Fig. 1. Novel heat-sensitive neurons do not express TRPA1.

A. Proportions of DRG neurons expressing TRPV1, TRPM3 and TRPA1, and of novel heat sensitive neurons expressing none of these TRP channels, from increases in F₃₄₀/F₃₈₀ in response to application of selective agonists (see Methods for details). Experimental protocol shown in Fig. 2A (except that TRPM2 blocker 2-APB was not applied). Pie chart shows data from 3 experiments as follows: neurons expressing only TRPV1 (19.6 ± 2.0%, mean ± SEM); only TRPM3 (13.5 ± 0.2%); both TRPV1 and TRPM3 but not TRPA1 (16.2 ± 3.5%); TRPA1 together with either TRPV1 or TRPM3 (24.4 ± 1.6%); TRPA1 alone, and responding to heat (0.2 ± 0.1%); TRPA1 alone and not responding to heat (0.49 ± 0.2%); novel heat-sensitive neurons responding to heat but not to agonists for any of TRPV1, TRPM3 or TRPA1 (9.5 ± 0.2%); and neurons not responding to heat nor to any of the TRP agonists but identified as viable from response to final pulse of KCl (16.1 ± 2.6%). See criteria in Methods for identifying positive heat and agonist responses. Data from 3 separate experiments on a total of 1563 neurons (13 cover slips from 3 male wild-type mice).

B. Similar experiment performed on DRG neurons from TRPM2^-/- mice. There is no significant difference in the proportions of any of the agonist-sensitive neurons from WT (p>0.05, multiple t test with Bonferroni correction). However, the proportion of novel heat-sensitive neurons is significantly reduced by deletion of TRPM2, from 9.5 ± 0.2% in WT to 2.5 ± 0.8% in TRPM2^-/-(p = 0.009, multiple t test with Bonferroni correction). Data from 3 separate experiments on a total of 660 neurons (14 cover slips from 3 male TRPM2^-/- mice).

C. Proportion of DRG neurons responding to heat in the presence of inhibitor cocktail blocking TRPV1, TRPM3 and TRPA1. See Extended data Fig. 1C, top trace, for protocol. In 4 separate experiments percentage of heat-responsive neurons = 38.2 ± 4.4%, and Δ F₃₄₀/F₃₈₀ = 1.90 ± 0.06 (mean ± SEM). Results from 1315 WT neurons.

D. Similar experiment to that shown in C, on DRG neurons from TRPM2^-/- mouse. Mean percentage of “unidentified” heat-responsive neurons in n = 4 separate experiments was 6.9 ± 2.2% (mean ± SEM, significant difference from WT, p = 0.0007, unpaired t-test) and ΔF₃₄₀/F₃₈₀ = 1.59 ± 0.16 (mean ± SEM, significant difference from WT, p = 0.0237, unpaired t-test). Results from 1794 neurons.

E. Amplitudes of ΔF₃₄₀/F₃₈₀ in response to heat ramp to 48°C from the experiments shown in C (WT neurons) and D (TRPM2^-/- neurons). Threshold for accepting a heat response shown by vertical dotted line giving intrinsic heat-sensitivity of the fura2 dye obtained from heat ramp applied in Ca-free solution (see details in Methods). Mean ΔF₃₄₀/F₃₈₀ in response to heat (48°C) in all WT neurons (i.e. both heat-sensitive and heat-insensitive) was 1.24 ± 0.04 (n = 1315) and in all TRPM2^-/- neurons was 0.46 ± 0.01 (n = 1794). Difference significant, p <0.0001, unpaired t-test.