Figure 2.

TRPV1 mutant F660S ablates proton activation but not capsaicin or heat activation. (A)An amino-acid sequence alignment of part of the pore region, including the upper third of TMD6 of human (h), mouse (m), and rat (r) TRPV1 and TRPV3. Residues reported to be specifically required for temperature activation of TRPV3 are indicated by purple arrows (I644, P651, L657, and Y661). Amino acids found to be required for temperature activation of both rTRPV1 and mTRPV3 are indicated by green arrows. The blue arrow indicates amino acid involved in heat activation of mTRPV3 and proton activation and potentiation of hTRPV1. The brown arrow indicates amino acid involved in heat activation of mTRPV3 and proton activation of hTRPV1. (B, C) Concentration response curves obtained from Fluo-4-based calcium flux experiments using HEK293 cells transiently expressing hTRPV1 wild-type or TRPV1 (F660S) mutant. Representative pH dose–response measurement (B) using MES-buffered solutions adjusted to different pH values. MES-buffered solutions were added to cells loaded with Fluo-4 calcium indicator dye in standard buffer solution without HEPES (adjusted to pH 7.4) resulting in final pH values of 5.6, 5.8, 6.2, 6.6, 7.0, and 7.4. Representative capsaicin dose–response measurement (C) performed in standard buffer solution containing 20 mM HEPES, adjusted to pH 7.4. Average EC₅₀ values (mean±s.d.) of 25±12 nM were measured for F660S versus 12±6 nM for wild-type TRPV1 (n=10, each). (D) Steady-state current–voltage relationships obtained from whole-cell patch-clamp experiments with HEK293 cells transiently expressing hTRPV1 wild-type or F660S mutant. Cells were stimulated subsequently with protons (pH 5.5), capsaicin (1 μM), or heat (45°C). (E) Representative whole-cell current traces (at +60 mV) obtained from HEK293 cells transiently expressing hTRPV1 or F660S mutant after subsequent stimulation with protons (pH 5.5), capsaicin (100 nM), or heat (45°C). (F) Bar diagram summarizing data obtained from experiments as described in B and C. (G) Bar diagram showing ratio values calculated based on data as described in D and E. Statistical analysis was performed using one-way ANOVA followed by Tukey's post test; ^**P<0.001.