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.     

Bidirectional Modulation of Thermal and Chemical Sensitivity of TRPM8 Channels by the Initial Region of the N-terminal Domain

TRPM8, a nonselective cation channel activated by cold, voltage, and cooling compounds such as menthol, is the principal molecular detector of cold temperatures in primary sensory neurons of the somatosensory system. The N-terminal domain of TRPM8 consists of 693 amino acids, but little is known about its contribution to channel function. Here, we identified two distinct regions within the initial N terminus of TRPM8 that contribute differentially to channel activity and proper folding and assembly. Deletion or substitution of the first 40 residues yielded channels with augmented responses to cold and menthol. The thermal threshold of activation of these mutants was shifted 2 °C to higher temperatures, and the menthol dose-response curve was displaced to lower concentrations. Site-directed mutagenesis screening revealed that single point mutations at positions Ser-26 or Ser-27 by proline caused a comparable increase in the responses to cold and menthol. Electrophysiological analysis of the S27P mutant revealed that the enhanced sensitivity to agonists is related to a leftward shift in the voltage dependence of activation, increasing the probability of channel openings at physiological membrane potentials. In addition, we found that the region encompassing positions 40–60 is a key element in the proper folding and assembly of TRPM8. Different deletions and mutations within this region rendered channels with an impaired function that are retained within the endoplasmic reticulum. Our results suggest a critical contribution of the initial region of the N-terminal domain of TRPM8 to thermal and chemical sensitivity and the proper biogenesis of this polymodal ion channel.     

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.     

Discovery and development of a novel class of phenoxyacetyl amides as highly potent TRPM8 agonists for use as cooling agents

The paper presents the activity trends for a novel series of phenoxyacetyl amides as human TRPM8 receptor agonists. This series encompasses in vitro activity values ranging from the micromolar to the picomolar levels. Sensory evaluation of these molecules highlights their relevance as cooling agents for oral applications. The positive outcome of the complete evaluation of N-(1H-pyrazol-3-yl)-N-(thiophen-2-ylmethyl)-2-(p-tolyloxy)acetamide resulted in its approval for Generally Recognized As Safe (GRAS) status by the Flavor & Extract Manufacturer Association (FEMA) as FEMA 4809.     

Inhibition of transient receptor potential melastatin 7 (TRPM7) protects against Schwann cell trans-dedifferentiation and proliferation during Wallerian degeneration

ABSTRACT

Irreversible peripheral neurodegenerative diseases such as diabetic peripheral neuropathy are becoming increasingly common due to rising rates of diabetes mellitus; however, no effective therapeutic treatments have been developed. One of main causes of irreversible peripheral neurodegenerative diseases is dysfunction in Schwann cells, which are neuroglia unique to the peripheral nervous system (PNS). Because homeostasis of calcium (Ca²⁺) and magnesium (Mg²⁺) is essential for Schwann cell dynamics, the regulation of these cations is important for controlling peripheral nerve degeneration and regeneration. Transient receptor potential melastatin 7 (TRPM7) is a non-selective ion (Ca²⁺ and Mg²⁺) channel that is expressed in Schwann cells. In the present study, we demonstrated in an ex vivo culture system that inhibition of TRPM7 during peripheral nerve degeneration (Wallerian degeneration) suppressed dedifferentiable or degenerative features (trans-dedifferentiation and proliferation) and conserved a differentiable feature of Schwann cells. Our results indicate that TRPM7 could be very useful as a molecular target for irreversible peripheral neurodegenerative diseases, facilitating discovery of new therapeutic methods for improving human health.     

Short isoforms of the cold receptor TRPM8 inhibit channel gating by mimicking heat action rather than chemical inhibitors

Transient receptor potential (TRP) channels couple various environmental factors to changes in membrane potential, calcium influx, and cell signaling. They also integrate multiple stimuli through their typically polymodal activation. Thus, although the TRPM8 channel has been extensively investigated as the major neuronal cold sensor, it is also regulated by various chemicals, as well as by several short channel isoforms. Mechanistic understanding of such complex regulation is facilitated by quantitative single-channel analysis. We have recently proposed a single-channel mechanism of TRPM8 regulation by voltage and temperature. Using this gating mechanism, we now investigate TRPM8 inhibition in cell-attached patches using HEK293 cells expressing TRPM8 alone or coexpressed with its short sM8-6 isoform. This is compared with inhibition by the chemicals N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide (BCTC) and clotrimazole or by elevated temperature. We found that within the seven-state single-channel gating mechanism, inhibition of TRPM8 by short sM8-6 isoforms closely resembles inhibition by increased temperature. In contrast, inhibition by BCTC and that by clotrimazole share a different set of common features.     

