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Current Pharmaceutical Biotechnology, 2011, 12, 000-000 1

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TRPM8, a Sensor for Mild Cooling in Mammalian Sensory Nerve Endings

Alexandru Babes*, Alexandru Cristian Ciobanu, Cristian Neacsu and Ramona-Madalina Babes

Department of Anatomy, Physiology and Biophysics, Faculty of Biology, University of Bucharest Splaiul Independentei

91-95, 050095 Bucharest, Romania

Abstract: Temperature sensing is a crucial feature of the nervous system, enabling organisms to avoid physical danger

and choose optimal environments for survival. TRPM8 (Transient Receptor Potential Melastatin type 8) belongs to a se-

lect group of ion channels which are gated by changes in temperature, are expressed in sensory nerves and/or skin cells

and may be involved in temperature sensing. This channel is activated by a moderate decrease in temperature, with a

threshold of 25 °C in heterologous expression systems, and by a variety of natural and synthetic compounds, including

menthol. While the physiological role of TRPM8 as a transducer of gentle cooling is widely accepted, its involvement in

acute noxious cold sensing in healthy tissues is still under debate. Although accumulating evidence indicates that TRPM8

is involved in neuropathic cold allodynia, in some animal models of nerve injury peripheral and central activation of

TRPM8 is followed by analgesia. A variety of inflammatory mediators, including bradykinin and prostaglandin E2, modu-

late TRPM8 by inhibiting the channel and shifting its activation threshold to colder temperatures, most likely counteract-

ing the analgesic action of TRPM8. While important progress has been made in unraveling the biophysical features of

TRPM8, including the revelation of its voltage dependence, the precise mechanism involved in temperature sensing by

this channel is still not completely understood. This article will review the current status of knowledge regarding the

(patho)physiological role(s) of TRPM8, its modulation by inflammatory mediators, the signaling pathways involved in

this regulation, and the biophysical properties of the channel.

Keywords: Cold, pain, inflammation, neuropathy, cancer.

1. INTRODUCTION

Detection of changes in ambient temperature is an essen-tial feature that enables organisms to seek optimal conditions for survival and reproduction. In recent years the understand-ing of the molecular logic of temperature sensing has devel-oped at a fast pace, mainly due to the cloning and characteri-zation of several thermally gated ion channels expressed in specialized sensory nerve endings and belonging to the TRP (Transient Receptor Potential) super-family [1]. Among these, TRPM8 (TRP Melastatin type 8) is a cold-activated, non-selective cation channel with a temperature threshold of

25 °C in heterologous expression systems, expressed in both neural and non-neural tissue [2, 3]. TRPM8 appears to be polymodal, in that is also activated by chemical agents such as menthol and a variety of other naturally occurring or artificial compounds [2, 4]. Recent biophysical work has provided evidence towards a role of membrane voltage in channel gating [5], and the molecular determinants of chan-nel function, including the voltage sensor, the menthol bind-ing site, or the domain involved in tetramerization, have been recently identified [6, 7]. The crucial role of TRPM8 in cold sensing (in a certain range of temperatures, from skin tem-perature to 10 °C) has been substantiated by several studies on null mutant mice [8-10], and new evidence has been pro-vided for a physiological role of TRPM8 in non-neural tis-sue: sperm, smooth muscle, prostate, lung epithelia [11-14].

*Address correspondence to this author at the Department of Anatomy,

Physiology and Biophysics, Faculty of Biology, University of Bucharest,

Splaiul Independentei 91-95, 050095 Bucharest, Romania; Tel/Fax: +40-21-3181570; E-mails: [email protected], [email protected]

The involvement of TRPM8 in acute and chronic pain is a very important research topic and still a matter of debate. TRPM8 undergoes substantial modulation upon initiation of the major cellular signaling cascades and this process may contribute to the alterations in cold sensing in pathological pain states [15, 16]. The finding that TRPM8 is functionally down-regulated by inflammatory mediators may be related to a proposed analgesic action of this channel in neuropathic and other chronic pain models [17], but a contribution of this channel to cold allodynia and hyperalgesia is not to be ruled out. Finally, TRPM8 is associated with a number of tumors (skin, prostate, breast, colon, lung) and its function appears to be related to the viability and malignancy of tumor cells [18, 19]. Thus, interfering with TRPM8 expression or func-tion may prove to be a promising cancer therapy. All these aspects of TRPM8 physiology and pathophysiology will be addressed in detail in this review.

2. FEATURES OF TRPM8

2.1. Cloning and Tissue Distribution

The first hint of the existence of a non-selective cation channel expressed in rodent sensory neurons and activated by cooling and menthol came from the work of Reid and Flonta. Patch-clamp recordings on cultured rat dorsal root ganglion (DRG) neurons voltage-clamped at -60 mV re-vealed an inward current activated by cooling and sensitized by (-)-menthol in a dose-dependent manner [20]. The same authors published a follow up analysis of single-channel cur-rents activated by cold and menthol in excised outside-out patches from DRG neurons and speculated on the molecular

wasim

Final

2 Current Pharmaceutical Biotechnology, 2011, Vol. 12, No. 1 Babes et al.

identity of the channel that it may belong to the TRP (Tran-sient Receptor Potential) super-family [21]. Not much later this hypothesis was confirmed when two groups working independently and using different cloning strategies were able to report the cloning of TRPM8, also known as CMR1 (Cold and Menthol Receptor 1), a non-selective outwardly-rectifying cation channel expressed in a subpopulation (5-10%) of DRG and trigeminal ganglion (TG) neurons, which was activated by cooling with a temperature threshold of

