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TRPM3 is a member of the melastatin family of the transient receptor potential (TRP) channels and was described as a nociceptor. It can be activated by a multitude of stimuli, including chemical compounds such as pregnenolone sulphate (PS) or nifedipine (Nif), as well as by physical signals like temperature. Activation of TRPM3 by all these different type of stimuli leads to the opening of a central pore that is formed by the transmembrane domains five (S5) and six (S6) and the interconnecting pore-loop, resulting in an outward rectifying current-voltage relationship. Recently, we have described the existence of an alternative ion permeation pathway in TRPM3. This alternative pathway could be opened by simultaneous application of PS and the antifungal drug clotrimazole (Clt), resulting in a current shape that showed an in- and outward rectification. Very recently, we have described a novel synthetic agonist of TRPM3, CIM0216, which is remarkable by the fact that it is able to open as single compound both, the central pore and the alternative ion permeation pathway.By the use of CIM0216, we were able to further investigate the biophysical properties of the alternative ion permeation pathway and to achieve further evidence of the existence of an alternative ion permeation pathway in TRPM3. In addition, new mutagenesis studies of TRPM3 provided further evidence for the location of the alternative ion permeation pathway within the region of S4 of the voltage sensing domain.All together, these data provide more evidence for the existence of an alternative ion permeation pathway in TRPM3 that will open upon displacement of the voltage sensing domain S4.

The Ca(2+)-permeable cation channel TRPA1 acts as an ionotropic receptor for various pungent compounds and as a noxious cold sensor in sensory neurons. It is unclear what proportion of the TRPA1-mediated current is carried by Ca(2+) ions and how the permeation pathway changes during stimulation. Here, based on the relative permeability of the nonstimulated channel to cations of different size, we estimated a pore diameter of approximately 11 A. Combined patch-clamp and Fura-2 fluorescence recordings revealed that with 2 mM extracellular Ca(2+), and at a membrane potential of -80 mV, approximately 17% of the inward TRPA1 current is carried by Ca(2+). Stimulation with mustard oil evoked an apparent dilatation of the pore of 3 A and an increase in divalent cation selectivity and fractional Ca(2+) current. Mutations in the putative pore that reduced the divalent permeability and fractional Ca(2+) current also prevented mustard-oil-induced increases in Ca(2+) permeation. It is interesting that fractional Ca(2+) currents for wild-type and mutant TRPA1 were consistently higher than values predicted based on biionic reversal potentials using the Goldman-Hodgkin-Katz equation, suggesting that binding of Ca(2+) in the pore hinders monovalent cation permeation. We conclude that the pore of TRPA1 is dynamic and supports a surprisingly large Ca(2+) influx.

Transient receptor potential melastatin 8 (TRPM8) is a cold sensitive member of transient receptor potential ion channels. It is expressed by a subset of primary sensory neurons of the dorsal root and trigeminal ganglia and plays an important role in temperature sensation. Beyond cold temperature, several cooling agents, like menthol or icilin, also activate the channel. Here we report a chemically new class of agonists on the basis of macrocyclic lactone ring. In patch clamp and calcium imaging we found that the investigated compounds can activate the human recombinant TRPM8 overexpressed in HEK293T cells which activity was blocked by the TRPM8 antagonist N-(3-aminopropyl)-2-{[(3-methylphenyl) methyl]oxy}-N-(2-thienylmethyl)benzamide hydrochloride (AMTB). Some of the tested compounds were even more effective than the prototypic TRPM8 agonist menthol. Investigating various analogs, we identified a key motif of the compounds crucial in TRPM8 activation. Moreover, testing various mutants, we also identified amino acid residues forming the binding sites for these novel agonists on TRPM8. Investigating the native TRPM8 expressed by mouse sensory neurons we concluded that the most effective model compound is not only more potent but even more selective than menthol. Our results identified excellent chemical tools to study TRPM8 related functions and also highlight their potential pharmacological use.

TRPM3 is a calcium-permeable nonselective cation channel in the melastatin (TRPM) subfamily of TRP channels. It represents a typical example of a polymodally gated TRP channel, in that it can be activated by chemical ligands such as nifedipine and the neurosteroid pregnenolone sulphate (PS), as well as by physical stimuli such as heat and membrane depolarization. Recently, we have found evidence for an alternative ion permeation pathway distinct from the central pore, which can be gated by combined application of PS and exogenous chemicals such as clotrimazole. This alternative ion permeation pathway is preserved following desensitization, blockade, mutagenesis and chemical modification of the central pore, and enables massive Na+ influx at negative voltages. By screening a compound library, we identified CIM021600 as potent agonist of TRPM3. The compound exhibited a marked specificity for TRPM3 and induced [Ca2+]I signals in somatosensory neurons. Intriguingly, single application of CIM021600 was able to activate both the central pore ion permeation pathway and the alternative ion permeation pathway. Since physiological functions of TRPM3 and the alternative ion permeation pathway of TRPM3 are still poorly defined, the identification of a potent and selective activator is expected to contribute to clarifying the role of TRPM3 in vivo.