Selective Activation of Nociceptor TRPV1 Channel and Reversal of Inflammatory Pain in Mice by a Novel Coumarin Derivative Muralatin L from Murraya alata

Coumarin and its derivatives are fragrant natural compounds isolated from the genus Murraya that are flowering plants widely distributed in East Asia, Australia, and the Pacific Islands. Murraya plants have been widely used as medicinal herbs for relief of pain, such as headache, rheumatic pain, toothache, and snake bites. However, little is known about their analgesic components and the molecular mechanism underlying pain relief. Here, we report the bioassay-guided fractionation and identification of a novel coumarin derivative, named muralatin L, that can specifically activate the nociceptor transient receptor potential vanilloid 1 (TRPV1) channel and reverse the inflammatory pain in mice through channel desensitization. Muralatin L was identified from the active extract of Murraya alata against TRPV1 transiently expressed in HEK-293T cells in fluorescent calcium FlexStation assay. Activation of TRPV1 current by muralatin L and its selectivity were further confirmed by whole-cell patch clamp recordings of TRPV1-expressing HEK-293T cells and dorsal root ganglion neurons isolated from mice. Furthermore, muralatin L could reverse inflammatory pain induced by formalin and acetic acid in mice but not in TRPV1 knock-out mice. Taken together, our findings show that muralatin L specifically activates TRPV1 and reverses inflammatory pain, thus highlighting the potential of coumarin derivatives from Murraya plants for pharmaceutical and medicinal applications such as pain therapy.     

Transient Receptor Potential Canonical 1 (TRPC1) Channels as Regulators of Sphingolipid and VEGF Receptor Expression: IMPLICATIONS FOR THYROID CANCER CELL MIGRATION AND PROLIFERATION*

The identity of calcium channels in the thyroid is unclear. In human follicular thyroid ML-1 cancer cells, sphingolipid sphingosine 1-phosphate (S1P), through S1P receptors 1 and 3 (S1P₁/S1P₃), and VEGF receptor 2 (VEGFR2) stimulates migration. We show that human thyroid cells express several forms of transient receptor potential canonical (TRPC) channels, including TRPC1. In TRPC1 knockdown (TRPC1-KD) ML-1 cells, the basal and S1P-evoked invasion and migration was attenuated. Furthermore, the expression of S1P₃ and VEGFR2 was significantly down-regulated. Transfecting wild-type ML-1 cells with a nonconducting TRPC1 mutant decreased S1P₃ and VEGFR2 expression. In TRPC1-KD cells, receptor-operated calcium entry was decreased. To investigate whether the decreased receptor expression was due to attenuated calcium entry, cells were incubated with the calcium chelator BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid). In these cells, and in cells where calmodulin and calmodulin-dependent kinase were blocked pharmacologically, S1P₃ and VEGFR2 expression was decreased. In TRPC1-KD cells, both hypoxia-inducible factor 1α expression and the secretion and activity of MMP2 and MMP9 were attenuated, and proliferation was decreased in TRPC1-KD cells. This was due to a prolonged G₁ phase of the cell cycle, a significant increase in the expression of the cyclin-dependent kinase inhibitors p21 and p27, and a decrease in the expression of cyclin D2, cyclin D3, and CDK6. Transfecting TRPC1 to TRPC1-KD cells rescued receptor expression, migration, and proliferation. Thus, the expression of S1P₃ and VEGFR2 is mediated by a calcium-dependent mechanism. TRPC1 has a crucial role in this process. This regulation is important for the invasion, migration, and proliferation of thyroid cancer cells.     

