Characterisation of TRPM8 as a pharmacophore receptor

Some proteins of the transient receptor potential (TRP) family form temperature sensitive ion channels. One member of the melastatin (M) group, namely TRPM8 is activated by cold and cooling compounds such as menthol and icilin, and its gene is up-regulated in prostate cancer and other malignancies. Here we characterise the effects of the carboxamides WS-12, CPS-113, CPS-369, the carboxylic acid WS-30 and the phosphine oxide WS-148 by Ca²⁺ imaging experiments and whole-cell patch-clamp recordings on TRPM8 expressing human embryonic kidney (HEK), lymph node prostate cancer (LNCaP) and dorsal root ganglia (DRG) cells. The cooling compounds introduced in this study, show a dose-dependent and reversible activation of TRPM8 with EC₅₀ values in the nM to low μM range. The carboxamide WS-12 is most potent in activating TRPM8. It is selective, since other TRP proteins are not stimulated at μM concentrations and its efficacy with respect to TRPM8 is similar to the one of icilin. In summary, the compounds described in this study represent new tools to dissect TRPM8 functions and may serve as chemical leads for the development of additional TRPM8 agonists and novel antagonists. Such compounds may be beneficial for preventing noxious cold perception. They could also be useful in diagnosis and treatment of most common cancers in which the TRPM8 gene is up-regulated in comparison to the corresponding normal tissue.     

Prospects for prostate cancer imaging and therapy using high-affinity TRPM8 activators

One of the best-studied temperature-gated channels is transient receptor potential melastatin 8 (TRPM8), which is activated by cold and cooling agents, such as menthol. Besides inducing a cooling sensation in sensory neurons, TRPM8 channel activation also plays a major role in physiopathology. Indeed, TRPMP8 expression increases in early stages of prostate cancer and its involvement in prostate cell apoptosis has recently been demonstrated. Thus, as TRPM8 is a tumor marker with significant potential use in diagnosis, as well as a target for cancer therapy, there is a need for new TRPM8-specific ligands. In this study, we investigated the action of "WS" compounds on TRPM8 channels. We compared the affinity of these molecules to that of menthol and icilin. This enabled us to identify new TRPM8 agonists. The menthol analog with the highest affinity, WS-12, had an EC(50) value about 2000 times lower than that of menthol and is, therefore, the highest-affinity TRPM8 ligand known to date. Finally, incorporating a fluorine atom in the WS-12 retained 75% of the activity of the parent compound. The high affinity of this new TRPM8 ligand and the possibility of incorporating a radiohalogen could thus be useful for diagnosis, monitoring and, perhaps, even therapy of prostate cancer.     

瞬时受体电位M8离子通道功能及其翻译后修饰调节机制

瞬时受体电位M8(transient receptor potential melastatin 8, TRPM8)又称冷及薄荷醇感受器,位于细胞膜或细胞器膜上,是瞬时受体电位(transient receptor potential, TRP)通道超家族中的一员。TRPM8通道分布广泛,是一个非选择性阳离子通道,可作为冷热传感器和冷痛传感器进行信号传导,参与众多生物过程的调节,在维持细胞内外稳态、控制离子进出细胞方面具有重要作用。研究发现,蛋白质翻译后修饰(post-translational modification, PTM)通过调控TRPM8通道的功能,进而影响多种疾病的发生和发展。因此,探究TRPM8的翻译后修饰的过程,对深入了解TRPM8的功能及调控机制是十分必要的。目前,已报道的TRPM8翻译后修饰包括磷酸化、泛素化和糖基化等,它们能够调控蛋白质的相互作用和改变TRPM8离子通道的活性,从而调控细胞增殖、迁移和凋亡。值得注意的是,TRPM8的表达与前列腺癌、膀胱癌和乳腺癌等多种癌症密切相关。本文将从TRPM8离子通道的结构出发,系统地阐述TRPM8蛋白翻译后修饰和激动剂、...     

The super-cooling agent icilin reveals a mechanism of coincidence detection by a temperature-sensitive TRP channel

TRPM8, a member of the transient receptor potential family of ion channels, depolarizes somatosensory neurons in response to cold. TRPM8 is also activated by the cooling agents menthol and icilin. When exposed to menthol or cold, TRPM8 behaves like many ligand-gated channels, exhibiting rapid activation followed by moderate Ca(2+)-dependent adaptation. In contrast, icilin activates TRPM8 with extremely variable latency followed by extensive desensitization, provided that calcium is present. Here, we show that, to achieve full efficacy, icilin requires simultaneous elevation of cytosolic Ca2+, either via permeation through TRPM8 channels or by release from intracellular stores. Thus, two stimuli must be paired to elicit full channel activation, illustrating the potential for coincidence detection by TRP channels. Determinants of icilin sensitivity map to a region of TRPM8 that corresponds to the capsaicin binding site on the noxious heat receptor TRPV1, suggesting a conserved molecular logic for gating of these thermosensitive channels by chemical agonists.     

