Abstract
In mammals, the main molecular entity involved in innocuous cold transduction is TRPM8. This polymodal ion channel is activated by cold, cooling compounds such as menthol and voltage. Despite its relevance, the molecular determinants involved in its activation by cold remain elusive. In this study we explored the use of TRPM8 orthologs with different cold responses as a strategy to identify new molecular determinants related with their thermosensitivity. We focused on mouse TRPM8 (mTRPM8) and chicken TRPM8 (cTRPM8), which present complementary thermosensitive and chemosensitive phenotypes. Although mTRPM8 displays larger responses to cold than cTRPM8 does, the avian ortholog shows a higher sensitivity to menthol compared with the mouse channel, in both HEK293 cells and primary somatosensory neurons. We took advantage of these differences to build multiple functional chimeras between these orthologs, to identify the regions that account for these discrepancies. Using a combination of calcium imaging and patch clamping, we identified a region encompassing positions 526-556 in the N terminus, whose replacement by the cTRPM8 homolog sequence potentiated its response to agonists. More importantly, we found that the characteristic cold response of these orthologs is due to nonconserved residues located within the pore loop, suggesting that TRPM8 has evolved by increasing the magnitude of its cold response through changes in this region. Our results reveal that these structural domains are critically involved in cold sensitivity and functional modulation of TRPM8, and support the idea that the pore domain is a key molecular determinant in temperature responses of this thermo-transient receptor potential (TRP) channel.
Highlights
In mammals, the main molecular entity involved in innocuous cold transduction is TRPM8
We found that mTRPM8transfected cells displayed a large cold-induced response and a menthol-evoked response at 34 °C that correspond to nearly 60% of the maximal response (Fig. 1A, left panel; Fig. 1B)
The cumulative population of chicken TRPM8 (cTRPM8)-expressing cells recruited during a cold ramp show a significant shift in the curve toward lower temperatures compared with mouse TRPM8 (mTRPM8) cells (Fig. 1E)
Summary
The chicken TRPM8 ortholog (cTRPM8) was first characterized in a study where its insensitivity to icilin was exploited to identify the residues involved in the activation of rat TRPM8 (rTRPM8) by this compound [24]. It is noteworthy that when these values were normalized to the maximal response of the channel induced by menthol at 20 °C (i.e. cold plus menthol stimulus), the current at the lowest temperature is close to 40% of the maximal response observed in mTRPM8 cells, and only 10% of that observed in cTRPM8 cells (Fig. 1K, lower panel). This observation indicates that the current variation during a cold drop is similar between both channels, there is an important difference in the proportion that the cold-induced response represents compared with the
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