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. Author manuscript; available in PMC: 2022 Nov 7.
Published in final edited form as: Nature. 2020 Aug 12;584(7820):E5–E12. doi: 10.1038/s41586-020-2510-7

Fig. 2. Pharmacological block of TRPM2 inhibits the heat response of novel heat-sensitive neurons.

Fig. 2

A. Effect of TRPM2 blocker 2-APB (25μM) on identified somatosensory neurons. Neurons expressing TRPV1, TRPM3 and/or TRPA1 identified by brief application of the specific agonists capsaicin (1μM), pregnenolone sulphate (PS, 100μM) and allyl isothiocyanate (AITC, 50 μM), respectively, followed by heat ramp to 48°C to activate all heat-sensitive neurons. Effect of TRPM2 blocker 2-aminoethoxydiphenyl borate (2-APB, 25μM) on heat-activated neurons is tested, followed by a pulse of KCl (50mM) to test neuronal viability. At end, heat pulse repeated in Ca-free solution, to identify changes in F340/F380 fluorescence ratio (ΔF340/F380) arising from sensitivity of the fura2 dye to temperature. Response to heat in a novel heat-sensitive neuron not expressing any of TRPV1, TRPM3 or TRPA1 is strongly suppressed by the TRPM2 blocker 2-APB (25μM).

B. Increase in calcium-dependent fluorescence ratio (ΔF340/F380) in response to heat in novel heat-sensitive neurons (n = 149 neurons from 3 wild-type mice) is strongly suppressed by 2-APB (25μM). Increase in ΔF340/F380 corrected for each neuron by subtracting the increase in ΔF340/F380 measured from heat ramp applied in Ca-free solution.

C. Similar experiment on neurons from 3 TRPM2-/- mice. The few novel-heat-sensitive neurons observed (see Fig. 1B) show no significant effect of 2-APB on response amplitude.

D. Effect of terminating temperature ramp at 48°C1 and at an earlier time point when temperature had reached 45°C2. Reducing final temperature had a dramatic effect on proportion of novel heat-sensitive neurons, from 9.5 ± 0.2% in response to heat to 48°C to 2.2 ± 0.2% in response to heat to 45°C (p<0.0001, unpaired t-test). n = 1563 neurons from 3 WT mice.

E. Amplitudes of ΔF340/F380 in response to heat ramp reaching 48°C and 45°C from the experiment shown in B. Threshold for accepting a heat response shown by vertical dotted lines showing intrinsic heat-sensitivity of the fura2 dye obtained from heat ramp applied in 0Ca solution (see details in Methods). Mean ΔF340/F380 in response to heat to 48°C was 2.04 ± 0.11 and to 45°C was 0.67± 0.03 (n = 149 neurons, p<0.0001, unpaired t-test).