25 °C and also by the natural compounds menthol and eucalyptol and the artificial agent icilin [2, 3]. Interestingly, the new channel sequence was almost identical to a cDNA corresponding to a prostate-specific transcript, up-regulated in prostate cancer and also expressed in a variety of primary tumors (colon, skin, lung and breast) but lacking in the cor-responding normal tissue [22]. More recent studies also iden-tified either TRPM8 mRNA or protein in lung epithelial cells [13], vascular smooth muscle [12], liver [23], gastric fundus [24], bladder and different tissues of the male genital tract [25] and human sperm [14]. Within the PNS, TRPM8 ap-pears to be mainly expressed in the DRG and TG, but evi-dence was provided for TRPM8 expression in nodose gan-glia [26] and geniculate ganglia [27]. Very little evidence exists regarding the presence of TRPM8 at protein or mRNA level in the CNS. To our best knowledge, there is only one very recent report describing immunostaining for TRPM8 in cerebrospinal fluid-contacting neurons in rat brain [28]. In what follows we shall focus on the role of TRPM8 as a cold sensor in the PNS, and therefore we shall mainly discuss anatomical, (patho)physiological and biophysical aspects relevant to this issue and only briefly summarize the most recent knowledge concerning the role and properties of TRPM8 in non-sensory tissue. Regarding the subcellular distribution of TRPM8 in sensory neurons, it was already established that the cold and menthol receptor was function-ally expressed in cutaneous nerve endings of cold receptors, but anatomical evidence was provided only recently by two independent studies using mice genetically modified to ex-press green fluorescent protein (GFP) controlled by the TRPM8 promoter [29, 30]. According to these authors, TRPM8 is expressed in free nerve endings which terminate in the epidermis (stratum spinosum and stratum granulo-sum), but also in deeper skin layers, which agrees with a hypothetically superficial localization of innocuous cold-sensing nerve endings. The same studies demonstrated the presence of TRPM8 in the central terminals in the most su-perficial layer (lamina I) of the dorsal horn, the main target of primary afferent nociceptors and thermoreceptors [31]. These results are in agreement with previous work carried out in vitro in a primary afferent-dorsal horn co-culture sys-tem showing functional expression of TRPM8 in pre-synaptic terminals where it can modulate glutamate release in a Ca

2+-dependent manner [32] and also with more recent

experiments carried out on spinal cord slices [33]. Localiza-tion of TRPM8 was observed not only in the plasma mem-brane but also in the endoplasmic reticulum (ER) membrane, where it mediates menthol-induced Ca

2+ release from stores

[32] (see also TRPM8 and cancer, section 4.3). An important issue that was raised in relation to the putative role of TRPM8 in noxious cold sensing was its co-expression (or lack of) with molecules involved in pain signaling. It was initially reported that TRPM8 in rodents defines a unique

sub-population of trkA-dependent small C-fibre neurons devoid of nociceptive markers such as TRPV1 and calcitonin gene-related peptide (CGRP) [3]. In a follow up study, the same group proposed that although normally not co-expressed with these nociceptive molecules, TRPM8 positive neurons begin to express TRPV1 in inflammatory condi-tions, which may be involved in abnormal thermal sensitivity in pathological pain states [34]. However, other investigators were able to detect co-expression of TRPM8 and TRPV1 using functional assays (calcium imaging) [2, 35-38] or dou-ble labeling experiments (in situ hybridization for TRPM8 mRNA coupled with immunostaining for TRPV1) [39]. Re-cent work from two different laboratories, using a genetic approach of expressing GFP from the TRPM8 locus and immunostaining for TRPV1, confirmed that the two channels are indeed co-expressed in normal tissue, such that 25% of DRG neurons and 40% of TG neurons that are positive for TRPM8 also express TRPV1 (similar numbers were reported for co-expression between TRPM8 and CGRP) [29, 30]. Another very recent report described immunohistochemical co-localization of TRPM8 and CGRP and substance P in human cutaneous unmyelinated nerve fibres. The same study showed that patients with Norrbottnian congenital insensitiv-ity to pain have a markedly reduced number of TRPM8-expressing C-fibres in their skin, [40]. These results raise the intriguing hypothesis that a sub-population of TRPM8-expressing cells may serve a nociceptive function, even in healthy tissue.

2.2. Structure, Trafficking and Tetramerization

TRPM8 has 1014 aminoacids (in the rat) and a predicted topology of 6 membrane spanning domains (TM1-6) with a pore loop between TM5 and 6 and intracellular N- and C-terminal domains [2]. This type of topology is shared by TRP channels, voltage-gated potassium channels, Ca

