In light of the potential involvement of TRPM8 channels in the pathophysiology of cold nociception and cold allodynia [reviewed by ], there is a strong interest in the pursuit of novel modulators of TRPM8 channels. In this study, we investigated the effect of different compounds at the TRPM8-Y745H mutant channel, focusing on the differential effects of several antagonists on the gating properties.
Cold and menthol activate wild-type TRPM8 by shifting its voltage-dependent activation towards more negative potentials [3, 6]. We confirmed a previous report showing that the tyrosine at position 745, on the putative S2 transmembrane segment, is essential for the agonist effects of menthol . Menthol is a small and fairly lipophilic compound which easily partitions in the lipid bilayer and could thus be expected to affect TRPM8 channel function from various interaction sites. However, during menthol application to the menthol-insensitive TRPM8-Y745H mutant, the parameters describing voltage gating: V1/2 and g/gctrl underwent no changes whatsoever, suggesting that menthol exerts its full effect by specific interaction with the binding site governed by the tyrosine residue 745. In the absence of menthol, the apparent gating charge, zapp, was slightly lower in the TRPM8-Y745H mutant, indicating that this site is to some extent communicated with the voltage sensor of the channel. This finding is supported by recent work on TRPM8 voltage sensor mutants  and a fragmental model of the channel structure .
While TRPM8 and TRPV1 share only around 20% protein sequence homology , the channels have several phenotypic characteristics in common. Both are activated by temperature changes, and C-terminal chimeras between the channels exhibit reversed temperature sensitivity . Furthermore, both TRPM8 and TRPV1 are inhibited by compounds such as BCTC, CTPC, SB-452533, capsazepine and ruthenium red [24, 29]. All TRPV1 antagonists, with the exception of pore blockers such as ruthenium red, seem to bind at the vanilloid binding pocket . However, no reports exist on the binding site of TRPM8 antagonists.
In this study, we demonstrate the variable inhibitory effect of several compounds at the TRPM8-Y745H mutant channel. This finding is unexpected since all of them affect gating of the channel in a similar fashion, shifting voltage-dependence to more positive potentials and exerting an allosteric negative modulation of the channel during cold- or chemically evoked activation . Our results reveal that the tyrosine residue 745 at the menthol binding site is critical for inhibition mediated by SKF96365. In contrast, the inhibition by other antagonists is unaffected (BCTC) or only partially reduced (capsazepine, clotrimazole, econazole) by the mutation, suggesting that at least one other binding site exists on the TRPM8 channel from where the inhibitors exert their negative allosteric modulation. Furthermore, the results imply that not all TRPM8 blockers should be considered competitive antagonists of menthol, even though the allosteric inhibitory effect they mediate lies in competition with the activating effect of cooling agents .
The experimental observations of differential interaction of Y745 with the antagonists SKF96365 and BCTC were further confirmed by molecular docking studies. Interestingly, in these simulations SKF96365 exhibited strong interactions with both Y745 and an asparagine residue, N799, that also interacts with icilin . Looking at Figure 8C, SKF96365 can be hypothesized to lock the S2 and S3 domains into a fixed position, thereby preventing conformational shifts of the S4 domain induced by menthol (or cooling) that would lead to channel activation. This idea is further supported by our finding that SKF96365 inhibits the TRPM8-wt current at 33°C, a condition in which the channel is only activated by voltage, whereas no effect is seen at the Y745H mutant. BCTC, in contrast, blocks both the wild-type and mutant channels in this condition with similar potency, indicating the presence of an alternative binding site. Overall, our data suggest a sequential model of TRPM8 gating where chemical modulators can favour (e.g. menthol) or hinder (e.g. BCTC or SKF96365) the energetics of subsequent channel opening by cold temperature or voltage, from different binding sites.
Our results provide direct clues for the identification of different structural motifs required for antagonist binding, aiding in the design of new candidate molecules for specific inhibition of TRPM8 channels. They also provide tools for current efforts to resolve the crystal structure of TRP channels [43–45]. Further mutagenesis work is required to identify the remaining binding site(s) of the various TRPM8 blockers. One potential site is formed by residues in the S2-S3 linker region, known to be important for the sensitivity to icilin . Another plausible location is centred on residues within the TRP domain. This domain is known to be important in the energetics of channel opening, i.e. translating drug binding into channel opening [35, 46, 47]. In this regard, we consider a very powerful approach to subject random generated libraries of TRPM8 mutant channels  to the inhibitory actions of the different antagonists characterized in this study. In this way, one could obtain unbiased structural information on the action of different inhibitors.
Finally, we also studied for the first time the inhibitory capacity of two members of the imidazole family: the parent compound itself, and the antimycotic agent econazole, at the cooling-activated TRPM8 channel. Imidazole was unable to inhibit neither of the two TRPM8 constructs, while econazole, similarly to its relative clotrimazole, proved to be a potent antagonist of the wild-type channel. Both econazole and clotrimazole lost potency at the Y745H mutant channel. This identification of a novel TRPM8 antagonist prompts further screening of imidazole-based compounds in the quest for new TRPM8 blockers, and offers indications for the design of more potent derivatives.