Regulation of activity of transient receptor potential melastatin 8 (TRPM8) channel by its short isoforms

One important mechanism of the regulation of membrane ion channels involves their nonfunctional isoforms generated by alternative splicing. However, knowledge of such isoforms for the members of the transient receptor potential (TRP) superfamily of ion channels remains quite limited. This study focuses on the TRPM8, which functions as a cold receptor in sensory neurons but is also expressed in tissues not exposed to ambient temperatures, as well as in cancer tissues. We report the cloning from prostate cancer cells of new short splice variants of TRPM8, termed short TRPM8α and short TRPM8β. Our results show that both variants are in a closed configuration with the C-terminal tail of the full-length TRPM8 channel, resulting in stabilization of its closed state and thus reducing both its cold sensitivity and activity. Our findings therefore uncover a new mode of regulation of the TRPM8 channel by its splice variants.     

TRPM8 is required for cold sensation in mice

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; however, it is unclear whether either ion channel is required for thermosensation in vivo. We show that mice lacking TRPM8 have severe behavioral deficits in response to cold stimuli. In thermotaxis assays of temperature gradient and two-temperature choice assays, TRPM8-deficient mice exhibit strikingly reduced avoidance of cold temperatures. TRPM8-deficient mice also lack behavioral response to cold-inducing icilin application and display an attenuated response to acetone, an unpleasant cold stimulus. However, TRPM8-deficient mice have normal nociceptive-like responses to subzero centigrade temperatures, suggesting the presence of at least one additional noxious cold receptor. Finally, we show that TRPM8 mediates the analgesic effect of moderate cooling after administration of formalin, a painful stimulus. Therefore, depending on context, TRPM8 contributes to sensing unpleasant cold stimuli or mediating the effects of cold analgesia.     

Contribution of the S5-Pore-S6 Domain to the Gating Characteristics of the Cation Channels TRPM2 and TRPM8

The closely related cation channels TRPM2 and TRPM8 show completely different requirements for stimulation and are regulated by Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ and the single-channel properties in TRPM2 are not determined by the S5-pore-S6 region.     

Voltage- and cold-dependent gating of single TRPM8 ion channels

Transient receptor potential (TRP) channels play critical roles in cell signaling by coupling various environmental factors to changes in membrane potential that modulate calcium influx. TRP channels are typically activated in a polymodal manner, thus integrating multiple stimuli. Although much progress has been made, the underlying mechanisms of TRP channel activation are largely unknown. The TRPM8 cation channel has been extensively investigated as a major neuronal cold sensor but is also activated by voltage, calcium store depletion, and some lipids as well as by compounds that produce cooling sensations, such as menthol or icilin. Several models of TRPM8 activation have been proposed to explain the interaction between these diverse stimuli. However, a kinetic scheme is not yet available that can describe the detailed single-channel kinetics to gain further insight into the underlying gating mechanism. To work toward this goal, we investigated voltage-dependent single-channel gating in cell-attached patches at two different temperatures (20 and 30 °C) using HEK293 cells stably expressing TRPM8. Both membrane depolarization and cooling increased channel open probability (P(o)) mainly by decreasing the duration of closed intervals, with a smaller increase in the duration of open intervals. Maximum likelihood analysis of dwell times at both temperatures indicated gating in a minimum of five closed and two open states, and global fitting over a wide range of voltages identified a seven-state model that described the voltage dependence of P(o), the single-channel kinetics, and the response of whole-cell currents to voltage ramps and steps. The major action of depolarization and cooling was to accelerate forward transitions between the same two sets of adjacent closed states. The seven-state model provides a general mechanism to account for TRPM8 activation by membrane depolarization at two temperatures and can serve as a starting point for further investigations of multimodal TRP activation.     