2+-

activated potassium channels (of small and intermediate conductance) and cyclic-nucleotide-gated channels. All these channels are thought to form homo- or heterotetramers. Se-quence homology studies have placed the cold and menthol receptor in the TRPM (TRP Melastatin) sub-family, in which members are also known as long-TRP channels, due to their long N- and C-terminals. The functional diversity of TRPM channels is wide, as they appear to be involved in sensory processes (taste – TRPM5; thermoreception – TRPM8), im-mune cell activation (TRPM4), oxidative stress (TRPM2, which is also structurally the closest related to TRPM8) and magnesium balance (TRPM6 and TRPM7) (for recent re-views on TRP channel structure-function see [41, 42]). TRPM8 differs from the other members of the TRPM sub-family in that it has a rather short C-terminal domain (120 residues), in which the only structural motif is a coiled-coil domain proposed to be involved in tetramerization of the channel [43, 44] (Fig. (1)), albeit a more recent study has shown that the transmembrane region of TRPM8 is sufficient for tetramerization, and a truncated variant of TRPM8 lack-ing precisely the C-terminal domain was able to translocate to the plasma membrane as a tetramer [7]. The C-terminal domain is nevertheless essential for channel function, as C-terminal deleted TRPM8 could not be activated by cold or chemical agonists [7]. This finding is in agreement with the identification of the proximal C-terminal domain as having a

TRPM8, a Sensor for Mild Cooling in Mammalian Sensory Nerve Endings Current Pharmaceutical Biotechnology, 2011, Vol. 12, No. 1 3

Fig. (1). Structural determinants of TRPM8 function and modulation by intracellular signaling cascades. The membrane topology model of

TRPM8 is illustrated, indicating residues involved in the channel’s sensitivity to cold, menthol, icilin and voltage, and also the protein do-

mains related to channel function. Bradykinin (BK) activates its B2 receptors (B2R), stimulating phospholipase C (PLC), followed by cleav-

age of phosphatidylinositol 4,5-bisphosphate (PIP2) and generation of second messengers diacyl glycerol and inositol triphosphate (IP3). The

ensuing drop in membrane PIP2 levels leads to TRPM8 desensitization. IP3 binds to its intracellular receptor and initiates Ca2+

release from

stores. DAG and Ca2+

ions activate protein kinase C (PKC) and promote its translocation to the plasma membrane, followed by activation of

protein phosphatase 1 (PP1) and dephosphorylation of TRPM8, leading to inhibition of channel function. Activation of EP receptor sybtypes

by prostaglandin E2 is followed by stimulation of adenylate cyclase, cAMP production and activation of protein kinase A (PKA), leading to

inhibition of TRPM8 via an unknown mechanism. Finally, activation of PLA2 leads to generation of lysophospholipids and functional

upregulation of TRPM8. Some TRPM8 agonists and antagonists are also illustrated.

crucial role in the thermal sensitivity of TRPM8: swapping these domains between TRPM8 and TRPV1 also led to a switch in the thermal gating of the channels [45]. TRPM8 is glycosylated on asparagine 934, located in the pore loop, between TM5 and TM6 (Fig. (1)) but this event appears to

have no consequence on channel gating and possibly a minor effect on its translocation to the plasma membrane [46]. A structural motif with an important role in channel function is a disulfide bond between two cysteines C929 and C940, (Fig. (1)) also located in the pore loop, flanking the glycosy-


lation site [46]: mutation of either of the two cysteine resi-dues led to inactive TRPM8 channels, unable to respond to cold, menthol or icilin (the deficit was not related to traffick-ing, as both mutants were able to reach the plasma mem-brane). Finally, a region within the N-terminal domain which is conserved between TRPM channels appears to be the criti-cal determinant for correct localization in the plasma mem-brane [7]. Interestingly, a truncated version of TRPM8 which lacks a part of this conserved N-terminal domain was found in prostate cancer cells, where it seems to be confined to the endoplasmic reticulum, mediating calcium release [47].

2.3. Pharmacology

The first two papers reporting TRPM8 cloning also in-vestigated the channel’s pharmacology, showing that it is activated by the natural compounds (-)-menthol (or l-menthol) and eucalyptol and by the artificial agent icilin [2, 3]. Among TRPM8 agonists are natural odorants such as linalool, geraniol and hydroxycitronellal, as well as synthetic compounds used as cooling agents by the cosmetics industry, like WS-3, WS-12, WS-30, CPS-369, Coolact P, Cooling-agent 10 and PMD-38, while capsazepine and BCTC were identified as antagonists [4, 48]. Although papers describing agents which interfere with TRPM8 function followed at a fast pace, the list of truly specific agonists or antagonists is still very restricted. This reflects a more general issue, namely the lack of selectivity of many agents acting on thermoTRP (thermally gated TRP) channels. Thus, menthol not only activates TRPM8, but also activates the warm re-ceptor TRPV3 and has a bi-modal action on the noxious cold-activated channel TRPA1 (agonist al low concentration and antagonist at higher ones), while cinnamaldehyde, ini-tially described as a TRPA1 agonist, also inhibits TRPM8 [49, 50]. Icilin, initially described as a TRPM8 agonist [2], was found to activate TRPA1 as well, albeit at higher con-centrations [34]. Moreover, it was recently shown that plant-derived cannabinoids (cannabidiol and

9-tetrahydro-

cannabinol) act as antagonists of recombinant TRPM8 (but also as agonists of TRPA1) [51] and a similar action was reported for the antifungal agent clotrimazole [52]. Similarly, the few TRPM8 antagonists identified so far are either shared with the heat receptor TRPV1 (capsazepine, BCTC and SB-452533 [53]) or are non-selective blockers of cal-cium permeable channels (SKF96365 [54, 55]). However, recent developments raise new possibilities of identifying selective tools to discriminate between TRP-mediated re-sponses: a novel TRPM8 blocker (AMTB) was described recently, which lacks any antagonistic activity on TRPV1 and TRPV4 channels [56]; however, its effect on TRPA1 has not been yet tested.

