Two out of three S1–S2 linker mutants do not respond to heat. (A) Representative Ca²⁺ imaging traces. (B) Summary of the amplitude ratios between heat- and capsaicin-induced responses. n = 59 (wild type), 37 (Rs₁s₂_1), 45 (Rs₁s₂_2), and 52 (Rs₁s₂_3). *, P < 0.05; ***, P < 0.001. (C and D) Representative single-channel traces recorded at +80 mV from Rs₁s₂_1 (C) and Rs₁s₂_2 (D) at room temperature (RT), elevated temperatures, and in the presence of 10 µM capsaicin. The recording shown in D was from a patch containing two channels. (E) Representative current trace recorded from Rs₁s₂_3 in response to heating and 3 µM capsaicin. (F) Current–temperature relationship of Rs₁s₂_3. Dotted lines represent linear fits to the leak and channel currents.