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.     

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.     

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、...     

5-benzyloxytryptamine as an antagonist of TRPM8

5-Benzyloxytryptamine 19 was found to act as an antagonist of the TRPM8 ion-channel. For example, 19 had an IC(50) of 0.34 μM when menthol was used as the stimulating agonist. Related commercially-available tryptamine derivatives showed diminished, or no antagonist activity at TRPM8. The structural similarity of 5-benzyloxytryptamine to other literature TRPM8 antagonists was noted.     

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 design and synthesis of novel, phosphonate-containing transient receptor potential melastatin 8 (TRPM8) antagonists

A series of benzothiophene-based phosphonates was synthesized and many analogs within the series were shown to be potent antagonists of the TRPM8 channel. The compounds were obtained as a racemic mixture in 5 synthetic steps, and were tested for TRPM8 antagonist activity in a recombinant, canine TRPM8-expressing cell line using a fluorometric imaging plate reader (FLIPR) assay. Structure-activity relationships were developed initially by modification of the core structure and subsequently by variation of the aromatic substituents and the phosphonate ester. Compound 9l was administered intraperitoneally to rats and demonstrated engagement of the TRPM8 target in both prevention and reversal-modes in an icilin-induced 'wet-dog' shake model.     

Discovery of vinylcycloalkyl-substituted benzimidazole TRPM8 antagonists effective in the treatment of cold allodynia

Thermosensitive transient receptor potential melastatin 8 (TRPM8) antagonists are considered to be potential therapeutic agents for the treatment of cold hypersensitivity. The discovery of a new class of TRPM8 antagonists that shows in vivo efficacy in the rat chronic constriction injury (CCI)-induced model of neuropathic pain is described.     

Effect of TRPM8 ion channel activation on thermoregulatory response to cooling

In rats, the effect of activation of the cold- and menthol-sensitive TRPM8 ion channel on different thermoregulatory parameters: total oxygen consumption, carbon dioxide release, respiration coefficient, constriction response of skin blood vessels, muscle activity, was studied. Activation of TRPM8 with menthol even in thermoneutral conditions produces an increase in oxygen consumption and a decrease in respiratory coefficient, which may suggest enhanced non-shivering thermogenesis and lipolysis. Rapid cooling against the background of TRPM8 activation is characterized by a decrease in the temperature thresholds of all thermoregulatory responses without associated changes in sequences of their initiation as well as in enhancement of metabolic component of emergency thermogenesis which leads to improved maintenance of core temperature in conditions when external cold acts on the organism. The obtained data on the effect of TRPM8 activation on metabolic parameters in thermoneutral conditions and under cooling suggest acontinuous involvement of this receptor in regulation of total metabolism and, possibly, in determination of the type of organism's metabolism as well as in determination of organism's response to external cooling.     

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.     

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 microM range. The carboxamide WS-12 is most potent in activating TRPM8. It is selective, since other TRP proteins are not stimulated at muM 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.     

Human TRPM8 and TRPA1 pain channels,including a gene variant with increased sensitivity to agonists (TRPA1 R797T), exhibit differential regulation by SRC-tyrosine kinase inhibitor

TRPM8 (transient receptor potential M8) and TRPA1 (transient receptor potential A1) are cold-temperature-sensitive nociceptors expressed in sensory neurons but their behaviour in neuronal cells is poorly understood. Therefore DNA expression constructs containing human TRPM8 or TRPA1 cDNAs were transfected into HEK (human embryonic kidney cells)-293 or SH-SY5Y neuroblastoma cells and G418 resistant clones analysed for effects of agonists and antagonists on intracellular Ca²⁺ levels. Approximately 51% of HEK-293 and 12% of SH-SY5Y cell clones expressed the transfected TRP channel. TRPM8 and TRPA1 assays were inhibited by probenecid, indicating the need to avoid this agent in TRP channel studies. A double-residue mutation in ICL-1 (intracellular loop-1) of TRPM8 (SV762,763EL, mimicking serine phosphorylation) or one in the C-terminal tail region (FK1045,1046AG, a lysine knockout) retained sensitivity to agonists (WS 12, menthol) and antagonist {AMTB [N-(3-Aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide]}. SNP (single nucleotide polymorphism) variants in TRPA1 ICL-1 (R797T, S804N) and TRPA1 fusion protein containing C-terminal (His)₁₀ retained sensitivity to agonists (cinnamaldehyde, allyl-isothiocyanate, carvacrol, eugenol) and antagonists (HC-030031, A967079). One SNP variant, 797T, possessed increased sensitivity to agonists. TRPA1 became repressed in SH-SY5Y clones but was rapidly rescued by Src-family inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine]. Conversely, TRPM8 in SH-SY5Y cells was inhibited by PP2. Further studies utilizing SH-SY5Y may identify structural features of TRPA1 and TRPM8 involved in conferring differential post-translational regulation.     

TRPM8 ion channel ligands for new therapeutic applications and as probes to study menthol pharmacology

Since the discovery of the TRPM8 gene in 2001, the TRPM8 ion channel, better known as the ‘cold receptor’ has been the target of a significant effort from the pharmaceutical industry to produce small-molecule agonists and antagonists of this receptor for various therapeutic applications ranging from cancer and urological disorders to the treatment of cold hypersensitivity and pain. Recently, a number of clinical studies have implicated menthol, the natural ligand of TRPM8, in facilitating and maintaining cigarette smoking behavior, possibly through its counter-irritant effects. However, a pharmacological link between menthol's action via TRPM8 and nicotine addiction has not been yet been investigated. This review gives an overview of reported small-molecule TRPM8 agonists and antagonists and discusses their efficacy in models of various disease states. These compounds may be useful pharmacological tools to investigate the effect of menthol on nicotine addiction.     