The biophysical mechanism of agonist (or antagonist) action on TRPM8 is beginning to be unraveled, as it was shown that it involves a leftward (or rightward) shift in the voltage-dependence of channel gating ([57]; see below about the voltage gating of TRPM8 in section 2.4). The mechanism of action of icilin appears to be unique among TRPM8 ago-nists, in that it requires an elevation of cytoplasmic Ca

2+

concentration to achieve full efficacy. Interestingly, chicken TRPM8 is icilin-insensitive, and for this reason rat-avian TRPM8 chimeras were used to identify the molecular deter-minants of icilin sensitivity. Finally, the rat vs. chicken dif-

ference was found to reside in one aminoacid: replacement of glycine in position 805 (rat) to an alanine (chicken) com-pletely abolished icilin sensitivity [58], and conversely, in-troducing the rat residue glycine at position 796 in chicken TRPM8 (replacing the alanine) rendered the avian channel icilin-sensitive. Mutation of two other residues (asparagine 799 and aspartate 802) also eliminates icilin evoked currents in rat TRPM8 [58]. A different approach was used to find the menthol binding site of TRPM8, as all TRPM8 known orthologues are menthol sensitive to a similar degree: a TRPM8 mutant library was generated by random mutagene-sis and was later screened for mutants with impaired menthol sensitivity and intact cold activation. Two regions were iden-tified as interfering with menthol-activation of TRPM8: the transmembrane 2 (TM2) domain and particularly tyrosine 745, located in the middle of TM2, and the TRP domain (leucine 1009), in the proximal C-terminal region. Careful analysis revealed that while the TRP domain is involved in modulating a downstream event from agonist binding, Y745 appears to be involved in the actual menthol binding to the channel, without interfering with cold-, voltage- or PIP2-mediated activation of TRPM8 [6]. Interestingly, mutation of the same residues, Y745 and L1009, was found to abolish the icilin sensitivity of TRPM8.

2.4. Biophysical Properties and Modulation

Recombinant TRPM8-mediated currents were activated by cooling with a temperature threshold of 25 °C, which was shifted to warmer temperatures in the presence of men-thol [2, 3, 57]. All these features closely resembled those measured on the native cold- and menthol-activated current in cultured DRG neurons [21, 54]. Single-channel events activated by cooling and menthol, most likely through TRPM8, were recorded in outside-out patches excised from cultured DRG neurons, indicating that cold activation of TRPM8 is a membrane-delimited process and does not de-pend on the release of a soluble second messenger [21]. In-terestingly, native channels are strongly desensitized to cold following patch excision: the activation threshold is shifted to cooler temperatures by 10-15 °C, suggesting that the dis-ruption of the cell membrane leads to the loss of a regulatory interaction which is involved in setting the temperature threshold [21]. Similar results were later obtained using re-combinant TRPM8 channels expressed in HEK293 cells [59]. TRPM8 was first described as an outwardly rectifying, non-selective cation channel, with high calcium permeabil-ity: PCa/PNa = 3.2 [2] or PCa/PNa = 1.4 [3]. The outward recti-fication appears to be associated with intrinsic voltage de-pendence, causing a marked decrease in open probability at negative potentials: while the tail currents recorded follow-ing a pulse to +120 mV showed a linear voltage-dependence, indicating an ohmic behavior of the open channel, the steady-state currents were strongly outwardly rectifying [59]. An interesting observation was that TRPM8 could be acti-vated at 32 °C, in the absence of any chemical agonist, just by depolarizing the membrane close to +100 mV. Thermal and electrical activation of TRPM8 appear to be strongly linked, in that lowering the temperature leads to a progres-sive shift in the voltage dependence of activation to more negative potentials, closer to the physiological range. This behavior could be fitted using a two state model with volt-


age- and temperature-dependent rate constants, of which only the closing rate had steep temperature dependence (a temperature coefficient Q10 of 10; [59]). In close analogy to the structurally related Shaker potassium channels, the gating charges responsible for the voltage dependence of TRPM8 were identified by the same authors in the S4 trans-membrane segment and the S4-S5 linker. Using site-directed mutagenesis, arginines in positions 842, 851 and 862, his-tidine 845 and lysine 856 (Fig. (1)) were found to alter the voltage dependence of TRPM8 [5]. However, this issue was not devoid of controversy, as other investigators claimed that a simple two-state model could not account for the two ex-ponential components of TRPM8 deactivation following a depolarizing step. Instead, they proposed a more complex model based on an allosteric interaction between a voltage sensor and a separate temperature sensor, which they later localized in the C-terminal domain of TRPM8 [45,60]. In an elegant series of experiments, these authors have switched the C-terminal domains between the cold receptor TRPM8 and the noxious heat receptor TRPV1 and found that this procedure completely reverses the temperature sensitivity of the chimeras, such that TRPV1 with a TRPM8 C-terminal domain became cold-sensitive and TRPM8 with a C-terminal domain from TRPV1 was activated by heat [45].

