A hot-sensing cold receptor: C-terminal domain determines thermosensation in transient receptor potential channels

Abstract

Temperature transduction in mammals is possible because of the presence of a set of temperature-dependent transient receptor potential (TRP) channels in dorsal root ganglia neurons and skin cells. Six thermo-TRP channels, all characterized by their unusually high temperature sensitivity (Q10 > 10), have been cloned: TRPV1-4 are heat activated, whereas TRPM8 and TRPA1 are activated by cold. Because of the lack of structural information, the molecular basis for regulation by temperature remains unknown. In this study, we assessed the role of the C-terminal domain of thermo-TRPs and its involvement in thermal activation by using chimeras between the heat receptor TRPV1 and the cold receptor TRPM8, in which the entire C-terminal domain was switched. Here, we demonstrate that the C-terminal domain is modular and confers the channel phenotype regarding temperature sensitivity, channel gating kinetics, and PIP2 (phosphatidylinositol-4,5-bisphophate) modulation. Thus, thermo-TRP channels contain an interchangeable specific region, different from the voltage sensor, which allows them to sense temperature stimuli.

Figure 1.

Design and expression of thermo-TRP C-terminal chimeras. A, Sequence alignments between rTRPV1 and rTRPM8. The cut–paste limit for chimera construction is marked by a double arrow. Several important features are highlighted in the alignment: the TM6 segment, TRPbox, and TRPV1 PIP₂ binding domain. B, Chimeras and their expression in HEK-293 cells. Each of the top schemes corresponds to the immunochemistry below (scale bar, 20 μm). It is clear that the chimeric channels are able to reach the membrane, but large amounts of them are retained on intracellular compartments.

Figure 2.

Temperature sensitivity of chimeric channels. A, VRctCR chimera normalized current as a function of temperature. B, CRctVR chimera normalized current as a function of temperature. Data obtained using slow temperature ramps (0.25°C/s) and a holding potential of 80 mV. The solid lines are the best fits to a Boltzmann function. Each point represents an average of at least four different experiments. Error bars indicate SD.

Figure 3.

Electrophysiological analysis of the chimeras. A, B, Plots showing the normalized conductance (for details, see Materials and Methods) in function of voltage at the indicated temperatures for TRPM8 and TRPV1, respectively. The solid lines correspond to the best fit to Boltzmann functions. C, Whole-cell recordings of a cell expressing the VRctCR chimera and exposed to the indicated temperatures. The voltage protocol is shown in the top of the figure. D, Whole-cell recordings of a cell expressing the CRctVR chimera and exposed to the indicated temperatures. E, F, Plots showing the normalized conductance in function of voltage at the indicated temperatures for VRctCR and CRctVR, respectively. The solid lines correspond to the best fit to Boltzmann functions. G, H, Arrhenius plots for activation and deactivation process of VRctCR and CRctVR chimeras, respectively (each data point represent at least three experiments). Activation (α) and deactivation (β) rates were calculated from double exponential function fits (see Materials and Methods). Activation energy was calculated directly from the slope of the Arrhenius plots (slope = −E_a/R). Error bars indicate SD.

Figure 4.

Pharmacological properties of the chimeras. Current records obtained at 100 mV. Temperature, 25°C. A, PIP₂ (10 μm) activates the VRctCR channel. B, PIP₂ (10 μm) inhibits the CRctVR channel. C, Bar plot of several experiments exposing the CRctVR and VRctCR channels to PIP₂ (10 μm). Unpaired t test showed that both chimeras were affected by PIP₂ addition. D, Current traces showing menthol-induced activation and La³⁺ blocking of CRctVR chimera. F, Current traces for capsaicin-induced activation and La³⁺ blocking of VRctCR chimera. E, G, A summary of the experiments performed for the indicated chimera. La³⁺ was added to check whether the chimeras were able to be blocked (the addition was always performed after a maximal activation of the respective channel). Capsaicin (Cap) (200 μm) and menthol (Men) (500 μm) were added to CRctVR and VRctCR, respectively, as control experiments. One-way ANOVA and Bonferroni’s statistical analysis were performed for the two data sets in E and G, and in both cases the populations were found to be statistically different (p < 0.05). H, Capsaicin dose–response curves for TRPV1 (IC₅₀, 0.6 μm) and VRctCR (IC₅₀, 7.8 μm). I, Menthol dose–response curves for TRPM8 (IC₅₀, 22 μm) and CRctVR (IC₅₀, 28 μm). Current values were normalized and expressed as percentage of maximal response to the agonist. Each point represents mean values ± SD from at least three different patches. The Hill equation was used to fit the data.