Menthol is best known to elicit a cooling sensation in humans (Eccles, 1994
). In mice, TRPM8 has recently been demonstrated to underlie temperature sensation at cool temperatures (Bautista et al., 2007
; Colburn et al., 2007
; Dhaka et al., 2007
). In addition to its cooling properties, menthol can produce a burning and painful sensation (Green, 1992
). The molecular mechanism underlying this pro-nociceptive effect remains unknown. Here we report that menthol is an efficacious agonist of human TRPA1, suggesting that TRPA1 could contribute to the menthol-induced burning sensation in humans. Furthermore, we found striking species-specific variation of menthol effects on TRPA1. In contrast to human TRPA1, TRPA1 orthologs from fly, mosquito and fugu are insensitive to menthol, while murine TRPA1 reveals a bell-shaped concentration dependence as previously reported (Karashima et al., 2007
). While the biological significance of acquiring menthol sensitivity(with different pharmacological profiles) in mammalian TRPA1 remains unclear, the unique menthol response profiles of various TRPA1 orthologs enabled a chimeric structure-function approach from which three major conclusions can be drawn. First, the TM5 domain critically determines the ability of TRPA1 to sense menthol. Secondly, switching nine pore domain residues in human TRPA1 to the mouse residues reverses how hTRPA1 responds to menthol, turning menthol from a pure agonist to an apparent antagonist. Thirdly, the identified structural domains required for menthol sensitivity also determine the sensitivity of mammalian TRPA1 to an array of TRPA1 modulators, including thymol, FTS, AP18, and AMG5445.
Initial studies identified menthol as an inhibitor of mTRPA1 when applied at high concentrations (≥ 250 μM) (Macpherson et al., 2006
). Subsequent studies further identified agonist activity of menthol at low concentrations (Karashima et al., 2007
). The mechanisms underlying this bimodal effect of menthol on mTRPA1 have not been determined. Nilius and colleagues have proposed that menthol may have two separate binding sites on mTRPA1: one responsible for activation, the other for inhibition (Karashima et al., 2007
). An alternative interpretation postulates a single binding site where block is observed as a consequence of activation. It is possible that menthol at high concentrations initially activates mTRPA1 but the channel rapidly switches into a non-conducting state (e.g., inactivation state) which has access to the open state upon washout of menthol (“off” response). The response of mTRPA1 to menthol is reminiscent of voltage dependent activation of human ether-a-go-go-related gene (hERG) potassium channels: strong depolarizations cause hERG to transit through the open state to an inactivated state and, upon repolarization, return to the open state before closing (Sanguinetti et al., 1995
; Trudeau et al., 1995
Regardless of the complexity of menthol effects on mTRPA1, it is striking that mTRPA1-dTM5 and mTRPA1-S876V/T877L are neither activated nor inhibited by menthol. How could specific residues in the TM5 domain determine the complicated menthol sensitivity of mouse TRPA1? One hypothesis is that the TM5 domain may constitute a binding site for menthol. Our modeling and docking studies reveal a putative menthol-binding site in TM5 regions. However, it is important to note that there is no direct evidence that menthol specifically binds to TRPA1. It is entirely possible that the effects of menthol are indirect, through modifications of the lipid bilayer, for example. At present, a cross-linkable menthol analogue does not exist, and thus the sites of contact with the TRPA1 channel cannot be directly identified.
The existence of a ligand binding pocket in TM5 appears to be inconsistent with the general observation that most thermoTRPs have putative ligand binding sites within TM2 through TM4, particularly TM2 (Gavva et al., 2005
; Jordt and Julius, 2002
; Voets et al., 2007
; Vriens et al., 2004
). Interestingly, Bandell et al., using a high-throughput random mutagenesis screen, identified Tyr-745 in TM2 as a critical determinant of menthol sensitivity of mTRPM8 (Bandell et al., 2006
). Given this precedence, it is possible that the region from TM2 to TM4 in TRPA1 may contain a common binding site for nonreactive TRPA1 modulators, whereas the TM5 domain is involved in translating initial binding to channel gating. To test whether a conserved role of tyrosine in determining menthol sensitivity exists in mTRPA1, we individually mutated the two tyrosine residues (Tyr-788 and Tyr-875) located in the TM2 and TM3 of mTRPA1 to alanine. We found that these mutations do not affect mTRPA1 menthol sensitivity (data not shown). We also made a series of mTRPA1-dTRPA1 chimeras. Unfortunately, most of these resulted in non-functional channels, and specific conclusion about the involvement of TM2-TM4 was not addressed. On the other hand, we are able to conclude that TM1-TM4 region is not involved in the distinct responses of mTRPA1 and hTRPA1 to menthol. Regardless of its direct involvement in menthol binding or in gating, the TM5 domain critically determines the ability of TRPA1 to sense menthol.