TRPM8 activation by cold and chemical agonists is sub-ject to modulation by second messenger cascades, which may be relevant for the abnormal thermal sensitivity in some pathological states. While the noxious heat receptor TRPV1 is sensitized by post-translational modifications upon activa-tion of G-protein coupled receptors (GPCRs) or tyrosine kinase receptors (reviewed by [61]), the same action appears to mainly desensitize TRPM8. Work on primary DRG cul-tures from the rat demonstrated that acute treatment with pro-inflammatory mediators bradykinin and prostaglandin E2 (PGE2) leads to a desensitization of cold- and menthol-sensitive (most likely TRPM8-expressing) neurons mediated by protein kinase C (PKC) and protein kinase A (PKA) re-spectively [16] (Fig. (1)). Experiments carried out on recom-binant channels produced strong evidence that PKC activa-tion is indeed followed by a decrease in TRPM8 activity. The actual target of PKC phosphorylation could not be iden-tified, but instead it was shown that PKC activation is fol-lowed by stimulation of protein phosphatase 1 (PP1), which in turn dephosphorylates TRPM8 [15] (Fig. (1)). Just as in the case of TRPV1, but acting in the opposite direction, a key regulatory factor which regulates TRPM8 is phosphati-dylinositol 4,5-bisphosphate (PIP2) (Fig. (1)). Two inde-pendent groups demonstrated that PIP2 maintains TRPM8 in an active state and signaling events coupled to the activation of phospholipase C (PLC) and subsequent PIP2 degradation lead to functional downregulation of TRPM8 [62, 63]. One of the groups went on to identify the molecular determinants underlying TRPM8 modulation by PIP2 and found that the TRP domain located at the C-terminus of the channel serves as a PIP2-interacting domain, as neutralization of three posi-tive residues in these region led to a markedly decreased PIP2 sensitivity [63]. Interestingly, while activating the heat receptor TRPV1, ethanol inhibits the cold receptor TRPM8, and the mechanism appears to be a weakening of the stimu-latory binding of PIP2 to the channel [64]. Another important signaling molecule which is implicated in the action of sev-

eral pro-inflammatory mediators, phospholipase A2 (PLA2), has a more complex action on TRPM8. PLA2 enzymes break down the sn-2 ester of glycerophospholipids and generate a polyunsaturated fatty acid (PUFA, including, but not only, arachidonic acid) and a lysophospholipid. While PUFAs inhibit TRPM8, lysophospholipids (lysophosphatidylcholine, lysophosphatidylserine, lysophosphatidylinositol) activate the channel even at normal body temperature, by shifting the temperature threshold to higher values in a dose-dependent manner [65]. This important finding sheds light on the issue of identifying endogenous agonists of TRPM8 and the re-lated question concerning the gating of this channel in tissues which are not subject to significant changes in temperature (see below). Even though the products of PLA2 have oppos-ing effects on TRPM8, at equimolar concentrations the effect of lysophospholipids prevails, such that the overall effect of PLA2 activation on TRPM8 is a positive one [65]. Finally, TRPM8 activity induced by cold and chemical agonists is subject to modulation by intracellular pH. Acidification in-hibits icilin-evoked currents through TRPM8 (at pH 6 the response is completely abolished) and shifts the cold activa-tion temperature to lower values. Conversely, alkalinization of the internal pH from 7.3 to 8 enhances both thermal and icilin activation. Interestingly, the activity induced by men-thol in TRPM8 appears to be pH-independent, at least in the range between pH 6.5 and 8 [66].

3. PHYSIOLOGY OF TRPM8

3.1. Role in Cold Sensing and Cold Nociception

Propagated action potentials evoked by cooling of cuta-neous receptive fields have been recorded in several species, but significant progress has been made with recordings in primates in the 1970’s. More recent work revealed several classes of cold-sensitive primary afferent fibers in humans: low threshold A and C fibers activated by gentle cooling on the one hand, and C-fibre polymodal nociceptors activated by stronger cooling (reviewed by [67, 68]). Understanding the precise molecular events involved in cold transduction by peripheral thermoreceptors was hampered by the technical difficulties in recording from intact cutaneous bare nerve endings. An alternative solution was to use the soma of DRG or TG neurons in culture as a model of their own terminals [69]: in vitro the soma expresses channels and receptors that are normally present in peripheral terminals, including trans-duction channels, and their activity can be recorded using calcium microfluorimetry and the patch clamp technique. This approach was used by Reid and Flonta (2001), who were able to record inward currents activated by cooling (with a temperature threshold of 29 °C) and sensitized by menthol in a subpopulation of cultured DRG neurons from the rat, voltage clamped at -60 mV. Following this develop-ment, and the cloning of TRPM8, the DRG or TG culture was used by many investigators as a system for investigating cold sensing in mammals. A plethora of in vitro studies iden-tified TRPM8 as the main somatic innocuous cold sensor, in a temperature range between 30 and 12-17 °C [21, 36, 38, 39, 54, 70-72]. These results should be carefully interpreted, as one study found that although TRPM8 appears to be chiefly responsible for cold-sensing in cultured DRG neu-rons, the response to cooling of intact corneal cold receptors was not impaired by the TRPM8 blocker BCTC [73]. How-


