McKemy et al (2002) used whole-cell patch clamping and calcium imaging to record the responses of cultured rat trigeminal ganglion neurons to cold temperatures and various cooling compounds. They found that the cells respond to menthol and cold with an increase in intracellular calcium ion concentration, and that these stimuli activate non-selective cation channels which are highly permeable to calcium. The currents measured were also found to be outwardly rectifying (i.e. much larger at positive than at negative holding potentials). Similar results were obtained from DRG neurons.
They then cloned TRPM8 from a trigeminal ganglion cDNA library and expressed it in Xenopus oocytes. Electrophysiological recordings showed that oocytes expressing TRPM8 channel were sensitive to cold, confirming that the channel is indeed a cold receptor. The cloned channel was found to have a temperature threshold of 8-28 degrees Celcius; it also conferred upon the oocytes sensitivity to menthol and eucalyptol, with the strongest response elicited by the super-cooling compound icilin.
To further investigate the role of TRPM8 in cold thermosensation, Dhaka et al (2007) generated TRPM8 knockout mice by replacing amino acid residues 2-29 of the TRPM8 gene with enhanced green fluorescent protein (EGFP). Calcium imaging showed that only 7.6% of DRG neurons from TRPM8-deficient mice, respond to a cold stimulus of 10 degrees Celcius, compared to 14.9% of cells from wild type (WT) animals.
The temperature sensitivity of the mutants was then assayed, using an apparatus consisting of multiple compartments to produce a surface temperature gradient ranging from 15-53 degrees Celcius. In this assay, both WT and TRPM8-deficient mice largely avoided severe cold (16-20 degrees Celcius) and hot (41-53 degrees Celcius) temperature compartments. However, whereas WT mice spent twice as much time in the compartment with a surface of ~35 degrees Celcius than in other the compartments, the TRPM8-/- animals spent significantly more time in the cooler zones (23-30 degrees Celcius).
A two-temperature choice assay was then performed, in which the mice were placed on a platform consisting of two identical surfaces set at different temperatures. Whereas WT mice strongly preferred warm over cold temperature surfaces, TRPM8-/- animals showed no preference to a platform set at 31 degrees Celcius to one set at 18 degrees Celcius.
The ability of TRPM8-deficient mice to detect noxious cold temperatures was then examined. When TRPM8-deficient mice were placed on a cold plate of temperature -1 degrees Celcius, their behaviour was identical to that of WTs. When placed on the cold plate following an injection of icilin into the hindpaw, the WT mice responded by rapidly by withdrawing the paw from the cold surface; this behaviour was completely abolished in the TRPM-/- mice, as was the vigorous body shaking that is normally induced by intraperitoneal injection of icilin.
Finally, it was demonstrated that TRPM8 mediates cooling-induced analgesia. A 2% formalin injection into the paw produced the same nociceptive response in both mutant and wild-type animals. However, when the animals were placed on a 17 degrees Celcius cold plate following the formalin injection, the mutant mice spent significantly more time licking and lifting the injected paw than the wild-types.
Thus, mice lacking TRPM8 have a severely impaired sensitivity to innocuous cool temperatures and have a partially impaired ability to detect noxious cold temperatures. These findings are corroborated by two independent studies in which TRPM8-/- mice were generated (Bautista et al, 2007; Colburn et al, 2007).