N-glycosylation of TRPM8 ion channels modulates temperature sensitivity of cold thermoreceptor neurons

Abstract

TRPM8 is a member of the transient receptor potential ion channel superfamily, which is expressed in sensory neurons and is activated by cold and cooling compounds, such as menthol. Activation of TRPM8 by agonists takes place through shifts in its voltage activation curve, allowing channel opening at physiological membrane potentials. Here, we studied the role of the N-glycosylation occurring at the pore loop of TRPM8 on the function of the channel. Using heterologous expression of recombinant channels in HEK293 cells we found that the unglycosylated TRPM8 mutant (N934Q) displays marked functional differences compared with the wild type channel. These differences include a shift in the threshold of temperature activation and a reduced response to menthol and cold stimuli. Biophysical analysis indicated that these modifications are due to a shift in the voltage dependence of TRPM8 activation toward more positive potentials. By using tunicamycin, a drug that prevents N-glycosylation of proteins, we also evaluated the effect of the N-glycosylation on the responses of trigeminal sensory neurons expressing TRPM8. These experiments showed that the lack of N-glycosylation affects the function of native TRPM8 ion channels in a similar way to heterologously expressed ones, causing an important shift of the temperature threshold of cold-sensitive thermoreceptor neurons. Altogether, these results indicate that post-translational modification of TRPM8 is an important mechanism modulating cold thermoreceptor function, explaining the marked differences in temperature sensitivity observed between recombinant and native TRPM8 ion channels.

FIGURE 1.

Expression of N-glycosylated forms of recombinant and native TRPM8 channels. A, Western blots of cell lysates from TRPM8-transfected HEK293 cells (HEK) and mouse TG neurons probed with an anti-TRPM8 antibody. B, non-N-glycosylated TRPM8-N934Q mutant and the wild type TRPM8 channel from tunicamycin-treated cells migrates faster than wild type channels. C, TRPM8 expression in trigeminal ganglia in control condition and 48 h after treatment with tunicamycin. D, time course of treatment with tunicamycin (Tunic.) (5 μg/ml) at 24 and 48 h. E, effect of treatment with brefeldin A (BFA) (5 μg/ml) for 20 h. TRPM8 protein was detected by an anti-TRPM8 antibody (1:500). F, representative Western blots from biotinylation assays using HEK293 cells transfected with the TRPM8-WT and the TRPM8-N934Q mutant. Biotinylated proteins were precipitated with Neutravidin beads (avidin-bound fraction, ABF) from cell lysates (CL) to measure the surface expression of TRPM8. Total expression of the ion channel was assessed by directly immunoblotting 15 μg of each cell lysate. The left panel corresponds to transfected HEK293 cells with TRPM8-N934Q mutant that were not subjected to cell surface biotinylation (No biotinylation). PKC-α was used as a control for cell membrane integrity. The normalized TRPM8 cell surface expression was calculated by expressing the intensities of the TRPM8 bands in the avidin-bound fraction as a fraction of those in the corresponding cell lysates. The lower panel shows TRPM8 cell surface expression, normalized to total TRPM8 expression. Data are means ± S.E. of three experiments from different batches of transfected cells. The two mean values did not differ from each other at the p = 0.05 level (Student's t test).

FIGURE 2.

Responses to cold and menthol are reduced in the nonglycosylated TRPM8 mutant. A, representative intracellular calcium imaging traces showing responses to cold and 100 μm menthol in nontransfected HEK293 cells (gray) and cells expressing TRPM8-WT (black) or TRPM8-N934Q (red). The protocol was performed under normal (2.4 mm) and zero external Ca²⁺ conditions. Note the lack of response to cold or menthol in untransfected cells. B, summary histogram of the results obtained for the experimental protocol shown in A. Intracellular calcium increases for TRPM8-WT and TRPM8-N934Q were compared using an unpaired Student t test: ***, p < 0.001; TRPM8-WT, n = 56; TRPM8-N934Q, n = 54. C, representative traces showing intracellular calcium elevations to cold and 100 μm menthol in HEK293 cells expressing TRPM8-WT (black) in control and after 48 h of exposure to 5 μg/ml tunicamycin (red). The stimulation protocol was carried out with and without external Ca²⁺. D, summary histogram of the results obtained with the experimental protocol shown in C. Intracellular calcium elevations in control and tunicamycin-treated cells were compared using an unpaired Student's t test: **, p < 0.01; ***, p < 0.001; control, n = 81; tunicamycin-treated, n = 83.

FIGURE 3.

