Heat detection by the TRPM2 ion channel

Nature

a, Neurons from the TG were tested for heat and agonist sensitivity. Left, percentage distributions of heat-sensitive and agonist-sensitive neurons. TRPA1 is strongly co-expressed with TRPV1 or TRPM3 (39%), with few neurons expressing TRPA1 alone (2.5%). Heat-sensitive neurons that respond to heat but do not express any of TRPV1, TRPM3 or TRPA1 can be clearly identified (8%). n = 388 neurons from 1 wild-type mouse. Right, similar experiment on n = 382 TG neurons from 1 Trpm2−/− mouse. The proportion of heat-sensitive neurons with responses that are not attributable to TRPV1, TRPM3 or TRPA1 is significantly reduced compared with the wild type (from 8% to 3%, P = 0.0077, Fisher’s exact test). The mean response amplitude of heat-sensitive neurons with responses that are not attributable to TRPV1, TRPM3 or TRPA1 was not significantly reduced by deletion of Trpm2F340/F380 = 1.13 ± 0.05, n = 31 for wild type to 0.96 ± 0.08, n = 13 for Trpm2−/−, difference not significant, P = 0.0539, unpaired t-test). b, Neurons from the DRG were tested for heat and agonist sensitivity using the protocol shown in Fig. 2a, but without the application of 2-APB and with agonists presented after heat rather than before. Left, neurons were cultured for 12 h in NGF1. See Methods for details. The pie chart shows data from 3 experiments on a total of 1,312 neurons from 3 wild-type mice on 12 coverslips as follows: neurons expressing only TRPV1 (32.2 ± 4.2%, mean ± s.e.m.); only TRPM3 (8.2 ± 1.2%); both TRPV1 and TRPM3 but not TRPA1 (13.3 ± 0.6%); TRPA1 together with either TRPV1 or TRPM3 (13.3 ± 0.9%); TRPA1 alone and responding to heat (0.2 ± 0.2%); TRPA1 alone and not responding to heat (0.4 ± 0.2%); heat-sensitive neurons responding to heat but not to agonists for any of TRPV1, TRPM3 or TRPA1 (7.6 ± 2.0%); and neurons not responding to heat nor to any of the TRP agonists but that were identified as viable from their response to a final pulse of KCl (24.8 ± 1.8%). The application of agonists before (Fig. 1a) or after the heat pulse (this Figure) does not significantly affect the proportion of heat-sensitive neurons with responses that are not attributable to TRPV1, TRPM3 or TRPA1 (9.5 ± 0.2% when agonists are applied before (Fig. 1a) and 7.6 ± 2.0% when applied after, P = 0.3740, unpaired t-test). The proportions of neurons in other categories are also not significantly affected, apart from a small reduction in the proportion of neurons expressing TRPA1 in combination with TRPV1 and/or TRPM3 (compare with Fig. 1a). Right, neurons cultured for 12 h in medium containing glial cell line-derived neurotrophic factor (GDNF) at 2 ng ml−1 and neurotrophin 4 (NT4) at 10 ng ml−1, the same culture protocol used by Vandewauw et al.2. n = 1,521 neurons from 3 wild-type mice on 12 coverslips. The altered culture conditions had no significant effect on the proportions of heat-sensitive and agonist-sensitive neurons (P > 0.05, multiple t-test with Bonferroni correction). c, Left, protocols for determining the total proportion of DRG neurons in which heat responses are partly driven by a mechanism independent of TRPV1, TRPM3 or TRPA1. The heat ramp is first applied in a Ca-free solution, to identify changes in the F340/F380 fluorescence ratio (ΔF340/F380) arising from sensitivity of the fura2 dye to temperature, followed by an identical ramp in the presence of a cocktail of inhibitors of TRPV1 (AMG9810, 5 μM), TRPM3 (naringenin, 10 μM) and TRPA1 (HC-030031, 100 μM) (see details in Methods). A final pulse of KCl confirms viability of the neurons. Right, top: heat application in which the temperature was increased from 33 °C to 48 °C over 180 s (ref. 1). n = 710 neurons from 2 wild-type mice on 3 coverslips. Middle, heat application in which the temperature was increased from 33 °C to 48 °C over 25 s (ref. 2). n = 99 neurons from 1 wild-type mouse on 2 coverslips. Bottom, heat application in which the temperature was increased from 23 °C to 48 °C over 180 s (ref. 2). n = 657 neurons from 1 wild-type mouse on 2 coverslips. Reducing the rise time of heat application or the starting temperature does not reduce the total proportion of heat-sensitive neurons with responses that are not attributable to TRPV1, TRPM3 or TRPA1. Differences not significant, P = 0.9197 (middle) and 0.3339 (bottom) respectively, unpaired t-test compared to top panel.

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