The TRPM8 Protein Is a Testosterone Receptor: I. BIOCHEMICAL EVIDENCE FOR DIRECT TRPM8-TESTOSTERONE INTERACTIONS*

The transient receptor potential ion channel of the melastatin subfamily, TRPM8, is a major cold receptor in the peripheral nervous system. Along with the sensory neurons, the TRPM8 protein is highly expressed in the prostate epithelial cells, and this expression is regulated by androgens. Here we investigated the expression and intracellular localization of the TRPM8 channel in relationship to androgens. We performed experiments using human prostate tissues obtained from healthy individuals and patients with prostate cancer at various stages of the disease as well as in cultured cells. Using an immunohistochemistry approach, we detected an intensive colocalization pattern of the TRPM8 protein with endogenous androgens in all tissues tested, suggesting possible interactions. Co-immunoprecipitation experiments performed using cultured prostate epithelial cells, prostate cancer cells, and HEK-293 cells stably expressing TRPM8 further confirmed direct binding of the steroid hormone, testosterone, to the TRPM8 protein. Applications of picomolar concentrations of testosterone to the primary human prostate cells, endogenously expressing TRPM8, elicited Ca²⁺ responses and channel currents, and those were inhibited in the presence of TRPM8 antagonist, N-(2-aminoethyl)-N-(4-(benzyloxy)-3-methoxybenzyl)thiophene-2-carboxamide hydrochloride. These results indicate that the TRPM8 channel is physically associated with testosterone and suggest that, in addition to a genomic role, testosterone plays a role in direct regulation of the TRPM8 channel function.     

The TRPM8 Protein Is a Testosterone Receptor: II. FUNCTIONAL EVIDENCE FOR AN IONOTROPIC EFFECT OF TESTOSTERONE ON TRPM8*

Testosterone is a key steroid hormone in the development of male reproductive tissues and the regulation of the central nervous system. The rapid signaling mechanism induced by testosterone affects numerous behavioral traits, including sexual drive, aggressiveness, and fear conditioning. However, the currently identified testosterone receptor(s) is not believed to underlie the fast signaling, suggesting an orphan pathway. Here we report that an ion channel from the transient receptor potential family, TRPM8, commonly known as the cold and menthol receptor is the major component of testosterone-induced rapid actions. Using cultured and primary cell lines along with the purified TRPM8 protein, we demonstrate that testosterone directly activates TRPM8 channel at low picomolar range. Specifically, testosterone induced TRPM8 responses in primary human prostate cells, PC3 prostate cancer cells, dorsal root ganglion neurons, and hippocampal neurons. Picomolar concentrations of testosterone resulted in full openings of the purified TRPM8 channel in planar lipid bilayers. Furthermore, acute applications of testosterone on human skin elicited a cooling sensation. Our data conclusively demonstrate that testosterone is an endogenous and highly potent agonist of TRPM8, suggesting a role of TRPM8 channels well beyond their well established function in somatosensory neurons. This discovery may further imply TRPM8 channel function in testosterone-dependent behavioral traits.     

Antibodies to the Extracellular Pore Loop of TRPM8 Act as Antagonists of Channel Activation

The mammalian transient receptor potential melastatin channel 8 (TRPM8) is highly expressed in trigeminal and dorsal root ganglia. TRPM8 is activated by cold temperature or compounds that cause a cooling sensation, such as menthol or icilin. TRPM8 may play a role in cold hypersensitivity and hyperalgesia in various pain syndromes. Therefore, TRPM8 antagonists are pursued as therapeutics. In this study we explored the feasibility of blocking TRPM8 activation with antibodies. We report the functional characterization of a rabbit polyclonal antibody, ACC-049, directed against the third extracellular loop near the pore region of the human TRPM8 channel. ACC-049 acted as a full antagonist at recombinantly expressed human and rodent TRPM8 channels in cell based agonist-induced ⁴⁵Ca²⁺ uptake assays. Further, several poly-and monoclonal antibodies that recognize the same region also blocked icilin activation of not only recombinantly expressed TRPM8, but also endogenous TRPM8 expressed in rat dorsal root ganglion neurons revealing the feasibility of generating monoclonal antibody antagonists. We conclude that antagonist antibodies are valuable tools to investigate TRPM8 function and may ultimately pave the way for development of therapeutic antibodies.     

