Figure 1.

Heat activation profile of VdTRPA1L. (A) The upper traces indicate the changes of intracellular calcium level in VdTRPA1L- and VdTRPA1S-expressing cells (84 and 54 DsRed-positive cells on one cover glass, respectively) on temperature fluctuation in the absence [white bar, Ca²⁺ (−)] or presence [black bar, Ca²⁺ (+)] of extracellular calcium. Each line represents the intracellular calcium level in the individual cell measured by calcium imaging. The arrows show the time points when 5 μM ionomycin was added. The lower traces show the changes of bath temperature by time (sec, second). (B) Heat activation of VdTRPA1L analyzed by whole-cell patch clamp recording. (C) Heat-activated current density of mock-transfected cells, VdTRPA1L-, and VdTRPA1S-expressing cells measured by patch-clamp recording. The mean value with error bar (± SEM) is indicated for each sample (n = 6 for VdTRPA1L and n = 4 for mock-transfected and VdTRPA1S). p-value for VdTRPA1L-expressing cells compared to mock-transfected cells is < 0.04 (^*). (D) The representative trace indicates the activation currents of VdTRPA1L elicited by two different heat applications, 16–42°C (the first stimulation) and 24–42°C (the second stimulation). The red dotted lines show the apparent initiation points of the currents. The upper and lower traces show the changes of inward current and the changes of bath temperature by time. (E) Arrhenius plots for the heat-evoked VdTRPA1L currents shown in (D). Blue and black lines represent the current amplitudes generated by heat stimulation from 16°C (Cold to heat) to 24°C (RT to heat), respectively. (F) Current–voltage relationship of heat (red line)-, 0.5 mM α-terpineol (black line)-, or 0.1 mM carvacrol (blue line)-evoked current exhibits a positive reversal potential. We corrected the liquid junction potential (ΔJPH) between extracellular and intracellular solution but not the heat-dependent shifts of ΔJPH in the plot. The data of α-terpineol and carvacrol were reproduced from previous report (Peng et al., 2015b).