Contribution of the S5-pore-S6 domain to the gating characteristics of the cation channels TRPM2 and TRPM8.
ABSTRACT: The closely related cation channels TRPM2 and TRPM8 show completely different requirements for stimulation and are regulated by Ca(2+) in an opposite manner. TRPM8 is basically gated in a voltage-dependent process enhanced by cold temperatures and cooling compounds such as menthol and icilin. The putative S4 voltage sensor of TRPM8 is closely similar to that of TRPM2, which, however, is mostly devoid of voltage sensitivity. To gain insight into principal interactions of critical channel domains during the gating process, we created chimeras in which the entire S5-pore-S6 domains were reciprocally exchanged. The chimera M2-M8P (i.e. TRPM2 with the pore of TRPM8) responded to ADP-ribose and hydrogen peroxide and was regulated by extracellular and intracellular Ca(2+) as was wild-type TRPM2. Single-channel recordings revealed the characteristic pattern of TRPM2 with extremely long open times. Only at far-negative membrane potentials (-120 to -140 mV) did differences become apparent because currents were reduced by hyperpolarization in M2-M8P but not in TRPM2. The reciprocal chimera, M8-M2P, showed currents after stimulation with high concentrations of menthol and icilin, but these currents were only slightly larger than in controls. The transfer of the NUDT9 domain to the C terminus of TRPM8 produced a channel sensitive to cold, menthol, or icilin but insensitive to ADP-ribose or hydrogen peroxide. We conclude that the gating processes in TRPM2 and TRPM8 differ in their requirements for specific structures within the pore. Moreover, the regulation by extracellular and intracellular Ca(2+) and the single-channel properties in TRPM2 are not determined by the S5-pore-S6 region.
Project description:For mammalian TRPM8, the amino acid residues asparagine-799 and aspartate-802 are essential for the stimulation of the channel by the synthetic agonist icilin. Both residues belong to the short sequence motif N-x-x-D within the transmembrane segment S3 highly conserved in the entire superfamily of voltage-dependent cation channels, among them TRPM8. Moreover, they are also conserved in the closely related TRPM2 channel, which is essentially voltage-independent. To analyze the differential roles of the motif for the voltage-dependent and voltage-independent gating, we performed reciprocal replacements of the asparagine and aspartate within the S3 motif in both channels, following the proposed idea that specific electrostatic interactions with other domains take place during gating. Wild-type and mutant channels were heterologeously expressed in HEK-293 cells and channel function was analyzed by whole-cell patch-clamp analysis as well as by Ca(2+)-imaging. Additionally, the expression of the channels in the plasma membrane was tested by Western blot analysis, in part after biotinylation. For the mutations of TRPM8, responses to menthol were only compromised if also the expression of the glycosylated channel isoform was prevented. In contrast, responses to cold were consistently and significantly attenuated but not completely abolished. For TRPM2, surface expression was not significantly affected by any of the mutations but channel function was only retained in one variant. Remarkably, this was the variant of which the corresponding mutation in TRPM8 exerted the most negative effects both on channel function and expression. Furthermore, we performed an exchange of the inner pair of residues of the N-x-x-D motif between the two channels, which proved deleterious for the functional expression of TRPM8 but ineffective on TRPM2. In conclusion, the N-x-x-D motif plays specific roles in TRPM8 and TRPM2, reflecting different requirements for voltage-dependent and voltage-independent channel gating.
