Detection and characterization of the structural determinants of temperature sensitivity in TRPM8

Transient Receptor Potential Melastatin 8 (TRPM8) is a Ca2+ permeable non-selective cation channel activated by cold temperatures, by membrane depolarization and chemical agonists, such as menthol. The channel is largely closed at 28 °C and is mainly open at 10 °C at the +60 mV holding potential. Their conductance increases about 24 times when the temperature drops 10 °C, indicating that the gating process in TRPM8 channels is 8 times more sensitive to changes in temperature that, for example, voltage-dependent K+ channels (Q10 ~3). Analysis of the conductance-voltage curves in the TRPM8 wild type, showed that values of the voltage at which the open probability is 0.5 (V0.5) saturate at high and low temperatures. This is a demonstration of the existence of a temperature sensor and a voltage sensor that determine the open probability of the channel. Furthermore, the quantification of the open probability at different temperatures shows that the voltage is a partial activator of channel. Analysis of the limiting slope of the open channel probability as a function of voltage and deactivation time constant at very negative potential values shows that the opening of the channel is independent of voltage. Finally, analyses of the activation kinetics suggest that TRPM8 channels undergo one or more transitions along a series of closed states before opening. The C-terminal of TRPM8 is an important molecular domain that defines the channel sensitivity to temperature. In order to determine which regions of the C-terminal are the molecular determinants of cold sensitivity of TRPM8, a series of deletions were performed in the distal region of this structure. The temperature sensitivity of each mutant was determined using electrophysiological techniques. A thermodynamic analysis of the results indicates that the coiled-coil structure, located between amino acids 1064 and 1104, plays a key role in temperature sensing of TRPM8 channels. Thus, all these data allow us to demonstrate: first the existence of independent temperature and voltage sensors coupled through allosteric interactions to channel gating and second, that the coiled-coil structure confers specific thermal sensitivity in TRPM8.