(a) Sequence comparison between rat (r) and chicken (Ck) TRPV1 vanilloid-binding pockets (VBPs). Yellow denotes conserved residues. The arrow denotes residues T550 in rat and A558 in CkTRPV1. (b,c) Representative TRPV1 currents evoked from a pH 5.5 solution (black traces) and after 5 μM OA in an outside-out patch for CktRPV1 (blue traces) (b) or rTRPV1 (orange traces) (c) expressing HEK293 cells. (d) Fraction of remaining currents in CkTRPV1 (Ck) (blue bar; n=30) and rTRPV1 (R) channels (orange bar; n=30). Ck (Ctrl, n=10) and R (Ctrl, n=8) are CkTRPV1 and rTRPV1 outside-out patches exposed to vehicle (recording solution+0.14% ethanol (EtOH) for 5 min and reactivated by pH 5.5. *P<0.001 between groups as compared by brackets. Grey bars represent control experiments with vehicle solution for Ck and rTRPV1. (e) Overlay assay for LPA and OA with rTRPV1 protein preincubated in the absence (−) and presence of 0.5 μmol capsaicin (+). LPA (0.2 μmol) signals were not affected by capsaicin, while OA (2 μmol) signals were fainter when TRPV1 was preincubated with capsaicin (top panel). (f) The percentage of interaction of TRPV1 protein with OA or LPA was obtained by densitometric analysis of the spots and normalized to the positive control, which was LPA itself. For OA in the absence of capsaicin (−) per cent of interaction was 99.6±0.2 and in the presence of capsaicin (+) per cent of interaction was 42.8±4.1, n=6 for all groups. *P<0.0001, t-test.