ever, the hypothesis that TRPM8 is the major cold sensor at least in a range of temperatures was confirmed recently by work carried out by three independent groups on TRPM8 null mutant mice: deletion of the TRPM8 gene led to pro-found deficits in cold sensing in a temperature preference test between 27 and 10 °C [8-10]. While there appears to be a consensus concerning its role in detecting mild cooling in the innocuous temperature range, the involvement of TRPM8 in sensing cold pain is still under debate. This issue was not resolved by the investigation of TRPM8 knockout mice. While two groups failed to find any difference in pain behavior between wild type and TRPM8-/- animals in the cold plate test [8, 9], the other group reported significantly longer response latencies for the null mutants in the same behavioral test [10]. A more recent behavioral study also described an increased latency for paw withdrawal in TRPM8-/- mice on a cold plate set at 10 °C [74]. The reason for this discrepancy is not yet clear, but it was suggested that lightly restraining the animals such that their paws are in constant contact with the cold plate results in less variable responses and thus a more accurate measurement of with-drawal latencies, as carried out by Gentry and colleagues [74]. While these studies on TRPM8 null mutant mice yielded controversial results, a different experimental ap-proach provided evidence that may implicate TRPM8 in noxious cold sensing. Two types of cold-sensitive C-fibres were described using microneurography in healthy subjects: a) C-fibre polymodal nociceptors activated by cold with a high threshold ( 19 °C) and, b) the so called type 2 C-fibres (or C2) which are activated by gentle cooling (in a similar manner to the A cold afferents thought to be responsible for the sensation of innocuous cold in humans) and also respond strongly to heating [75, 76]. The sensory function of these C2 fibres was until recently unclear, but it has been hypothe-sized by Campero and colleagues [76] that they might ac-count for what has been described in the literature as the paradoxical heat sensation (induced by cooling heat spots; [77]), the painful “thermal grill illusion” [78] and the so-called “innocuous cold nociception” (a sensation of burning pain evoked by mild cooling; [79]). Thus, it has been pro-posed that under normal circumstances cold evoked input from C2 fibres is inhibited by the activity in A cold recep-tors, leading to an innocuous, purely thermal perception. At lower temperatures, activity in A fibres declines and the C2 input is disinhibited, signaling that ambient temperature is becoming uncomfortable [76]. The balance between the ac-tivity in A and C fibres can be also reversed by selective A fibre block: disinhibition occurs and just a few degrees of cooling from normal skin temperature are perceived as icy, stinging or burning pain [80]. Their low threshold to activa-tion by cooling as well as their menthol sensitivity strongly suggest that the temperature sensor in C2 fibres is TRPM8, endowing this channel with a subtle nociceptive function.

Outside of the somatic sensory system, TRPM8 protein has been identified in a variety of deep tissues such as lung [13], liver [23], vascular smooth muscle [81], prostate [22] and sperm [14], in which the physiological role of the chan-nel in not entirely understood. In prostate epithelial cells TRPM8 appears be present in two isoforms, one primarily located in the membrane of the ER and the other (classical) in the plasma membrane, and is supposed to be involved in

processes like proliferation, differentiation, secretion and apoptosis [11, 18]. TRPM8 is expressed at both mRNA and protein level in sperm cells, in which it mediates cold and menthol-induced increases in intracellular calcium concen-tration, but its actual role is not fully clear [14]. In the lung, a truncated but functional TRPM8 variant is expressed in epithelial cells lining the airways where it may be activated by cooling, leading to Ca

2+ entry and upregulation of pro-

inflammatory interleukins IL-6 and IL-8, possibly contribut-ing to the pathophysiology of asthma [13]. This process is enhanced by activation of vagal afferent fibres expressing TRPM8 which trigger autonomic responses to cooling such as cough, broncho-constriction and mucus secretion [82]. A similar sensory function of TRPM8 was described recently in bladder afferents involved in the micturition reflex; moreover, an increased number of TRPM8-positive afferent fibres was found in painful bladder syndrome patients, sug-gesting a role of TRPM8 in inflammatory pain [56, 83]. Fi-nally, TRPM8 was also found in smooth muscle cells in sev-eral large arteries including the pulmonary artery and the aorta [12]; when activated by menthol, TRPM8 produces vasodilatation of vessels precontracted with the -adrenergic agonist phenylephrine [81]. Most of the tissues discussed above are not exposed to significant variations in tempera-ture, which raises the intriguing issue of the existence of endogenous TRPM8 agonists. This question was recently answered when it was shown that lysophospholipids pro-duced following activation of PLA2 are able to activate TRPM8 at normal body temperature, which makes TRPM8 not only a thermally gated but also a receptor operated chan-nel [65].

4. PATHO-PHYSIOLOGY OF TRPM8

4.1. Inflammation

Inflammation is a complex response to injury or disease, in an attempt to protect the damaged tissue, but often the cause of chronic pain. This condition is associated with the recruitment and activation of many types of immunocompe-tent cells, such as macrophages, mast cells, leukocytes, which, themselves or upon stimulation of resident cells, re-lease a variety of pro-inflammatory mediators, including inteleukin-1 (IL-1 ), tumor necrosis factor (TNF ), NGF, histamine, serotonin, prostaglandin E2 (PGE2), bradykinin, etc… [84]. These factors bind to receptors ex-pressed in the membrane of peripheral nociceptive nerve endings and trigger intracellular signaling cascades, resulting in nociceptor sensitization (decreased activation threshold and enhanced response), through post-translational modifica-tion and/or changes in gene expression of key molecules in the pain pathway. Inflammatory pain is manifested as allody-ina (pain produced by innocuous stimuli) and hyperalgesia (increased sensitivity to painful stimuli). Several TRP chan-nels, particularly TRPV1 and TRPA1, have been linked to inflammatory pain (reviewed by [85]). TRPM8 appears to be involved in inflammation as well, but its actions are not en-tirely understood.

