Figure 2.

Heat activation of SiHsTRPA and the effects of introduction on the temperature-related phenotypes of D. melanogaster trpA¹ mutant. A, The upper traces indicate the changes of intracellular calcium level in SiHsTRPA-expressing cells by high (left) and low (right) temperatures. The lower traces show the changes of bath temperature by time (sec, second). Each line represents the intracellular calcium level in the individual cell measured by calcium imaging. Decanoic acid (C10) was added at 180–300 s (blue bar) after cold stimulation to confirm that the channels expressed in the cells were functional. The arrows show the time points when 5 μm ionomycin was added. The mean value of fold change of Fura-2 ratio with error bar (± SEM) is shown for the heat- or cold stimulated DsRed-positive (blue) and DsRed-negative (yellow) cells. The total numbers of cells analyzed were 79–228. Significant difference is shown by (**, P < 0.001, one-tailed Welch’s t test). B, The distribution of wild-type, trpA1¹, and trpA1¹ expressing either DmTRPA1 (trpA1 > DmTRPA1; trpA1¹) or SiHsTRPA (trpA1 > SiHsTRPA; trpA1¹) under trpA1-Gal4 was recorded along a thermal gradient (14–37°C). The recording was repeated three times for each group. The mean value with error bar (± SEM; n = 3) is shown for each temperature section (left). The percentage of fruit flies in the area of 28–37°C (wild type: 8.6 ± 5.9%; trpA1¹: 48.8 ± 8.9%; trpA1¹expressing DmTRPA1: 4.1 ± 0.9%; trpA1¹expressing SiHsTRPA: 13.7 ± 2.6%) of the thermal gradient. Only trpA1¹ (**) is significantly different from wild type (two-tailed Dunnett test, P < 0.003; right). C, Wild type (n = 92), trpA1¹ (n = 90), and trpA1¹ expressing either DmTRPA1 (ppk1 > DmTRPA1; trpA1¹, n = 83) or SiHsTRPA (ppk1 > SiHsTRPA; trpA1¹, n = 125) under ppk1.9-Gal4 were tested for their response to high temperature using a 46°C probe. The percentage of responding larvae for each genotype is shown at each second within 9 s and >10 s (mean value ± SEM). P-values (two-tailed Steel test) for wild type and trpA1¹ expressing either DmTRPA1 or SiHsTRPA compared to trpA1¹are <0.023, <0.023, and <0.038, respectively. P-value (two-tailed Steel test) for trpA1¹ expressing SiHsTRPA is <0.12 compared with wild type.