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.     

TRPM8 channel augments T-cell activation and proliferation

The transient receptor potential melastatin 8 (TRPM8) is an ion channel that has been widely studied as a cold-sensitive nociceptor. However, its importance in nonneuronal cells is mostly unexplored. Here, we describe the presence and functional significance of endogenous TRPM8, a nonselective Ca²⁺-channel in T cell functions. The major pool of TRPM8 resides at the T cell surface and its surface accumulation significantly increases in activated T cells. TRPM8 activation synergizes with T-cell receptor (TCR) stimulation to increase CD25, CD69 levels and enhances secretion of proinflammatory cytokine tumor necrosis factor. However, TRPM8 inhibition does not restrict TCR stimulation mediated activation of T cells, indicating that unlike the heat-sensitive TRPV1 and TRPV4 channels, the cold-sensitive TRPM8 channel may be dispensable during T-cell activation, at least in mice. In this study, we demonstrate that TRPM8 promotes TCR-induced intracellular calcium increase. TRPM8 activation is beneficial for T-cell activation and differentiation into effector cells. TRPM8 inhibition during the T-cell activation process may lead to altered phenotype and reduced proliferation, without affecting cell viability. These results collectively establish TRPM8 as a functional calcium channel whose activation may be utilized for mounting an effective immune response. The findings of this study will be relevant to the regulation and response of T cells during cell-mediated immunity. These results will likely further our understanding on the role of ion channels in T-cell activation.     

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.     

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.     

Arylglycine derivatives as potent transient receptor potential melastatin 8 (TRPM8) antagonists

A series of arylglycine-based analogs was synthesized and tested for TRPM8 antagonism in a cell-based functional assay. Following structure–activity relationship studies in vitro, a number of compounds were identified as potent TRPM8 antagonists and were subsequently evaluated in an in vivo pharmacodynamic assay of icilin-induced ‘wet-dog’ shaking in which compound 12 was fully effective. TRPM8 antagonists of the type described here may be useful in treating pain conditions wherein cold hypersensitivity is a dominant feature.     

Cooling-sensitive TRPM8 is thermostat of skin temperature against cooling

We have shown that cutaneous cooling-sensitive receptors can work as thermostats of skin temperature against cooling. However, molecule of the thermostat is not known. Here, we studied whether cooling-sensitive TRPM8 channels act as thermostats. TRPM8 in HEK293 cells generated output (y) when temperature (T) was below threshold of 28.4°C. Output (y) is given by two equations: At T >28.4°C, y = 0; At T <28.4°C, y  =  -k(T – 28.4°C). These equations show that TRPM8 is directional comparator to elicits output (y) depending on negative value of thermal difference (ΔT  =  T – 28.4°C). If negative ΔT-dependent output of TRPM8 in the skin induces responses to warm the skin for minimizing ΔT recursively, TRPM8 acts as thermostats against cooling. With TRPM8-deficient mice, we explored whether TRPM8 induces responses to warm the skin against cooling. In behavioral regulation, when room temperature was 10°C, TRPM8 induced behavior to move to heated floor (35°C) for warming the sole skin. In autonomic regulation, TRPM8 induced activities of thermogenic brown adipose tissue (BAT) against cooling. When menthol was applied to the whole trunk skin at neutral room temperature (27°C), TRPM8 induced a rise in core temperature, which warmed the trunk skin slightly. In contrast, when room was cooled from 27 to 10°C, TRPM8 induced a small rise in core temperature, but skin temperature was severely reduced in both TRPM8-deficient and wild-type mice by a large heat leak to the surroundings. This shows that TRPM8-driven endothermic system is less effective for maintenance of skin temperature against cooling. In conclusion, we found that TRPM8 is molecule of thermostat of skin temperature against cooling.     

Ethanol inhibits cold-menthol receptor TRPM8 by modulating its interaction with membrane phosphatidylinositol 4,5-bisphosphate

Ethanol has opposite effects on two members of the transient receptor potential (TRP) family of ion channels: it inhibits the cold-menthol receptor TRPM8, whereas it potentiates the activity of the heat- and capsaicin-gated vanilloid receptor TRPV1. Both thermosensitive cation channels are critically regulated by the membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP(2)). The effects of this phospholipid on TRPM8 and TRPV1 are also functionally opposite: PIP(2) is necessary for the activation of TRPM8 but it constitutively inhibits TRPV1. This parallel led us to investigate the possible role of PIP(2) in the ethanol-induced modulation of rat TRPM8, heterologously expressed in HEK293T cells. In this study, we characterize the effects of ethanol (0.1-10%) on whole-cell currents produced by menthol and by low temperature (< 17 degrees C). We show that the inclusion of PIP(2) in the intracellular solution results in a strong reduction in the ethanol-induced inhibition of menthol-evoked responses. Conversely, intracellular dialysis with anti-PIP(2) antibody or with the PIP(2) scavenger, poly L-lysine, enhanced the ethanol-induced inhibition of TRPM8. A 20 min pre-incubation with wortmannin caused a modest decrease in inhibition produced by 1% ethanol, indicating that the ethanol-induced inhibition is not mediated by lipid kinases. These findings suggest that ethanol inhibits TRPM8 by weakening the PIP(2)-TRPM8 channel interaction; a similar mechanism may contribute to the ethanol-mediated modulation of some other PIP(2)-sensitive TRP channels.