N-glycosylation of TRPM8 ion channels modulates temperature sensitivity of cold thermoreceptor neurons

TRPM8 is a member of the transient receptor potential ion channel superfamily, which is expressed in sensory neurons and is activated by cold and cooling compounds, such as menthol. Activation of TRPM8 by agonists takes place through shifts in its voltage activation curve, allowing channel opening at physiological membrane potentials. Here, we studied the role of the N-glycosylation occurring at the pore loop of TRPM8 on the function of the channel. Using heterologous expression of recombinant channels in HEK293 cells we found that the unglycosylated TRPM8 mutant (N934Q) displays marked functional differences compared with the wild type channel. These differences include a shift in the threshold of temperature activation and a reduced response to menthol and cold stimuli. Biophysical analysis indicated that these modifications are due to a shift in the voltage dependence of TRPM8 activation toward more positive potentials. By using tunicamycin, a drug that prevents N-glycosylation of proteins, we also evaluated the effect of the N-glycosylation on the responses of trigeminal sensory neurons expressing TRPM8. These experiments showed that the lack of N-glycosylation affects the function of native TRPM8 ion channels in a similar way to heterologously expressed ones, causing an important shift of the temperature threshold of cold-sensitive thermoreceptor neurons. Altogether, these results indicate that post-translational modification of TRPM8 is an important mechanism modulating cold thermoreceptor function, explaining the marked differences in temperature sensitivity observed between recombinant and native TRPM8 ion channels.     

TRPM8-independent menthol-induced Ca2+ release from endoplasmic reticulum and Golgi

Menthol, a secondary alcohol produced by the peppermint herb, Mentha piperita, is widely used in the food and pharmaceutical industries as a cooling/soothing compound and odorant. It induces Ca2+ influx in a subset of sensory neurons from dorsal root and trigeminal ganglia, due to activation of TRPM8, a Ca2+-permeable, cold-activated member of the TRP superfamily of cation channels. Menthol also induces Ca2+ release from intracellular stores in several TRPM8-expressing cell types, which has led to the suggestion that TRPM8 can function as an intracellular Ca2+-release channel. Here we show that menthol induces Ca2+ release from intracellular stores in four widely used cell lines (HEK293, lymph node carcinoma of the prostate (LNCaP), Chinese hamster ovary (CHO), and COS), and provide several lines of evidence indicating that this release pathway is TRPM8-independent: 1) menthol-induced Ca2+ release was potentiated at higher temperatures, which contrasts to the cold activation of TRPM8; 2) overexpression of TRPM8 did not enhance the menthol-induced Ca2+) release; 3) menthol-induced Ca2+ release was mimicked by geraniol and linalool, which are structurally related to menthol, but not by the more potent TRPM8 agonists icilin or eucalyptol; and 4) TRPM8 expression in HEK293 cells was undetectable at the protein and mRNA levels. Moreover, using a novel TRPM8-specific antibody we demonstrate that both heterologously expressed TRPM8 (in HEK293 cells) and endogenous TRPM8 (in LNCaP cells) are mainly localized in the plasma membrane, which contrast to previous localization studies using commercial anti-TRPM8 antibodies. Finally, aequorin-based measurements demonstrate that the TRPM8-independent menthol-induced Ca2+ release originates from both endoplasmic reticulum and Golgi compartments.     

TRP channels in cancer

The progression of cells from a normal differentiated state in which rates of proliferation and apoptosis are balanced to a tumorigenic and metastatic state involves the accumulation of mutations in multiple key signalling proteins and the evolution and clonal selection of more aggressive cell phenotypes. These events are associated with changes in the expression of numerous other proteins. This process of tumorigenesis involves the altered expression of one or more TRP proteins, depending on the nature of the cancer. The most clearly described changes are those involving TRPM8, TRPV6 and TRPM1. Expression of TRPM8 is substantially increased in androgen-dependent prostate cancer cells, but is decreased in androgen independent and metastatic prostate cancer. TRPM8 expression is regulated, in part, by androgens, most likely through androgen response elements in the TRPM8 promoter region. TRPM8 channels are involved in the regulation of cell proliferation and apoptosis. Expression of TRPV6 is also increased in prostate cancer and in a number of other cancers. In contrast to TRPM8, expression of TRPV6 is not directly regulated by androgens. TRPM1 is highly expressed in early stage melanomas but its expression declines with increases in the degree of aggressiveness of the melanoma. The expression of TRPV1, TRPC1, TRPC6, TRPM4, and TRPM5 is also increased in some cancers. The level of expression of TRPM8 and TRPV6 in prostate cancer, and of TRPM1 in melanomas, potentially provides a good prognostic marker for predicting the course of the cancer in individuals. The Drosophila melanogaster, TRPL, and the TRPV1 and TRPM8 proteins, have been used to try to develop strategies to selectively kill cancer cells by activating Ca(2+) and Na(+) entry, producing a sustained increase in the cytoplasmic concentration of these ions, and subsequent cell death by apoptosis and necrosis. TRPV1 is expressed in neurones involved in sensing cancer pain, and is a potential target for pharmacological inhibition of cancer pain in bone metastases, pancreatic cancer and most likely in other cancers. Further studies are required to assess which other TRP proteins are associated with the development and progression of cancer, what roles TRP proteins play in this process, and to develop further knowledge of TRP proteins as targets for pharmaceutical intervention and targeting in cancer.     

