Modulation of the cold-activated cation channel TRPM8 by surface charge screening

Abstract

TRPM8, a cation channel activated by cold and by cooling agents such as menthol and icilin, is critically involved in somatosensory cold sensation. Ion fluxes through TRPM8 are highly sensitive to changes in extracellular Ca(2+) and pH, but the mechanisms underlying this type of modulation are poorly understood. Here we provide evidence that inhibition of TRPM8 currents by extracellular divalent cations and protons is due to surface charge screening. We demonstrate that increasing concentrations of divalent cations or protons cause parallel shifts of the voltage dependence of TRPM8 activation towards positive potentials. These shifts were interpreted using the Gouy-Chapman-Stern theory, yielding an estimate for the density of fixed negative surface charge between 0.0098 and 0.0126 equivalent charges per A(2). These results represent the first description of the effects of surface charge screening on a TRP channel and provide a straightforward explanation for the known effects of extracellular Ca(2+) on cold-sensitive neurons.

Figure 1. Effects of extra- and intracellular Ca²⁺ on TRPM8 currents

A, whole-cell TRPM8 currents measured at −100 and +100 mV illustrating the activating effect of menthol (100 μm) and the inhibitory effect of Ca²⁺. The pipette solution contained 1 mm EGTA. Representative example for 5 similar experiments. B, simultaneous measurements of intracellular Ca²⁺ and whole-cell TRPM8 current at +100 mV under basal conditions and upon stimulation with menthol (100 μm) or icilin (2 μm). At the time points indicated by a red dotted line, a brief UV flash was applied causing photolysis of DM-nitrophen and rapid increases in intracellular Ca²⁺. C, currents measured during voltage steps to +100 mV at the time points indicated in B. The arrows indicate the exact time point at which the UV flash was applied. Representative example for 7 similar experiments.

Figure 2. Extracellular Ca²⁺ inhibits TRPM8 by shifting the voltage dependence of activation

A and B, time course of the menthol-stimulated (A) or basal (B) whole-cell TRPM8 currents measured at the end of 100 ms voltage steps to −80 and +100 mV illustrating the inhibitory effect of extracellular Ca²⁺. C, dose dependence of the effect of Ca²⁺ on the currents at +100 mV (circles) or −80 mV (squares) in the absence (open symbols) or presence (filled symbols) of 100 μm menthol. Lines represent fits using the Hill equation. D, activation curves showing the whole-cell conductance as a function of voltage for TRPM8 current at 25°C in the absence (open symbols) and presence (filled symbols) of 100 μm menthol, with the indicated concentrations of extracellular Ca²⁺. Continuous lines represent fits using the Boltzmann equation, where s and G_max were kept constant for all six experimental conditions, as expected in the case of surface charge screening.

Figure 3. TRPM8 voltage shifts with different divalent cations

A, non-stimulated whole-cell TRPM8 currents in response to 100 ms voltage steps to potentials between −120 and +160 mV measured in divalent cation-free solution (DVF) or in the presence of 20 mm Mg²⁺ or Ba²⁺. B, comparison of activation curves in DVF or in the presence of 20 mm of the indicated divalent cation. Continuous lines represent fits using the Boltzmann equation, where s and G_max were kept constant for all four experimental conditions, as expected in the case of surface charge screening. C, plot of the shift in V_1/2 (ΔV_1/2) as a function of the concentration of the indicated divalent cations. The lines represent the best fit using the Gouy–Chapman–Stern equation. D, plot of the minimal sum of squares of the residuals (SSR) as a function of σ. E, fitted dissociation constants for Ca²⁺ (red line) and Mg²⁺ (black line). The dotted square in D and E represents the area where SSR was within 20% of the minimum. See text for more details.

Figure 4. Extracellular protons shift the voltage dependence of TRPM8 activation

A, time course of menthol-stimulated whole-cell TRPM8 currents measured at the end of 100 ms voltage steps to −80 and +100 mV illustrating the inhibitory effect of extracellular acidification. B, dose dependence of the pH effect at +100 (black symbols) and −80 mV (red symbols). C, comparison of TRPM8 activation curves in the presence of 100 μm menthol at the indicated pH values. Continuous lines represent fits using the Boltzmann equation, where s and G_max were kept constant for all three experimental conditions, as expected in the case of surface charge screening. D, plot of the shift in V_1/2 (ΔV_1/2) as a function of pH, for TRPM8 currents at 15°C or at 25°C in the presence or absence of 100 μm menthol. The line represents the best fit using the Gouy–Chapman–Stern equation. The red symbol represents the effect of pH at an extracellular Mg²⁺ concentration of 20 mm. This data point was excluded from the fit. See text for more details.