Extended data Fig. 3. A fraction of TRPA1⁺ neurons from TRPM2^-/- mice respond to heat when TRPV1 and TRPM3 are blocked.

A. Upper: Protocol of solution changes used to identify neurons expressing TRPA1 and to test their activation by heat. TRPV1 and TRPM3 were blocked by AMG9810 (5μM) and naringenin (10μM) as shown. The calcium increase elicited by a heat ramp from 33°C to 48°C was tested in the absence (heat ramps 1 and 3) and presence (ramp 2) of the TRPA1 blocker HC030031 (100μM). Responses to the TRPA1-selective agonist AITC were tested as shown. Response to KCl (50mM) and heat ramp in absence of external Ca tested at end. Middle trace: increases in F₃₄₀/₃₈₀ in a neuron expressing TRPA1 in which the heat-activated Ca increase was suppressed by the TRPA1 blocker HC030031. TRPA1 was expressed in 31.2 ± 4.2% of the total population, but only a fraction of TRPA1⁺ neurons (34.7% ± 9.81%) gave a significant Ca increase in response to 48°C heat. Lower trace: a different neuron in same preparation is activated by heat but does not express TRPA1 (unidentified heat-responders). Data from 3 separate experiments on a total of 964 neurons.

B. Heat response in TRPA1-expressing neurons is suppressed by TRPA1 blocker HC030031 (protocol and example trace shown in A. ***, p<0.001, one-way ANOVA followed by Bonferroni’s post-hoc correction). Heat response in neurons not expressing TRPA1 is not significantly reduced by the TRPA1 blocker HC030031 (100μM). Proportion of unidentified heat-responders (5.5% ± 1.4%) is not significantly different from that in Fig. 1D.