There is functional and anatomical evidence that TRPM8 is co-expressed with receptors for pro-inflammatory media-tors, which makes it a putative target in inflammation. TRPM8 is co-expressed with the high affinity tyrosin kinase


receptor for NGF, trkA, a molecule with an important role in pain signaling [3]. In vitro studies using the DRG culture model have shown that cold and menthol sensitivity (most likely mediated by TRPM8) is up-regulated by exogenous NGF, but not BDNF (Brain-Derived Neurotrophic Factor) or GDNF (Glial cell line-Derived Neurotrophic Factor) [36, 54, 86]. Work from the same group found that cold and menthol sensitivity is co-expressed with sensitivity to the inflamma-tory mediators bradykinin, PGE2 and histamine in a sub-group of cultured sensory neurons from the rat [16]. How-ever, no change in TRPM8 expression at mRNA or protein level was found upon CFA (Complete Freund’s Adjuvant)-induced peripheral inflammation in rats or mice. The heat receptor TRPV1 is genetically up-regulated in inflammation and a higher fraction of TRPM8-positive neurons express TRPV1 following CFA-induced inflammation; it has been proposed that this mechanism is involved in inflammatory cold allodynia, in which gentle cooling, in the TRPM8 detec-tion range, is perceived as painful [30, 87, 88]. Strong evi-dence for a direct role of TRPM8 in inflammatory cold hy-persensitivity comes from work on TRPM8 null mutant mice, which show a profound reduction in the behavior evoked by plantar acetone application following CFA injec-tion [10].

Although the role of TRPM8 in inflammatory pain is not yet clearly defined, several groups have reported functional modulation of TRPM8 by pro-inflammatory mediators. Thus, TRPM8-mediated responses to menthol in cultured DRG neurons appear to be downregulated upon application of bradykinin [15] and cold-induced responses in TRPM8-expressing neurons were desensitized by both bradykinin and PGE2 [16]. Similarly, in a heterologous expression system, responses to cold and menthol in HEK293 cells co-expressing TRPM8 and trkA were strongly desensitized fol-lowing the application of NGF [62]. Moreover, as already mentioned, acidification of the external milieu (a known feature of inflammation) also leads to a reduction in TRPM8 activity [66]. At whole animal level, menthol-induced scratching behavior (presumably mediated by TRPM8) was attenuated by intrathecal administration of tachykinins sub-stance P and hemokinin-1, two neuropeptides involved in neurogenic inflammation [89]. Taken together, these results indicate that TRPM8 is inhibited following an acute inflam-matory challenge. How this correlates with the apparent re-duction in inflammatory pain behavior seen in TRPM8-/- mice is not quite clear.

4.2. Neuropathy

Neuropathic pain occurs following traumatic nerve lesion or disease (diabetic neuropathy), and in humans is associated primarily with hypersensitivity to mechanical and cold stim-uli. The role of TRPM8 in neuropathic pain has been thor-oughly investigated in the last few years, but the outcome of these studies was highly dependent on the animal model that was used, and not unambiguous. Concerning the plasticity in TRPM8 expression induced by nerve lesion, several groups that used the spinal nerve ligation (SNL), partial nerve injury (PNI) or spared nerve injury (SNI) models reported a reduc-tion of TRPM8 mRNA and/or protein levels in damaged (L5) neurons and no significant change in expression in un-injured (L4) cells [88, 90-92], suggesting that TRPM8 was

not accountable for the ensuing cold hypersensitivity in these animals. Accordingly, cold allodynia in SNL rats was pre-vented and reversed by intrathecally-delivered antisense oli-gonucleotides specific for TRPA1, but not for TRPM8 [88, 90]. However, another nerve injury model, the chronic con-striction injury (CCI), yielded entirely different results: TRPM8 transcription was upregulated, resulting in higher levels of TRPM8 protein in DRG neurons with both C- and A fibres, and also in superficial dorsal horn ipsilateral to the injury site [17, 93, 94]; but see also [95]. However, consen-sus was not reached regarding the pathophysiological out-come of this upregulation. While one group presented strong evidence that peripheral and central activation of TRPM8 by cold and icilin led to reduced pain behavior (heat and me-chanical hyperalgesia) indicating a profound analgesic action of this channel [17], another group was able to link this en-hanced expression to a role in the initiation and maintenance of neuropathic cold hypersensitivity [94]. Interestingly, in-vestigators using the SNL model in five different strains of mice found that regulation of TRPM8 was significantly cor-related with the increased sensitivity to heat in these strains, such that a larger decrease in TRPM8 levels was associated with a smaller degree of heat allodynia [91]. As heat and cold sensitivities appear to be genetically correlated in dif-ferent mouse strains [96], this would suggest a putative in-volvement of TRPM8 in mediating cold allodynia. Finally, adding to the complexity of this issue, other authors found no evidence to involve TRPM8 in the development of cold hypersensitivity in the CCI neuropathic pain model [95, 97]. One possible reason for these diverging or even conflicting results may be linked to the type of lesion produced in order to generate neuropathic pain. It has been shown that in loose nerve ligation (as in CCI models), damage is induced primar-ily to medium and large myelinated fibres and there is a more pronounced inflammatory component, while in models relying on axotomy or tight ligation (PNI, SNI or SNL) there is greater damage to small unmyelinated fibres, which may result in a different sensory phenotype [92]. Work in TRPM8 null mutant mice provided further evidence for a role of TRPM8 in generating cold hypersensitivity following nerve injury. Using the CCI model, Colburn and colleagues [10] investigated the ensuing cold allodynia (measured by the nocifensive response to evaporative cooling) and found that it failed to develop in TRPM8-/- mice. The significance of this plethora of animal studies for human disease is yet to be explored. However, one very recent study reported abnormal action potential firing correlated with a striking increase in menthol responsiveness in C-fibre nociceptors in one patient with small fibre polyneuropathy and cold allodynia, possibly due to alterations in TRPM8 expression and function [98].