Temperature-sensitive TRP channels (thermoTRPs) play a key role in somatosensory thermosensation. Several thermoTRPs, including the heat-activated TRPV1 and the cold-activated TRPM8, are not only sensitive to changes in temperature but also to ligands that evoke a thermal sensation, such as the ‘hot pepper’ compound capsaicin (TRPV1) and the cooling agent menthol (TRPM8). Recent data indicate that the binding site of these lipophilic compounds is located in the so-called ‘sensor domain’ made up of transmembrane domains S1-S4. For example, mutating S4 arginine at position 842 in TRPM8 to histidine (mutant R842H) produces a channel with unaltered cold and voltage sensitivity, but with a ∼100-fold reduction in menthol affinity. Given that TRP channels are tetramers and that the sensor domains are spatially apart, a functional TRPM8 channel can potentially bind four menthol molecules. However, the contribution of each individual menthol binding event to channel gating is unknown. To address this question, we made vectors encoding tandem tetramers with all possible combinations of WT and R842H subunits. Expression of these vectors in HEK293 cells exclusively produced protein of the expected tetrameric molecular weight, and whole-cell recordings further confirmed that the tandem linkage of subunits did not significantly affect channel gating. This approach will further be used to determine the exact stoichiometry of menthol-induced TRPM8 gating.

TRPM8, a transient receptor potential (TRP) cation channel expressed in sensory neurons, functions both as a cold sensor and as an ionotropic receptor for various natural and synthetic ligands, including menthol, eucalyptol, icilin and mustard oil (AITC). The mechanisms whereby TRPM8 agonists act on the channel are incompletely understood. Here we analyzed in detail the changes in kinetics of TRPM8 gating induced by different ligands, and identified two clearly distinct modes of agonist action. The majority of agonists (type I), including menthol, cause a prominent slowing of the channel relaxation kinetics in response to voltage steps, whereas AITC (type II) causes a clear acceleration. These results can be reproduced using a Monod-Wyman-Changeux model, where each subunit can bind a single ligand. In this model, type I agonists cause an stabilization of the open state, while type II ligands destabilize the closed state. These results indicate that agonists can exert energetically distinct effects on the TRPM8 channel protein, and suggest that “equipotent” concentrations of type I and type II agonists may differentially affect electrical activity in sensory neurons.

Mustard oil (MO, allyl isothiocyanate) is a highly electrophylic compound known to produce irritation, inflammation and pain. As such, it is used frequently in research and its effects were initially thought to be solely mediated by activation of the cation channel TRPA1. In contrast to this view, we have recently shown that MO also acts as an agonist of the capsaicin and heat receptor TRPV1, but the underlying molecular mechanisms remain unknown. We have found that MO effects on TRPV1 were rapidly reversible upon washout and were not significantly altered by the mutation of the residue C155, a residue which is essential for the action of the electrophile allicin. Hence, MO action on TRPV1 seems not to be mediated by long lasting covalent modification of cysteine residues. Notably, MO seems to act in a similar way as capsaicin, causing a shift of the voltage dependence of channel activation to more negative potentials. We therefore tested MO effects on TRPV1 mutants known to alter the sensitivity to capsaicin. We found that the substitution S512Y in the putative transmembrane domain 3 significantly reduces the ability of MO to induce stimulation and to shift of the voltage dependence of channel activation. In addition, we found that, similar to capsaicin, MO cross sensitize with other TRPV1 stimulators such as heat and extracellular protons. The molecular determinants of these interactions may help to understand the mechanisms of action of MO in real conditions (physiological temperatures and acidosis), as well as the mechanisms of MO-induced hyperalgesia to heat application.

TRPM3 is a cation conducting ion channel that belongs to the superfamily of transient receptor potential (TRP) channels. It is polymodally regulated by stimuli including heat, hypoosmolarity, the endogenous agonist pregnenolone sulphate (PS) as well as the synthetic agonist nifedipine. Recently, we have shown that combined application of PS and the antifungal drug clotrimazole (Clt) leads to the activation of TRPM3 by inducing the opening of two pores, the central pore and an alternative ion permeation pathway. We have further identified a selective activator of TRPM3, CIM0216, which is able to open both pathways independently of the presence of other chemicals. At the moment a tremendous amount of TRPM3 splice variants have been described. In this study, we have selected one splice variant TRPM3α1, that differs from the TRPM3α2 isoform only in the presumed pore region. Remarkably, the TRPM3α1 isoform did not show any increase in channel activity during stimulation by PS or nifedipine. However, HEK293 cells transfected by TRPM3α1 showed a robust increase in current amplitude during stimulation by CIM0216. Here, we have further investigated the existence of an alternative ion permeation pathway in the TRPM3α1 isoform. This study aims to understand the mechanism of activation of the alternative ion permeation pathway in TRPM3 and to determine the necessary structural features to open the alternative pathway.

Allyl isothiocyanate (AITC; mustard oil) confers the typical pungency to condiments such as wasabi and mustard, and has been widely used as a pharmacological tool to study pain and neurogenic inflammation. The somatosensory effects of AITC have been largely attributed to direct activation of TRPA1 and TRPV1, two excitatory Transient Receptor Potential (TRP) ion channels expressed in nociceptor neurons. Nevertheless, a subset of sensory neurons responds to AITC in a TRPA1- and TRPV1-independent manner. We show that AITC activates TRPA1/TRPV1-deficient sensory neurons through activation of TRPM8, a TRP channel involved in cool/cold sensation. Whole-cell and inside-out patch-clamp recordings revealed that AITC rapidly and reversibly activates heterologously expressed TRPM8 in a membrane-delimited manner. Moreover, we found that, at higher doses, AITC induced channel activation is followed by channel block, a process that is more pronounced in the human TRPM8 than in the mouse orthologue. Analysis of chimeric channels combining human and mouse TRPM8 orthologues cytosolic revealed domains that determine this AITC-induced channel blockade. TRPM8 may contribute to the complex psychophysical effects of AITC.