Remodeling of calcium signaling in tumor progression

Intracellular Ca²⁺ is one of the crucial signalings that modulate various cellular functions. The dysregulation of Ca²⁺ homeostasis has been suggested as an important event in driving the expression of the malignant phenotypes, such as proliferation, migration, invasion, and metastasis. Cell migration is an early prerequisite for tumor metastasis that has a significant impact on patient prognosis. During cell migration, the exquisite spatial and temporal organization of intracellular Ca²⁺ provides a rapid and robust way for the selective activation of signaling components that play a central role in cytoskeletal reorganization, traction force generation, and focal adhesion dynamics. A number of known molecular components involved in Ca²⁺ influx pathways, including stromal interaction molecule (STIM)/Orai-mediated store-operated Ca²⁺ entry (SOCE) and the Ca²⁺-permeable transient receptor potential (TRP) channels, have been implicated in cancer cell migration and tumor metastasis. The clinical significance of these molecules, such as STIM proteins and the TRPM7 channel, in tumor progression and their diagnostic and prognostic potentials have also been demonstrated in specific cancer types. In this review, we summarize the recent advances in understanding the important roles and regulatory mechanisms of these Ca²⁺ influx pathways on malignant behaviors of tumor cells. The clinical implications in facilitating current diagnostic and therapeutic procedures are also discussed.     

Role of TRPM2 and TRPV1 cation channels in cellular responses to radiation-induced DNA damage

Background

Radiation exposure causes DNA damage, and DNA repair systems are essential to rescue damaged cells. Although DNA damage or oxidative stress activates transient receptor potential melastatin 2 (TRPM2) and vanilloid 1 (TRPV1) cation channels, it has not been established whether these TRP channels are involved in cellular responses to radiation-induced DNA damage. Here, we investigated the contribution of TRPM2 and TRPV1 channels to γ-irradiation- and UVB-induced DNA damage responses in human lung cancer A549 cells.

Methods

A549 cells were irradiated with γ-rays (2.0 Gy) or UVB (5–10 mJ/cm²). γH2AX foci, ATM activation, 53BP1 accumulation and EGFR expression were evaluated by immunofluorescence staining. Extracellular ATP concentration was measured by luciferin–luciferase assay. Knockdown of TRPM2 and TRPV1 expression was done by siRNA transfection.

Results

γ-Irradiation-induced γH2AX focus formation, ATM activation, 53BP1 accumulation and EGFR nuclear translocation, which are all associated with DNA repair, were suppressed by knockdown of TRPM2 and TRPV1 channels in A549 cells. Release of ATP, which mediates DNA damage response-associated activation of P2Y receptors, was suppressed by pre-treatment with catalase or knockdown of TRPM2 channel, but not TRPV1 channel. Similarly, UVB-induced γH2AX focus formation was suppressed in TRPM2- and TRPV1-knockdown cells, while UVB-induced ATP release was blocked in TRPM2- but not TRPV1-knockdown cells.

Conclusion

Our results suggest that the activation of TRPM2 channel, which mediates ATP release, and TRPV1 channel plays significant roles in the cellular responses to DNA damage induced by γ-irradiation and UVB irradiation.

General significance

Our results provide a new insight into the function of TRP channels from the viewpoint of radiation biology.     

Monitoring ligand-mediated internalization of G protein-coupled receptor as a novel pharmacological approach

Agonist activation of a G protein-coupled receptor (GPCR) results in the redistribution of the receptor protein away from the cell surface into internal cellular compartments through a process of endocytosis known as internalization. Visualization of receptor internalization has become experimentally practicable by using fluorescent reagents such as green fluorescent protein (GFP). In this study, we examined whether the ligand-mediated internalization of a GPCR can be exploited for pharmacological evaluations. We acquired fluorescent images of cells expressing GFP-labeled GPCRs and evaluated the ligand-mediated internalization quantitatively by image processing. Using beta2-adrenoceptor and vasopressin V1a receptor as model GPCRs that couple to Gs and Gq, respectively, we first examined whether these GFP-tagged GPCRs exhibited appropriate pharmacology. The rank order of receptor internalization potency for a variety of agonists and antagonists specific to each receptor corresponded well with that previously observed in ligand binding studies. In addition to chemical ligand-induced internalization, this cell-based fluorescence imaging system successfully monitored the internalization of the proton-sensing GPCR TDAG8, and that of the free fatty acid-sensitive GPCR GPR120. The results show that monitoring receptor internalization can be a useful approach for pharmacological characterization of GPCRs and in fishing for ligands of orphan GPCRs.     

