Rapid Cooling Reveals the Closing Rate of TRPM8 to be Temperature-Dependent

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Thermal TRP channels are directly activated by ambient temperature changes and thus play important functions in thermosensation and pain. TRPM8, in particular, is sensitive to innocuous cold (<22°C) as well as chemicals that can produce cool sensation (e.g. menthol). Previous studies on cold sensitivity of the channel have been almost exclusively based on steady-state measurements. Single-channel recordings elucidate equilibrium gating kinetics showing that temperature has a most prominent effect on a type of long closures that separate opening bursts. However, they do not distinguish whether temperature drives channel opening or closing. To resolve such issues we have developed a rapid cooling apparatus and applied it in combination with rapid laser heating to time-resolve the activation and deactivation of TRPM8. Our data reveal that temperature mainly drives the deactivation rate of the channel, which has Q10∼10, while the activation rate has typical temperature dependence (Q10∼2-3). That is, cooling mainly increases the open probability of the channel by slowing down channel closing. Thus the cold-sensitive channels work in an opposite way to the heat-sensitive channels in which temperature drives the opening rate while the closing rate is insensitive to temperature. But similar to heat activation of TRPV1, the temperature dependence of cold activation of TRPM8 is also strongly dependent on membrane potentials. At hyperpolarization (−60 mV) the deactivation involves an enthalpy change of ∼40 kcal/mol, while at depolarization (+60 mV) the enthalpy is decreased to ∼20 kcal/mol. The reduction of enthalpy reaches ∼20 kcal/mol, which far exceeds the electrical energy due to charge movement (<2 kcal/mol for z∼0.8), suggesting that the mechanisms of voltage- and temperature-sensing of the channel are coupled.