The aim of this review is to provide a basic framework for understanding the function of mammalian transient receptor potential (TRP) channels, particularly as they have been elucidated in heterologous expression systems. Mammalian TRP channel proteins form six-transmembrane (6-TM) cation-permeable channels that may be grouped into six subfamilies on the basis of amino acid sequence homology (TRPC, TRPV, TRPM, TRPA, TRPP, and TRPML). Selected functional properties of TRP channels from each subfamily are summarized in this review. Although a single defining characteristic of TRP channel function has not yet emerged, TRP channels may be generally described as calcium-permeable cation channels with polymodal activation properties. By integrating multiple concomitant stimuli and coupling their activity to downstream cellular signal amplification via calcium permeation and membrane depolarization, TRP channels appear well adapted to function in cellular sensation. Our review of recent literature implicating TRP channels in neuronal growth cone steering suggests that TRPs may function more widely in cellular guidance and chemotaxis. The TRP channel gene family and its nomenclature, the encoded proteins and alternatively spliced variants, and the rapidly expanding pharmacology of TRP channels are summarized in online supplemental material.

An Introduction to TRP Channels

The clinical use of TRPV1 (transient receptor potential vanilloid subfamily, member 1; also known as VR1) antagonists is based on the concept that endogenous agonists acting on TRPV1 might provide a major contribution to certain pain conditions. Indeed, a number of small-molecule TRPV1 antagonists are already undergoing Phase I/II clinical trials for the indications of chronic inflammatory pain and migraine. Moreover, animal models suggest a therapeutic value for TRPV1 antagonists in the treatment of other types of pain, including pain from cancer. We argue that TRPV1 antagonists alone or in conjunction with other analgesics will improve the quality of life of people with migraine, chronic intractable pain secondary to cancer, AIDS or diabetes. Moreover, emerging data indicate that TRPV1 antagonists could also be useful in treating disorders other than pain, such as urinary urge incontinence, chronic cough and irritable bowel syndrome. The lack of effective drugs for treating many of these conditions highlights the need for further investigation into the therapeutic potential of TRPV1 antagonists.

The vanilloid receptor TRPV1: 10 years from channel cloning to antagonist proof-of-concept

TRPM8 (CMR1) is a Ca2+-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.


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.


The EC50 values of the agonists defined their relative potencies: icilin (0.2±0.1 μM)>FrescolatML (3.3±1.5 μM) > WS-3 (3.7±1.7 μM) >(−)menthol (4.1±1.3 μM) >frescolatMAG (4.8±1.1 μM) > cooling agent 10 (6±2.2 μM) >(+)menthol (14.4±1.3 μM) > PMD38 (31±1.1 μM) > WS-23 (44±7.3 μM) > Coolact P (66±20 μM) > geraniol (5.9±1.6 mM) > linalool (6.7±2.0 mM) > eucalyptol (7.7±2.0 mM) > hydroxycitronellal (19.6±2.2 mM).


Known VR1 antagonists (BCTC, thio-BCTC and capsazepine) were also able to block the response of TRPM8 to menthol (IC50: 0.8±1.0, 3.5±1.1 and 18±1.1 μM, respectively).


The Ca2+ 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.


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.





Keywords: TRPM8, CMR1, cold, menthol, VR1, odorants, proton activation, FLIPR, pain


Introduction
The recent cloning and characterization of the cold-menthol receptor (TRPM8; CMR1) (McKemy et al., 2002; Peier et al., 2002) was a major breakthrough in the study of thermosensation. TRPM8 is activated by menthol, eucalyptol and icilin, and by temperatures below ∼25°C. It belongs to the ‘long', or melastatin, subfamily of the transient receptor potential (TRP) family of ion channels (Montell et al., 2002), and shows pronounced outward rectification with a relatively high permeability for Ca2+ ions, and little selectivity between monovalent cations. The TRPM8 channel is expressed specifically in a subset of temperature-sensing trigeminal and dorsal root ganglion neurones (Peier et al., 2002; Reid et al., 2002a, 2002b; Nealen et al., 2003). Recently, a second cold receptor, ANKTM1, has been identified (Story et al., 2003), which, in contrast to TRPM8, is coexpressed with VR1 in a different subset of pain- and temperature-sensing trigeminal and dorsal root ganglion neurones. ANKTM1 is activated by icilin, but not menthol. These TRP channels play a major role in thermosensation (McKemy et al., 2002; Patapoutian et al., 2003).

Although treatment with menthol or eucalyptol, or with cold temperatures, is a traditional method of pain relief (Wright, 1870; Green & Mcanliffe, 2000; Davies et al., 2002; Galeotti et al., 2002; Shanghai Medicinal Herbs, Essential Balm), little is known about the underlying analgesic mechanisms. It has been demonstrated that menthol blocks Na+ and Ca2+ channels in dorsal root ganglion cells (Swandulla et al., 1987; Haeseler et al., 2002). Others have postulated that the analgesic activity of (–)menthol is mediated by selective activation of κ-opioid receptors (Galeotti et al., 2002).

The cold receptor TRPM8 is distantly related to the well-characterized heat-sensitive vanilloid receptor VR1 (or TRPV1). VR1 also belongs to the TRP channel family, but is activated by temperatures >42°C, or by ligands such as capsaicin and resiniferatoxin (RTX). Two endogenous VR1 agonists have been identified, anandamide (ANA) and N-arachidonoyl-dopamine (NADA) (Zygmunt et al., 1999; Di Marzo et al., 2001; Huang et al., 2002). Various VR1 antagonists have also been reported, for example, capsazepine, iodo-resiniferatoxin (I-RTX) and N-(4-tert.butyl-phenyl)-4-(3-chloropyridin-2-yl) tetrahydro-pyrazine-1(2H)-carboxamide (BCTC). These have analgesic effects in vivo (Bevan et al., 1992; Walpole et al., 1994; Catarina et al., 1997; 2000; Tominaga et al., 1998; Wahl et al., 2001; Pomonis et al., 2003; Rigoni et al., 2003).

