TRPA1 acts as a cold sensor in vitro and in vivo

Abstract

TRPA1 functions as an excitatory ionotropic receptor in sensory neurons. It was originally described as a noxious cold-activated channel, but its cold sensitivity has been disputed in later studies, and the contribution of TRPA1 to thermosensing is currently a matter of strong debate. Here, we provide several lines of evidence to establish that TRPA1 acts as a cold sensor in vitro and in vivo. First, we demonstrate that heterologously expressed TRPA1 is activated by cold in a Ca(2+)-independent and Ca(2+) store-independent manner; temperature-dependent gating of TRPA1 is mechanistically analogous to that of other temperature-sensitive TRP channels, and it is preserved after treatment with the TRPA1 agonist mustard oil. Second, we identify and characterize a specific subset of cold-sensitive trigeminal ganglion neurons that is absent in TRPA1-deficient mice. Finally, cold plate and tail-flick experiments reveal TRPA1-dependent, cold-induced nociceptive behavior in mice. We conclude that TRPA1 acts as a major sensor for noxious cold.

Fig. 1.

Ca²⁺-independent and Ca²⁺ store-independent activation of heterologously expressed TRPA1 by cold. (A) Time course of whole-cell TRPA1 currents at +50 and −75 mV during cooling in extracellular solution containing 2 mM Ca²⁺ (Left). Current–voltage relations were obtained at the indicated time points (Right). (B) Same as for A, but using Ca²⁺-free intracellular and extracellular solutions. (C) Same as for A, but now using a Ca²⁺-free extracellular solution and an intracellular solution containing 10 μM free Ca²⁺. (D) Average inward current amplitudes at −75 mV for the conditions shown in A, B, and C and for cells preincubated for 30 min with 10 μM CPA in Ca²⁺-free solution.

Fig. 2.

Effects of cooling on the voltage-dependent gating and kinetics of TRPA1. (A) Whole-cell currents in Ca²⁺-free intracellular and extracellular solutions in response to the indicated voltage step protocol applied at 26°C and 13°C. (B) Average peak inward tail currents at −150 mV (n = 8) at 26°C and 13°C. (C) Average time constants obtained from monoexponential fits to the time course of current relaxation at different voltages and temperatures. Solid lines in B and C represent a global fit of the 2-state model to the experimental data. (D) Model predictions of TRPA1 currents at 26°C and 13°C in response to the voltage step protocol in A. (E) Model predictions of TRPA1 currents during 400-ms voltage ramps, such as those used in Fig. 1. (F) Average TRPA1 currents at different temperatures and at −75 and +50 mV, normalized to the maximal current in the tested temperature range. Dotted lines represent the corresponding model prediction.

Fig. 3.

Combined effects of cold and MO on TRPA1 currents. (A) Time course of inward and outward TRPA1 currents during application of MO. Cooling leads to a decrease in the MO-activated current. The red dotted line represents the same data points normalized to the single-channel current amplitude at the corresponding temperature, which yields a direct measure of NP_open. Note the increase in NP_open upon cooling. (B) Time course of inward and outward TRPA1 currents cooling and subsequent stimulation with MO. (C) Average inward TRPA1 currents evoked by MO and/or cooling.

Fig. 4.

TRPA1-dependent cold responses in TG neurons. (A) Ratiometric measurement of changes in intracellular Ca²⁺ in response to cold, menthol (100 μM), MO (100 μM), or capsaicin (1 μM), illustrating the 3 types of cold-sensitive TG neurons. (B) Comparison of the percentage of cells responding to cold and chemical stimuli in preparations from WT and Trpa1^−/− mice. (C) Pie charts showing the percentage of cold-insensitive and the 3 types of cold-sensitive neurons in TG from WT and Trpa1^−/− mice. (D) Histogram comparing temperature thresholds in cold-sensitive TG from WT and Trpa1^−/− mice. The temperature threshold was defined as the temperature at which the ratio was increased by 10% of the maximal cold-induced increase. (E) Correlation of the amplitude of the MO and cold responses in MO-sensitive neurons from WT mice (n = 177). The solid line represents a linear fit to the data.

Fig. 5.

Comparison of TRPM8- and TRPA1-dependent, cold-sensitive neurons. (A) Histogram comparing the temperature threshold in MO-sensitive TG neurons (n = 88) and MO-insensitive, menthol-sensitive TG neurons (n = 65). (B) Histogram obtained from the same cells as for A, comparing the time needed to reach 80% of the maximal cold response (t_80%). (C) Comparison of the capsaicin sensitivity between MO-insensitive, menthol-sensitive (n = 52), and MO-sensitive (n = 133) cold-sensitive neurons.

Fig. 6.

Altered cold-induced nociceptive behavior in Trpa1^−/− mice. (A) Latency to the first behavior reaction to cold upon placement on a cold plate at 0°C in mice of different sex and genotype. The numbers of mice tested on the cold plate were as follows: WT male (n = 13), WT female (n = 12), Trpa1^−/− male (n = 15), and Trpa1^−/− female (n = 13). (B) Average number of jumps during a 2-min period on a cold plate at 0°C. No jumps were observed when the plate was set to 30°C. (C) Cumulative probability plot showing the latency to the first jump off the cold plate in WT and Trpa1^−/− mice. (D) Tail-flick latency upon tail immersion in a water–methanol mixture at −10°C in WT (n = 32) and Trpa1^−/− (n = 34) male mice.