How could TM5 mechanistically determine the menthol sensitivity of TRPA1? TRPA1 is predicted to have a similar structure as voltage-gated K+
channels, in which TM5 forms the outer helix of the pore and the TM4-TM5 linker helix is critical for electromechanical coupling by opening or closing the gate located in TM6 (Long et al., 2005
). In both mammalian TRPA1 channels, Ser and Thr residues located in the predicted inner side of TM5 were identified as critical for the sensitivity of mouse (and to a large extent, human) TRPA1 to menthol. Perhaps important for sensitivity to menthol as well as menthol's gating phenotype, the Ser and Thr residues are followed by a Gly residue (G878) in mTRPA1. Interestingly, all of these amino acids are known to break α-helical symmetry (Levitt, 1978
; Levitt and Greer, 1977
). Given the properties of these residues, it is possible that TM5 may form a hinge at this location. In line with this idea, our molecular dynamic simulation of mTRPA1 reveals a kink in TM5 helix at these Ser and Thr residues in mammalian TM5. Thus, it is conceivable that either direct menthol interactions or conformational changes sensed by these residues upon menthol binding to distant regions could be transmitted to TM6, leading to channel gating. The residues at these positions in TRPA1 from non-mammalian species are α-helix favoring (Val and Leu), which may preclude this proposed mechanism. Interestingly, Grimm et al. have found that a naturally occurring helix-breaking mutation in TM5 (Ala-419-Pro) of TRPML3 leads to constitutive channel activation. They further demonstrated that the inner third of TM5 from TRPML1, TRPML2, TRPV5, and TRPV6 is highly susceptible to proline-based kinks (Grimm et al., 2007
). Together with the present study, these data suggest that TM5 may play a critical role in TRP channel function.
Not only have we identified residues crucial for modulation of mammalian TRPA1 by menthol, we have also identified nine residues from the pore module that specifically accounts for the species-specific responses of TRPA1 to menthol (activator vs. inhibitor) (Gly878 and amino acids highlighted in red in ). It is possible that Gly878 immediately after Ser-Thr in mouse TM5 makes the hinge too floppy to confer full agonism in mouse TRPA1 and/or it alters other properties of the pore region that then confers the blocking effect of menthol at high concentrations. In support of this hypothesis, hTRPA1-V875G revealed a menthol “inhibitory phenotype”, albeit incomplete. Interestingly, Chen et al. have recently found that CMP1, an electrophilic reactive compound, activates mTRPA1 but inhibits hTRPA1 (Chen et al., 2008
). They identified the same residues in the upper part of TM6 as determinants of species-specific gating. Based on their findings, they proposed that covalent modification of TRPA1 can lead to different functional consequences of the channel. However, it is not clear how CMP1 could inhibit MO-activated hTRPA1 if both CMP1 and MO act through covalent modifications of cysteine residues. Chen et al. argued that the covalent modification of cysteines in TRPA1 by MO is rapidly reversible. However, the half-life of isothiocyanate-cysteine adducts was reported to be in the order of about one hour at physiological pH and temperature (Conaway et al., 2001
). Furthermore, the successful labeling of MO alkyne adducts on TRPA1 by click chemistry also suggests that MO adducts remain rather stable because the click reaction itself takes an hour to complete (Macpherson et al., 2007a
). Given these concerns, we raise another possibility: CMP1 inhibits hTRPA1 through a non-covalent mechanism. In support of this, mTRPA1 and hTRPA1 responsiveness to AMG5445, a structural analogue of CMP1, is essentially determined by the same residues as those for non-reactive menthol. This suggests that menthol and AMG5445 share a similar mechanism of action at TRPA1. These studies also raise the possibility that some reactive modulators of TRPA1 may simultaneously employ both the covalent and non-covalent activation mechanisms.
In summary, we have demonstrated that TM5 determines the sensitivity of TRPA1 to various chemical modulators and identified specific residues governing species-dependent gating of TRPA1. These findings not only reveal a novel mechanism of TRPA1 modulation, but also have important implications for efforts to identify specific small molecule TRPA1 inhibitors.