Protons act as endogenous activators and modulators of VR1 responses. Low pH enhances the apparent VR1-binding affinity of capsaicin, and potentiates the channel gating of VR1 receptors (Caterina et al., 1997; 2000; Tominaga et al., 1998; Olah et al., 2001; Ryu et al., 2003). Since inflammation leads to acidification of the inflamed tissue, VR1 is thought to play a major role in the transduction of inflammatory pain.

As VR1 and TRPM8 are distantly related, and no antagonists have been described for TRPM8, we tested the effects of VR1 antagonists on TRPM8. Further, we investigated whether the responses of VR1 and TRPM8 towards agonists are influenced by pH.

/pdf/characterization-of-the-mouse-cold-menthol-receptor-trpm8-cllgyeyun6.pdf

Characterization of the mouse cold-menthol receptor TRPM8 and vanilloid receptor type-1 VR1 using a fluorometric imaging plate reader (FLIPR) assay.

Depending on their primary structure, the 28 mammalian transient receptor potential (TRP) cation channels identified so far can be sorted into 6 subfamilies: TRPC (“Canonical”), TRPV (“Vanilloid”), TRPM (“Melastatin”), TRPP (“Polycystin”), TRPML (“Mucolipin”), and TRPA (“Ankyrin”). The TRPV subfamily (vanilloid receptors) comprises channels critically involved in nociception and thermosensing (TRPV1, TRPV2, TRPV3, and TRPV4), whereas TRPV5 and TRPV6 are involved in renal Ca 2 absorption/reabsorption. Apart from TRPV1, the pharmacology of these channels is still insufficiently known. Furthermore, only few small-molecule ligands for non-TRPV1 vanilloid receptors have been identified, and little is known of their endogenous ligands, resulting in a substantial “orphan” state for these channels. In this review, we summarize the pharmacological properties of members of the TRPV subfamily, highlighting the critical issues and challenges facing their “deorphanization” and clinical exploitation.

/pdf/pharmacology-of-vanilloid-transient-receptor-potential-3d6phmtznq.pdf

Pharmacology of vanilloid transient receptor potential cation channels.

Compounds having the general structure and compositions containing them, for the treatment of acute, inflammatory and neuropathic pain, dental pain, general headache, migraine, cluster headache, mixed-vascular and non-vascular syndromes, tension headache, general inflammation, arthritis, rheumatic diseases, osteoarthritis, inflammatory bowel disorders, inflammatory eye disorders, inflammatory or unstable bladder disorders, psoriasis, skin complaints with inflammatory components, chronic inflammatory conditions, inflammatory pain and associated hyperalgesia and allodynia, neuropathic pain and associated hyperalgesia and allodynia, diabetic neuropathy pain, causalgia, sympathetically maintained pain, deafferentation syndromes, asthma, epithelial tissue damage or dysfunction, herpes simplex, disturbances of visceral motility at respiratory, genitourinary, gastrointestinal or vascular regions, wounds, burns, allergic skin reactions, pruritus, vitiligo, general gastrointestinal disorders, gastric ulceration, duodenal ulcers, diarrhea, gastric lesions induced by necrotising agents, hair growth, vasomotor or allergic rhinitis, bronchial disorders or bladder disorders.

Vanilloid receptor ligands and their use in treatments

Vanilloids such as capsaicin have algesic properties and seem to mediate their effects via activation of the vanilloid receptor 1 (VR1), a ligand-gated ion channel highly expressed on primary nociceptors. Although blockade of capsaicin-induced VR1 activation has been demonstrated in vitro and in vivo with the antagonist capsazepine, efficacy in rat models of chronic pain has not been observed with this compound. Here, we describe the in vitro pharmacology of a highly potent VR1 antagonist, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC). Similar to capsazepine, this compound inhibits capsaicin-induced activation of rat VR1 with an IC50 value of 35 nM. Interestingly however, BCTC also potently inhibits acid-induced activation of rat VR1 (IC50 value of 6.0 nM), whereas capsazepine is inactive. Similarly, in the rat skin-nerve preparation both BCTC and capsazepine block capsaicin-induced activation, whereas the response to acidification is inhibited by BCTC, but not by capsazepine. Specificity for VR1 was demonstrated against 63 other receptor, enzyme, transporter, and ion channel targets. BCTC was orally bioavailable in the rat, demonstrating a plasma half-life of approximately 1 h and significant penetration into the central nervous system. Thus, BCTC is a high potency, selective VR1 antagonist that, unlike capsazepine, has potent blocking effects on low pH-induced activation of rat VR1. These properties make it a more suitable candidate than capsazepine for testing the role played by VR1 in rat models of human disease.

N-(4-Tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine -1(2H)-carbox-amide (BCTC), a Novel, Orally Effective Vanilloid Receptor 1 Antagonist with Analgesic Properties: I. In Vitro Characterization and Pharmacokinetic Properties

Shiazah Malik

Papers

N-(4-Tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine -1(2H)-carbox-amide (BCTC), a Novel, Orally Effective Vanilloid Receptor 1 Antagonist with Analgesic Properties: I. In Vitro Characterization and Pharmacokinetic Properties