TRPM8 activation by menthol, icilin, and cold is differentially modulated by intracellular pH

Abstract

TRPM8 is a nonselective cation channel activated by cold and the cooling compounds menthol and icilin (Peier et al., 2002). Here, we have used electrophysiology and the calcium-sensitive dye Fura-2 to study the effect of pH and interactions between temperature, pH, and the two chemical agonists menthol and icilin on TRPM8 expressed in Chinese hamster ovary cells. Menthol, icilin, and cold all evoked stimulus-dependent [Ca2+]i responses in standard physiological solutions of pH 7.3. Increasing the extracellular [H+] from pH 7.3 to approximately pH 6 abolished responses to icilin and cold stimulation but did not affect responses to menthol. Icilin concentration-response curves were significantly shifted to the right when pH was lowered from 7.3 to 6.9, whereas those with menthol were unaltered in solutions of pH 6.1. When cells were exposed to solutions in the range of pH 8.1-6.5, the temperature threshold for activation was elevated at higher pH and depressed at lower pH. Superfusing cells with a low subactivating concentration of icilin or menthol elevated the threshold for cold activation at pH 7.4, but cooling failed to evoke [Ca2+]i responses at pH 6 in the presence of either agonist. In voltage-clamp experiments in which the intracellular pH was buffered to different levels, acidification reduced the current amplitude of icilin responses and shifted the threshold for cold activation to lower values with half-maximal inhibition at pH 7.2 and pH 7.6. The results demonstrate that the activation of TRPM8 by icilin and cold, but not menthol, is modulated by intracellular pH in the physiological range. Furthermore, our data suggest that activation by icilin and cold involve a different mechanism to activation by menthol.

Figure 1.

Icilin, menthol, and cold evoke [Ca²⁺]_i responses and currents at pH 7.3. Concentration-response curves for icilin (A) and menthol (B) in TRPM8-expressing CHO cells. Data points represent mean maximal [Ca²⁺]_i response produced by each drug concentration during a 60 sec application, as illustrated in the insets (baseline ratios before the addition of drug has been subtracted; menthol, n = 11; icilin, n = 7). Drug was added after 17 sec. C, Cells exposed to a temperature ramp from 37 to 17°C were activated ∼22°C (average response in 25 cells). The individual responses in the same 25 cells are shown in the inset. Icilin (1 μm) and menthol (1 mm) evoked large inward currents (D,E). Currents in response to both agonists developed rapidly, but for icilin always after a short initial delay (at a holding potential of -60 mV). Icilin-evoked currents desensitized completely, whereas the desensitization of currents evoked by menthol varied. F, Current-temperature relationship in a cell exposed to a temperature ramp (at a holding potential of +60 mV). This particular cell had a threshold close to 24°C and a Q₁₀ of 35 (in the temperature range from 20 to 15°C).

Figure 2.

Low pH_o inhibits TRPM8 activation by cold and icilin but not menthol.A, Trace from a single cell, demonstrating that icilin is unable to evoke a response at pH_o 6.1 but evokes a large [Ca²⁺]_i response when pH_o is restored to pH 7.4 (experiment performed at 27°C). B, Cold stimulation (temperature ramp from 35 to 17°C) evokes a [Ca²⁺]_i response at pH_o 7.4 but not in solutions of pH_o 6.1; data from single cells. C, Low pH_o completely inhibits responses induced by 1 μm icilin, whereas responses to 1 mm menthol are not significantly affected. Each data point is the mean response in six wells; similar results were obtained in three experiments. D, Low pH_o completely inhibits [Ca²⁺]_i responses induced by cold (17°C). Data points are mean responses to 17°C in groups of cells monitored individually (n = 13-21).

Figure 3.

Effect of pH on concentration-response curves for icilin and menthol. A, Acidification of the extracellular solution caused a rightward shift of the responses to icilin, followed by insurmountable inhibition at lower pH_o (experiment shown is representative for n = 4). B, [Ca²⁺]_i responses evoked by menthol were not affected by acidification (p > 0.05 for E_max and pEC₅₀; one-way ANOVA; n = 4). C, Effect of menthol and icilin on [Ca²⁺]_i responses induced by cold stimulation. Cells were superfused at 37°C with a solution of pH_o 7.3 containing 25 μm menthol, 100 nm icilin (agonist concentrations that did not produce [Ca²⁺]_i responses on their own at this temperature) or vehicle for 1 min before they were stimulated by a temperature ramp down to 15°C. Responses to cold were significantly shifted to higher temperatures in the presence of either agonist (p < 0.001; n = 4 for menthol and n = 5 for icilin, in which each determination was the mean response of n = 21-31 individual cells). When the same experiments were performed in solutions of pH_o 6, no [Ca²⁺]_i response developed in the absence or presence of either agonist (n = 4).

Figure 4.

pH modulates TRPM8 activation at an intracellular site. A, Although icilin could still evoke inward currents after a 2 min exposure to pH_o 5.4, intracellular acidification reduced the current amplitude evoked by 1 μm icilin with an estimated half-maximal inhibition of pH 7.2 (n = 5-9). Inward currents evoked by 1 mm menthol were smaller than currents evoked by 1 μm icilin and not affected by intracellular pH (n = 12-16). B, The latency before icilin-evoked currents developed was increased by intracellular acidification. Data points are averages of cells responding within 120 sec (n = 5-9 cells, except for pH_i 6.5, where the only two responding cells are included individually); three of five cells recorded at pH_i 6.5 and two of seven cells recorded at pH_i 6.9 failed to respond during 120 sec. C, Intracellular acidification did not affect the time course of icilin-evoked currents. The rise time (time from 10 to 90% of current developed) and the τ (inactivation) was similar at all pH_i tested. Because only two cells responded at pH_i 6.5, the individual data points are shown (n = 5-9 for the other pH_i values). D, Representative recordings illustrating how pH_i affects icilin-evoked currents. As shown in A and B, the current amplitude is depressed and the latency is increased by intracellular acidification. The box demonstrates that inward currents evoked by menthol were unaffected by pH_i 6.5 (compare with Fig. 1 E). E, Extracellular acidification reduced the pH_i in a time- and pH-dependent manner. Cells were incubated in pH_o 7.3 (weakly buffered with 1 mm HEPES). After 22 sec, the pH_o was changed from 7.3 to the indicated values by adding a three times larger volume of strongly buffered solution (10 mm HEPES or MES). Error bars have been omitted for clarity.

Figure 5.

The activation threshold for temperature is modulated by pH. A, Cells were superfused with solutions of the pH indicated for at least 1 min before they were exposed to a temperature ramp from 37 to 12°C (each point is the average of 2-4 determinations of groups of individual cells; n = 14-31). No [Ca²⁺]_i responses were produced by superfusion alone at 37°C. Thresholds were determined as the intersection between two lines fitted to the log(Δratio) before and after [Ca²⁺]_i responses developed, as illustrated in the inset for an experiment performed at pH 7.1. B, The temperature threshold for cold activation of inward currents (-60 mV) is inhibited by intracellular acidification. The temperature at which a discernable current first developed was set as threshold (each point is the average of n = 5-7). C, Representative recordings demonstrating that the temperature threshold for cold activation is sensitive to the pH_i. Intracellular acidification also affects the slope of the temperature dependence.