Project description:1. TRPM8 (CMR1) is a Ca(2+)-permeable channel, which can be activated by low temperatures, menthol, eucalyptol and icilin. It belongs to the transient receptor potential (TRP) family, and therefore is related to vanilloid receptor type-1 (VR1, TRPV1). We tested whether substances which are structurally related to menthol, or which produce a cooling sensation, could activate TRPM8, and compared the responses of TRPM8 and VR1 to these ligands. 2. The effects of 70 odorants and menthol-related substances on recombinant mouse TRPM8 (mTRPM8), expressed in HEK293 cells, were examined using a FLIPR assay. In all, 10 substances (linalool, geraniol, hydroxycitronellal, WS-3, WS-23, FrescolatMGA, FrescolatML, PMD38, CoolactP and Cooling Agent 10) were found to be agonists. 3. The EC(50) values of the agonists defined their relative potencies: icilin (0.2+/-0.1 microM)>FrescolatML (3.3+/-1.5 microM) > WS-3 (3.7+/-1.7 microM) >(-)menthol (4.1+/-1.3 microM) >frescolatMAG (4.8+/-1.1 microM) > cooling agent 10 (6+/-2.2 microM) >(+)menthol (14.4+/-1.3 microM) > PMD38 (31+/-1.1 microM) > WS-23 (44+/-7.3 microM) > Coolact P (66+/-20 microM) > geraniol (5.9+/-1.6 mM) > linalool (6.7+/-2.0 mM) > eucalyptol (7.7+/-2.0 mM) > hydroxycitronellal (19.6+/-2.2 mM). 4. Known VR1 antagonists (BCTC, thio-BCTC and capsazepine) were also able to block the response of TRPM8 to menthol (IC(50): 0.8+/-1.0, 3.5+/-1.1 and 18+/-1.1 microM, respectively). 5. The Ca(2+) response of hVR1-transfected HEK293 cells to the endogenous VR1 agonist N-arachidonoyl-dopamine was potentiated by low pH. In contrast, menthol- and icilin-activated TRPM8 currents were suppressed by low pH. 6. In conclusion, in the present study, we identified 10 new agonists and three antagonists of TRPM8. We found that, in contrast to VR1, TRPM8 is inhibited rather than potentiated by protons.
Project description:Members of the transient receptor potential (TRP) ion channel family act as polymodal cellular sensors, which aid in regulating Ca(2+) homeostasis. Within the TRP family, TRPM8 is the cold receptor that forms a nonselective homotetrameric cation channel. In the absence of TRPM8 crystal structure, little is known about the relationship between structure and function. Inferences of TRPM8 structure have come from mutagenesis experiments coupled to electrophysiology, mainly regarding the fourth transmembrane helix (S4), which constitutes a moderate voltage-sensing domain, and about cold sensor and phosphatidylinositol 4,5-bisphosphate binding sites, which are both located in the C-terminus of TRPM8. In this study, we use a combination of molecular modeling and experimental techniques to examine the structure of the TRPM8 transmembrane and pore helix region including the conducting conformation of the selectivity filter. The model is consistent with a large amount of functional data and was further tested by mutagenesis. We present structural insight into the role of residues involved in intra- and intersubunit interactions and their link with the channel activity, sensitivity to icilin, menthol and cold, and impact on channel oligomerization.
Project description:Structure-activity relationship studies of a reported menthol-based transient receptor potential cation channel subfamily M member 8 channel (TRPM8) antagonist, guided by computational simulations and structure-based design, uncovers a novel series of TRPM8 antagonists with >10-fold selectivity versus related TRP subtypes. Spiro[4.5]decan-8-yl analogue <b>14</b> inhibits icilin-evoked Ca<sup>2+</sup> entry in HEK-293 cells stably expressing human TRPM8 (hTRPM8) with an IC<sub>50</sub> of 2.4 ± 1.0 nM, while in whole-cell patch-clamp recordings this analogue inhibits menthol-evoked currents with a hTRPM8 IC<sub>50</sub> of 64 ± 2 nM. Molecular dynamics (MD) simulations of compound <b>14</b> in our homology model of hTRPM8 suggest that this antagonist forms extensive hydrophobic contacts within the orthosteric site. In the wet dog shakes (WDS) assay, compound <b>14</b> dose-dependently blocks icilin-triggered shaking behaviors in mice. Upon local administration, compound <b>14</b> dose dependently inhibits cold allodynia evoked by the chemotherapy oxaliplatin in a murine model of peripheral neuropathy at microgram doses. Our findings suggest that <b>14</b> and other biphenyl amide analogues within our series can find utility as potent antagonist chemical probes derived from (-)-menthol as well as small molecule therapeutic scaffolds for chemotherapy-induced peripheral neuropathy (CIPN) and other sensory neuropathies.
