PMC

A, alignment of the S6 region (A) and pore helix (B) of dTRPA1 and hTRPA1. C, the mutation L1105A+I1106Q in S6 of HDH abolishes heat sensitivity. D, the HDH pore helix mutant, R1073Q is cold sensitive.

A, sequence alignment of dTRPA1 and hTRPA1. Replacement of the putative pore turret (B) or sequence between the filter and S6 (C) in HDH does not reduce heat sensitivity. D, substitution of HDH S6 with human S6 or replacement of the pore helix (E) reduces heat sensitivity. Currents were normalized to peak current at +100 mV. Insets show representative I–V curves. dTRPA1, Drosophila transient receptor potential A1; hTRPA1, human TRPA1.

A, voltage-dependent activation of H990A-R1004N (n = 5) (A) and H990Q-R1004A (n = 4) (B) at 32°C. (Insets show representative whole cell current traces at 32°C in response to the voltage protocol indicated in .) C, the H990A-R1004N (n = 4) and (D) H990Q-R1004A (n = 6) mutants have a higher temperature activation threshold.

A, schematic diagram of chimera construction. B, increasing temperature raises intracellular calcium concentration in HDH expressing cells. C, I–V curves of HDH channels. D, current traces of HDH chimeras at different temperatures in response to the voltage protocol indicated at the upper left. E, voltage-dependent activation of HDH at 16°C (n = 8), 20°C (n = 6) and 25°C (n = 7). dTRPA1, Drosophila transient receptor potential A1; hTRPA1, human TRPA1.

A, schematic diagram of dTRPA1 showing regions exchanged with human sequence in grey. B, deletion of the insertion between S1 and S2 in dTRPA1 does not alter temperature or voltage sensitivity. C, replacing S5–S6 in dTRPA1 with human counterpart abolishes heat activation (inset shows I–V curve). D, voltage-dependent activation of HDH-H56 is not altered by increasing temperatures (inset shows representative whole cell current traces at 24°C) (24°C, n = 7; 28°C, n = 8; 32°C, n = 7).

A, voltage ramps from −100 mV to +100 mV evoke an outward rectifying current in dTRPA1 that is potentiated at warm temperatures. B, dTRPA1 is activated by temperature in the range 20–35°C. Positive voltage shifts the temperature activation curve to the left (n = 11). C, current traces of dTRPA1 at indicated temperatures in response to the voltage protocol at the upper left. D, voltage-dependent activation of dTRPA1 at 24°C (n = 14), 28°C (n = 13) and 32°C (n = 8). Continuous lines are best fits to Boltzmann functions. Data are expressed as means ± SEM.

A, examples of dTRPA1 single channel currents recorded at +100 mV and 24°C and 32°C. B–E, histograms of dTRPA1 closed (B) and open distributions (C) at 24°C and 32°C (D and E). Histograms are superimposed with the probability density function (black line) and exponential components (red lines) estimated from fits of a five closed and two open state model. F and G, time constants (F) and areas (G) for the corresponding exponential components of closed (EC1–EC5) and open (EO1–EO2) distributions at 24°C and 32°C. H and I, highest ranking kinetic models of dTRPA1 single channel activity at 24°C and 32°C. Rates are in s⁻¹, red arrows indicate the principal transitions upon heating.

A, example of single channel currents of the S4 mutant (H990A-R1004N) recorded at +100 mV and 25°C. B–G, dwell-time histograms of closed (B and E), subconductance (C and F) and fully open states (D and G) for the H990A-R1004N mutant at 34°C and 40°C. The distributions are fitted with exponential components (red lines) derived from a five closed, two subconductance and two open state model and overlaid with the probability density function (black lines). H, highest ranking kinetic models of H990A-R1004N single channel activity at 34°C and 40°C. Rates are in s⁻¹, red arrows indicate the principal transitions upon heating. I, representative single channel trace of the dTRPA1–56H chimera at +100 mV and 25°C. J and K, histograms of dTRPA1–56H closed (J) and open distributions (K) fitted with exponential components (red lines) estimated from a four closed and four open state model (l).