Abstract
Expressed in somatosensory neurons of the dorsal root and trigeminal ganglion, the transient receptor potential melastatin 8 (TRPM8) channel is a Ca(2+)-permeable cation channel activated by cold, voltage, phosphatidylinositol 4,5-bisphosphate, and menthol. Although TRPM8 channel gating has been characterized at the single channel and macroscopic current levels, there is currently no consensus regarding the extent to which temperature and voltage sensors couple to the conduction gate. In this study, we extended the range of voltages where TRPM8-induced ionic currents were measured and made careful measurements of the maximum open probability the channel can attain at different temperatures by means of fluctuation analysis. The first direct measurements of TRPM8 channel temperature-driven conformational rearrangements provided here suggest that temperature alone is able to open the channel and that the opening reaction is voltage-independent. Voltage is a partial activator of TRPM8 channels, because absolute open probability values measured with fully activated voltage sensors are less than 1, and they decrease as temperature rises. By unveiling the fast temperature-dependent deactivation process, we show that TRPM8 channel deactivation is well described by a double exponential time course. The fast and slow deactivation processes are temperature-dependent with enthalpy changes of 27.2 and 30.8 kcal mol(-1). The overall Q10 for the closing reaction is about 33. A three-tiered allosteric model containing four voltage sensors and four temperature sensors can account for the complex deactivation kinetics and coupling between voltage and temperature sensor activation and channel opening.
Highlights
How voltage and temperature sensors are coupled to channel opening in transient receptor potential melastatin 8 (TRPM8) is still under debate
One of the problems with previous tail current measurements was the very fast TRPM8 channel deactivation at negative voltages, which made the determination of Itail(V) curves at high temperatures uncertain [2]
The fact that TRPM8 knock-out animals show a near-complete loss of cold sensitivity underscores the importance of this channel in both innocuous and noxious cold sensation in vivo [46, 47]
Summary
How voltage and temperature sensors are coupled to channel opening in transient receptor potential melastatin 8 (TRPM8) is still under debate. Previous experiments have shown that voltage is a partial activator of TRPM8 and that the half-activation voltage (V0.5) saturates at extreme temperatures, which suggests a lack of strict coupling between TRPM8 voltage and temperature activation [2, 13, 14] These results have been explained on the basis of an allosteric model with separate voltage and temperature sensors, which upon activation promote the opening of the pore domain independently from one another. This type of model has been criticized [10] in terms of the following: (a) inaccuracy in determining the maximum open probability (Po, max); (b) very rapid TPRM8 channel deactivation at negative voltages, leading to an underestimation of the open probability (Po) from tail currents; and (c) voltage clamp errors. These results rule out the possibility that both temperature and voltage sensors move as a single molecular entity
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