Regulation of transient receptor potential channels of melastatin type 8 (TRPM8): effect of cAMP, cannabinoid CB(1) receptors and endovanilloids

The transient receptor potential channel of melastatin type 8 (TRPM8), which is gated by low (<25 degrees C) temperature and chemical compounds, is regulated by protein kinase C-mediated phosphorylation in a way opposite to that observed with the transient receptor potential channel of vanilloid type 1 (TRPV1), i.e. by being desensitized and not sensitized. As TRPV1 is sensitized also by protein kinase A (PKA)-mediated phosphorylation, we investigated the effect of two activators of the PKA pathway, 8-Br-cAMP and forskolin, on the activity of menthol and icilin at TRPM8 in HEK-293 cells stably overexpressing the channel (TRPM8-HEK-293 cells). We also studied the effect on TRPM8 of: (1) a series of compounds previously shown to activate or antagonize TRPV1, and (2) co-stimulation of transiently co-expressed cannabinoid CB(1) receptors. Both 8-Br-cAMP (100 microM) and forskolin (10 microM) right-shifted the dose-response curves for the TRPM8-mediated effect of icilin and menthol on intracellular Ca(2+). The inhibitory effects of 8-Br-cAMP and forskolin were attenuated by the selective PKA inhibitor Rp-cAMP-S. Stimulation of human CB(1) receptors transiently co-expressed in TRPM8-HEK-293 cells also inhibited TRPM8 response to icilin. Finally, some TRPV1 agonists and antagonists, but not iodinated antagonists, antagonized icilin- and much less so menthol-, induced TRPM8 activation. Importantly, the endovanilloids/endocannabinoids, anandamide and NADA, also antagonized TRPM8 at submicromolar concentrations. Although these findings need to be confirmed by experiments directly measuring TRPM8 activity in natively TRPM8-expressing cells, they support the notion that the same regulatory events have opposing actions on TRPM8 and TRPV1 receptors and identify anandamide and NADA as the first potential endogenous functional antagonists of TRPM8 channels.     

Functional and Modeling Studies of the Transmembrane Region of the TRPM8 Channel

Members of the transient receptor potential (TRP) ion channel family act as polymodal cellular sensors, which aid in regulating Ca²⁺ 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.     

TRP channels in cancer

The progression of cells from a normal differentiated state in which rates of proliferation and apoptosis are balanced to a tumorigenic and metastatic state involves the accumulation of mutations in multiple key signalling proteins and the evolution and clonal selection of more aggressive cell phenotypes. These events are associated with changes in the expression of numerous other proteins. This process of tumorigenesis involves the altered expression of one or more TRP proteins, depending on the nature of the cancer. The most clearly described changes are those involving TRPM8, TRPV6 and TRPM1. Expression of TRPM8 is substantially increased in androgen-dependent prostate cancer cells, but is decreased in androgen independent and metastatic prostate cancer. TRPM8 expression is regulated, in part, by androgens, most likely through androgen response elements in the TRPM8 promoter region. TRPM8 channels are involved in the regulation of cell proliferation and apoptosis. Expression of TRPV6 is also increased in prostate cancer and in a number of other cancers. In contrast to TRPM8, expression of TRPV6 is not directly regulated by androgens. TRPM1 is highly expressed in early stage melanomas but its expression declines with increases in the degree of aggressiveness of the melanoma. The expression of TRPV1, TRPC1, TRPC6, TRPM4, and TRPM5 is also increased in some cancers. The level of expression of TRPM8 and TRPV6 in prostate cancer, and of TRPM1 in melanomas, potentially provides a good prognostic marker for predicting the course of the cancer in individuals. The Drosophila melanogaster, TRPL, and the TRPV1 and TRPM8 proteins, have been used to try to develop strategies to selectively kill cancer cells by activating Ca²⁺ and Na⁺ entry, producing a sustained increase in the cytoplasmic concentration of these ions, and subsequent cell death by apoptosis and necrosis. TRPV1 is expressed in neurones involved in sensing cancer pain, and is a potential target for pharmacological inhibition of cancer pain in bone metastases, pancreatic cancer and most likely in other cancers. Further studies are required to assess which other TRP proteins are associated with the development and progression of cancer, what roles TRP proteins play in this process, and to develop further knowledge of TRP proteins as targets for pharmaceutical intervention and targeting in cancer.