TRPC3-mediated Ca influx contributes to Rac1-mediated production of reactive oxygen species in MLP-deficient mouse hearts

Dilated cardiomyopathy (DCM) is a myocardial disorder that is characterized by dilation and dysfunction of the left ventricle (LV). Accumulating evidence has implicated aberrant Ca²⁺ signaling and oxidative stress in the progression of DCM, but the molecular details are unknown. In the present study, we report that inhibition of the transient receptor potential canonical 3 (TRPC3) channels partially prevents LV dilation and dysfunction in muscle LIM protein-deficient (MLP (−/−)) mice, a murine model of DCM. The expression level of TRPC3 and the activity of Ca²⁺/calmodulin-dependent kinase II (CaMKII) were increased in MLP (−/−) mouse hearts. Acitivity of Rac1, a small GTP-binding protein that participates in NADPH oxidase (Nox) activation, and the production of reactive oxygen species (ROS) were also increased in MLP (−/−) mouse hearts. Treatment with pyrazole-3, a TRPC3 selective inhibitor, strongly suppressed the increased activities of CaMKII and Rac1, as well as ROS production. In contrast, activation of TRPC3 by 1-oleoyl-2-acetyl-sn-glycerol (OAG), or by mechanical stretch, induced ROS production in rat neonatal cardiomyocytes. These results suggest that up-regulation of TRPC3 is responsible for the increase in CaMKII activity and the Nox-mediated ROS production in MLP (−/−) mouse cardiomyocytes, and that inhibition of TRPC3 is an effective therapeutic strategy to prevent the progression of DCM.     

Vascular smooth muscle cell proliferation as a therapeutic target. Part 1: molecular targets and pathways

Cardiovascular diseases are a major cause of human death worldwide. Excessive proliferation of vascular smooth muscle cells contributes to the etiology of such diseases, including atherosclerosis, restenosis, and pulmonary hypertension. The control of vascular cell proliferation is complex and encompasses interactions of many regulatory molecules and signaling pathways. Herein, we recapitulated the importance of signaling cascades relevant for the regulation of vascular cell proliferation. Detailed understanding of the mechanism underlying this process is essential for the identification of new lead compounds (e.g., natural products) for vascular therapies.     

Osmoregulation of leaf motor cells

"Osmotic Motors"--the best-documented explanation for plant leaf movements--frequently reside in specialized motor leaf organs, pulvini. The movements result from dissimilar volume and turgor changes in two oppositely positioned parts of the pulvinus. This Osmotic Motor is powered by a plasma membrane proton ATPase, which drives KCl fluxes and, consequently, water, across the pulvinus into swelling cells and out of shrinking cells. Light signals and signals from the endogenous biological clock converge on the channels through which these fluxes occur. These channels and their regulatory pathways in the pulvinus are the topic of this review.     

The dual face of connexin-based astroglial Ca communication: A key player in brain physiology and a prime target in pathology

For decades, studies have been focusing on the neuronal abnormalities that accompany neurodegenerative disorders. Yet, glial cells are emerging as important players in numerous neurological diseases. Astrocytes, the main type of glia in the central nervous system , form extensive networks that physically and functionally connect neuronal synapses with cerebral blood vessels. Normal brain functioning strictly depends on highly specialized cellular cross-talk between these different partners to which Ca^2 +, as a signaling ion, largely contributes. Altered intracellular Ca^2 + levels are associated with neurodegenerative disorders and play a crucial role in the glial responses to injury. Intracellular Ca^2 + increases in single astrocytes can be propagated toward neighboring cells as intercellular Ca^2 + waves, thereby recruiting a larger group of cells. Intercellular Ca²⁺ wave propagation depends on two, parallel, connexin (Cx) channel-based mechanisms: i) the diffusion of inositol 1,4,5-trisphosphate through gap junction channels that directly connect the cytoplasm of neighboring cells, and ii) the release of paracrine messengers such as glutamate and ATP through hemichannels (‘half of a gap junction channel’). This review gives an overview of the current knowledge on Cx-mediated Ca^2 + communication among astrocytes as well as between astrocytes and other brain cell types in physiology and pathology, with a focus on the processes of neurodegeneration and reactive gliosis. Research on Cx-mediated astroglial Ca^2 + communication may ultimately shed light on the development of targeted therapies for neurodegenerative disorders in which astrocytes participate. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.