Project description:Pirt is a two-transmembrane domain protein that regulates the function of a variety of ion channels. Our previous study indicated that Pirt acts as a positive endogenous regulator of the TRPM8 channel. The aim of this study was to investigate the mechanism underlying the regulation of TRPM8 channel by Pirt.HEK293 cells were transfected with TRPM8+Pirt or TRPM8 alone. Menthol (1 mmol/L) was applied through perfusion to induce TRPM8-mediated voltage-dependent currents, which were recorded using a whole-cell recording technique. PIP2 (10 μmol/L) was added into the electrode pipettes (PI was taken as a control). Additionally, cell-attached single-channel recordings were conducted in CHO cells transfected with TRPM8+Pirt or TRPM8 alone, and menthol (1 mmol/L) was added into the pipette solution.Either co-transfection with Pirt or intracellular application of PIP2 (but not PI) significantly enhanced menthol-induced TRPM8 currents. Furthermore, Pirt and PIP2 synergistically modulated menthol-induced TRPM8 currents. Single-channel recordings revealed that co-transfection with Pirt significantly increased the single channel conductance.Pirt and PIP2 synergistically enhance TRPM8 channel activity, and Pirt regulates TRPM8 channel activity by increasing the single channel conductance.
Project description:ThermoTRPs, a subset of the Transient Receptor Potential (TRP) family of cation channels, have been implicated in sensing temperature. TRPM8 and TRPA1 are both activated by cooling. TRPM8 is activated by innocuous cooling (<30 °C) and contributes to sensing unpleasant cold stimuli or mediating the effects of cold analgesia and is a receptor for menthol and icilin (mint-derived and synthetic cooling compounds, respectively). TRPA1 (Ankyrin family) is activated by noxious cold (<17 °C), icilin, and a variety of pungent compounds. Extensive amount of medicinal chemistry efforts have been published mainly in the form of patent literature on various classes of cooling compounds by various pharmaceutical companies; however, no prior comprehensive review has been published. When expressed in heterologous expression systems, such as Xenopus oocytes or mammalian cell lines, TRPM8 mediated currents are activated by a number of cooling compounds in addition to menthol and icilin. These include synthetic p-menthane carboxamides along with other class of compounds such as aliphatic/alicyclic alcohols/esters/amides, sulphones/sulphoxides/sulphonamides, heterocyclics, keto-enamines/lactams, and phosphine oxides. In the present review, the medicinal chemistry of various cooling compounds as activators of thermoTRPM8 channel will be discussed according to their chemical classes. The potential of these compounds to emerge as therapeutic agents is also discussed.
Project description:Transient receptor potential channels are a family of cation channels involved in diverse cellular functions. Most of these channels are expressed in the nervous system and play a key role in sensory physiology. TRPM8 (transient receptor potential melastatine 8), a member of this family, is activated by cold, cooling substances such menthol and icilin and voltage. Although TRPM8 is a thermosensitive channel highly expressed in cold sensory neurons, the mechanisms underlying its temperature sensitivity are still poorly understood. Here we show that, in sensory neurons, TRPM8 channel is localized in cholesterol-rich specialized membrane domains known as lipid rafts. We also show that, in heterologous expression systems, lipid raft segregation of TRPM8 is favored by glycosylation at the Asn(934) residue of the polypeptide. In electrophysiological and imaging experiments, using cold and menthol as agonists, we also demonstrate that lipid raft association modulates TRPM8 channel activity. We found that menthol- and cold-mediated responses of TRPM8 are potentiated when the lipid raft association of the channel is prevented. In addition, lipid raft disruption shifts the threshold for TRPM8 activation to a warmer temperature. In view of these data, we suggest a role for lipid rafts in the activity and temperature sensitivity of TRPM8. We propose a model wherein different lipid membrane environments affect the cold sensing properties of TRPM8, modulating the response of cold thermoreceptors.
Project description:The transient receptor potential melastatin 8 (TRPM8) ion channel is a major sensor of environmental cold temperatures. It is activated by cold and chemical agonists, such as menthol and icilin. The activation of these channels both by cold and cooling agents requires the presence of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)]. The mechanism of TRPM8 activation by physical and chemical factors is unknown, and the involvement of cellular signaling pathways has been considered. Here we have characterized the gating mechanism of the rat TRPM8 reconstituted in planar lipid bilayers and its activation by different stimuli. In this system, the influence of cellular signaling pathways can be excluded. We found that TRPM8 activated by cold exhibits steep temperature dependence [temperature coefficient (Q(10)) of ?40], and the channel openings are accompanied by large changes in entropy and enthalpy, suggesting a substantial conformation change. TRPM8 channel behavior upon menthol and icilin activation was distinguishable, and the effect of icilin depended on the presence of calcium on the intracellular side of the protein. Here we also demonstrate that PI(4,5)P(2) is the prime factor that impacts the gating of TRPM8 and that other phosphoinositides are less efficient in supporting channel activity. Menthol increases the potency of PI(4,5)P(2) to activate the channels and increases binding of phosphoinositides to the full-length channel protein. Our data demonstrate conclusively that TRPM8 is gated by cold and its chemical agonists directly, and that dependence of its gating on PI(4,5)P(2) is a result of direct specific interactions with the lipid.