Sensitivity of TRPM8 channels to agonists depends on the N-glycosylation. A, time course of [Ca²⁺]_i response in transfected HEK293 cell with TRPM8-WT (black) and TRPM8 N934Q (red) during cooling ramps and applications of menthol at different concentrations. B, dose-response curves of responses to menthol in transfected HEK293 cells. The solid lines represent a fit to the Hill equation that yielded an EC₅₀ of 70 ± 8 μm for TRPM8-WT and 176 ± 13 μm for TRPM8-N934Q (TRPM8-WT, n = 29; TRPM8-N934Q, n = 14). Responses were normalized to the amplitude obtained with maximal stimulation (menthol plus cold). C, percentage of active population recruited during a cooling ramp in HEK293 cells transfected with TRPM8-WT versus TRPM8-N934Q. D, histogram of temperature thresholds exhibited by HEK293 cells transfected with TRPM8-WT (n = 141) or TRPM8-N934Q (n = 162). Temperature thresholds were compared using a two-tailed unpaired Student's t test: ***, p < 0.001.

FIGURE 4.

Suppression of N-glycosylation causes a shift in the voltage activation of TRPM8 toward more positive potentials. A, whole-cell current recorded at −60 mV in HEK293 cells transfected with TRPM8-WT channels during two consecutive temperature drops and an application of 100 μm menthol. The spike-like currents are the truncated responses to voltage ramps (−100 to +180 mV, 1400 ms duration). Traces on the right are current-voltage relationship of TRPM8-transfected cells at 33 °C in control solution (control), at 20 °C in control solution (cold), 100 μm menthol at 33 °C (menthol), and during application of 100 μm menthol at 20 °C (cold plus menthol). B, same experimental protocol as in A but on mutated TRPM8-N934Q channels. C, bar graph of the mean results obtained in experiments like A, showing the maximal current at −60 mV in different conditions (cold, menthol, and cold plus menthol). D–E, mean values of V½ and g_max obtained from the fits of the currents to Equation 1. Black bars correspond to control and red bars to the unglycosylated channel. Statistical significance was assessed with a two-tailed unpaired Student's t test: *, p < 0,05; n = 7 for TRPM8-WT and n = 8 for TRPM8-N934Q.

FIGURE 5.

Addition of a negative charge at the position 934 partially recovers the responses to cold and menthol in the unglycosylated TRPM8 mutant. A and B, mean values of the [Ca²⁺]_i response amplitude to cold (A), and the temperature threshold (B) in wild type channels and in three different mutants. Statistical significance was assessed with a one-way analysis of variance test in combination with Bonferroni post hoc test: ***, p < 0.001 to TRPM8-WT value, and ###, p < 0.001 to TRPM8-N934D value. n = 141 (TRPM8-WT); n = 162 (TRPM8-N934Q); n = 180 (TRPM8-N934K); n = 190 (TRPM8-N934D). C, mean values of the [Ca²⁺]_i response amplitude to cold plus menthol. Statistical significance was assessed with a one-way analysis of variance test in combination with Bonferroni post hoc test: *, p < 0.05; ***, p < 0.001 to TRPM8-WT. n = 82 (TRPM8-WT); n = 60 (TRPM8-N934Q); n = 75 (TRPM8-N934K); n = 82 (TRPM8-N934D). D, dose-response curves for menthol in WT and different mutant TRPM8 channels expressed in HEK293 cells. The solid lines represent fits to Hill equation that yield an EC₅₀ 83 ± 13 μm for TRPM8-WT (n = 82), 356 ± 13 μm for TRPM8-N934Q (n = 60), 437 ± 71 μm for TRPM8-N934K (n = 75), and 232 ± 30 μm (n = 82) for N934D. For each condition, the response amplitude was normalized to that obtained with a combined pulse of cold and 300 μm menthol (i.e. the maximal response).

FIGURE 6.

N-Glycosylation of TRPM8 has a strong influence on the temperature threshold of cold-sensitive neurons. A and B, time course of ratiometric [Ca²⁺]_i responses in trigeminal cold-sensitive neurons during cooling ramps, applications of menthol at different concentrations and depolarization induced by 30 mm KCl, in control conditions and after 48 h of exposure to tunicamycin (5 μg/ml). C, mean evoked [Ca²⁺]_i responses for the experiments in A and B. Statistical significance was assessed with a two-tailed unpaired Student's t test. *, p < 0.05; ***, p < 0.001. n = 22 (control); n = 14 (tunicamycin). D, average temperature threshold of cold-sensitive neurons and HEK293 cells transfected with TRPM8-WT, in control condition and after tunicamycin treatment. Statistical significance was assessed with a one-way analysis of variance test in combination with Bonferroni post hoc correction, ***, p < 0.001 compared with their control value; ###, p < 0.001 comparing trigeminal neurons versus transfected human embryonic kidney cells in control conditions. n = 22 (TG control); n = 14 (TG tunicamycin); n = 95 (HEK293-TRPM8 WT, control); n = 105 (HEK293-TRPM8 WT, tunicamycin).