S512A-T550I (n=11) and CkTRPV1-A558T (n=15) *P<0.001 between groups as compared by brackets. (c) Surface representation of vanilloid-binding site (VBP) in the closed state structure (PDB 3J5R). A cavity is visible and the location of mutated residues is indicated: T550 (blue), Y511 (yellow) and S512 (magenta). (d) Docking calculation showing two representative protonated OA configurations with the lowest energy and highest occupancy binding to the VBP. Both conformations have −5.2 kcal mol−1 of binding energy. The average distance from the carbonyl oxygen in OA to the hydroxyl group in T550 is 2.5 Å, allowing for good hydrogen bonding (black dotted line, see arrow) between T550 and the carbonyl in OA. (e) Docking of OA to the triple mutant rTRPV1-Y511A-S512A-T550A. Mutations Y511A-S512A increase the size of the VBP and in combination with the absence of the hydrogen bond between OA and T550, reduce the binding energy to −4 kcal mol−1, allowing OA to bind in a more disordered conformation. Labels correspond to the S3, S4 and S4–S5 linker helices.