Project description:<h4>Background</h4>The cooling agents menthol and icilin act as agonists at TRPM8 and TRPA1. In vitro, activation of TRPM8 by icilin and cold, but not menthol, is dependent on the activity of a sub-type of phospholipase A2, iPLA2. Lysophospholipids (e.g. LPC) produced by PLA2 activity can also activate TRPM8. The role of TRPA1 as a primary cold sensor in vitro is controversial, although there is evidence that TRPA1 plays a role in behavioural responses to noxious cold stimuli. In this study, we have investigated the roles of TRPM8 and TRPA1 and the influence of iPLA2 on noxious cold sensitivities in naïve animals and after local administration of menthol, icilin and LPC. The roles of the channels in cold sensitivity were investigated in mice lacking either TRPM8 (Trpm8-/-) or TRPA1 (Trpa1-/-).<h4>Results</h4>Intraplantar administration of icilin evoked a dose-dependent increase in sensitivity to a 10 degrees C stimulus that was inhibited by iPLA2 inhibition with BEL. In contrast the cold hypersensitivities elicited by intraplantar menthol and LPC were not inhibited by BEL treatment. BEL had no effect on basal cold sensitivity and mechanical hypersensitivities induced by the TRPV1 agonist, capsaicin, and the P2X3 agonist alpha,beta-methylene ATP. Both Trpm8-/- and Trpa1-/- mice showed longer latencies for paw withdrawal from a 10 degrees C stimulus than wild-type littermates. Cold hypersensitivities induced by either icilin or LPC were absent in Trpm8-/- mice but were retained in Trpa1-/- mice. In contrast, cold hypersensitivity evoked by menthol was present in Trpm8-/- mice but was lost in Trpa1-/- mice.<h4>Conclusions</h4>The findings that iPLA2 inhibition blocked the development of cold hypersensitivity after administration of icilin but failed to affect menthol-induced hypersensitivity agree well with our earlier in vitro data showing a differential effect of iPLA2 inhibition on the agonist activities of these agents. The ability of LPC to induce cold hypersensitivity supports a role for iPLA2 in modulating TRPM8 activity in vivo. Studies on genetically modified mice demonstrated that the effects of icilin and LPC were mediated by TRPM8 and not TRPA1. In contrast, menthol-induced cold hypersensitivity was dependent on expression of TRPA1 and not TRPM8.
Project description:Expressed in somatosensory neurons of the dorsal root and trigeminal ganglion, the transient receptor potential melastatin 8 (TRPM8) channel is a Ca(2+)-permeable cation channel activated by cold, voltage, phosphatidylinositol 4,5-bisphosphate, and menthol. Although TRPM8 channel gating has been characterized at the single channel and macroscopic current levels, there is currently no consensus regarding the extent to which temperature and voltage sensors couple to the conduction gate. In this study, we extended the range of voltages where TRPM8-induced ionic currents were measured and made careful measurements of the maximum open probability the channel can attain at different temperatures by means of fluctuation analysis. The first direct measurements of TRPM8 channel temperature-driven conformational rearrangements provided here suggest that temperature alone is able to open the channel and that the opening reaction is voltage-independent. Voltage is a partial activator of TRPM8 channels, because absolute open probability values measured with fully activated voltage sensors are less than 1, and they decrease as temperature rises. By unveiling the fast temperature-dependent deactivation process, we show that TRPM8 channel deactivation is well described by a double exponential time course. The fast and slow deactivation processes are temperature-dependent with enthalpy changes of 27.2 and 30.8 kcal mol(-1). The overall Q10 for the closing reaction is about 33. A three-tiered allosteric model containing four voltage sensors and four temperature sensors can account for the complex deactivation kinetics and coupling between voltage and temperature sensor activation and channel opening.