4.3. Cancer

As already mentioned, TRPM8 was initially described as a prostate specific transcript upregulated in cancer (in pros-tate but also other tissues like colon, skin, breast), raising hopes that it may serve as a potential prognosis marker and target for immunotherapy [22, 99]. In the prostate, TRPM8 expression is initially up-regulated in the early stages of can-cer, but is then decreased in the transition to androgen-independence or in patients treated with anti-androgen ther-apy, and its loss is correlated with poor prognosis of patients with advanced prostate cancer [23]. The precise subcellular


localization and role of TRPM8 in cancer cells is not entirely understood, but progress has been made in recent years. The cold and menthol receptor appears to be expressed in both androgen-responsive and unresponsive prostate cancer cell lines, but only in the former is TRPM8 expression upregu-lated following androgen treatment. The same authors pro-vide evidence for a role of the channel in calcium homeosta-sis: they recorded TRPM8-mediated Ca

2+ entry, as well as

TRPM8-mediated Ca2+

mobilization from internal stores, which argues for the channel being present in both plasma and ER membrane [47]. However, using the same cell line (prostate cancer lymph node carcinoma, LNCaP), another group only found evidence for channel expression in the ER membrane and attributed menthol-induced Ca

2+ entry to a

store operated channel activated secondary to TRPM8 open-ing in the ER and store depletion [11]. A follow up study of the same group reported that the localization and activity of TRPM8 in prostate primary epithelial cells from human tis-sue specimens is linked to their differentiation. Two TRPM8 isoforms are differentially localized in the plasma and ER membrane respectively; while the ER isoform was functional independent of the differentiation status of prostate cells, the expression and activity of the plasma membrane TRPM8 was coupled to those of the androgen receptor, which in turn were linked to cellular differentiation and oncogenic state: plasma membrane TRPM8 was lost in androgen-independent metastatic prostate cancer cells [18]. TRPM8 appears to regulate prostate cancer cell survival: treatment with either capsazepine (a TRPM8 antagonist, see above) or siRNA against TRPM8 promoted apoptosis and reduced survival of androgen-sensitive cancer cells, indicating that the channel is required for survival. On the other hand, prolonged exposure to menthol (3 days in culture) decreased cellular viability in the same cell line, possibly through a Ca

2+-dependent

mechanism [47] (but see also [100]).

Human glioblastoma cells (DBTRG) express TRPM8 which mediates menthol-induced Ca

2+ entry that stimulates

cell migration. Functional large conductance Ca2+

-activated potassium (BK) channels are required to antagonize the de-polarization induced by TRPM8-mediated cation currents and thereby maintain a strong electrochemical gradient and subsequent Ca

2+ influx necessary for increasing the rate of

glioblastoma cell migration. As described in prostate cancer cells, DBTRG cells appear to express both the plasma mem-brane and the ER isoforms of TRPM8 [101, 102].

It should be stated that, in spite of this plethora of ex-perimental results, the actual role of TRPM8 in normal and cancer tissue outside the sensory system is not fully under-stood. Given the fact that they are not exposed to significant changes in temperature, a role of TRPM8 as a cold sensor in these tissues (prostate, brain) is unlikely. However, most tumors are associated with chronic inflammation and, de-pending on the signaling pathway, TRPM8 could be either activated at body temperature by lysophospholipids gener-ated by PLA2 or inhibited by PIP2 breakdown following PLC activation.

5. CONCLUSIONS

TRPM8 is a calcium-permeable, non-selective cation channel with a subunit structure consisting of 6 transmem-

brane segments and intracellular N- and C-terminal domains that most likely assembles and functions as a homo-tetramer. This channel is activated by gentle cooling, natural agonists like menthol and eucalyptol and endogenous agents like PIP2 and lysophospholipids and modulated following the activa-tion of intracellular signaling cascades. TRPM8 is also acti-vated by depolarizing potentials; temperature and chemical agonists act by shifting the voltage dependence of activation into the physiological range. It is expressed primarily in thermoreceptors and possibly noiceptors of the dorsal root and trigeminal ganglia, but also in non-sensory tissues such as prostate, lung, liver and vascular smooth muscle. In terms of subcellular localization, TRPM8 is found in the plasma membrane of peripheral and central terminals of sensory neurons, but also in the endoplasmic reticulum, where it can mediate calcium release. In the peripheral nervous system, TRPM8 is mainly expressed in a subpopulation of small neu-rons with unmyelinated or thinly myelinated fibres with a purely thermoreceptive function, where it acts as a sensor for innocuous cooling. It is also found together with nociceptive markers in a group of C-fibres responsive to both cooling and heating, in which it may serve a subtle nociceptive role. TRPM8 expression and function appears to be altered in inflammation and nerve injury, which may underlie the ab-normal thermal sensitivity in pathological pain states. Pe-ripheral and central activation of TRPM8 may also lead to analgesia, as it was shown in several chronic pain models. As more experimental results are accumulating, a clearer view of the (patho)physiological roles of TRPM8 is emerg-ing. However, a certain degree of uncertainty remains con-cerning the involvement of TRPM8 in noxious cold sensing and the function of TRPM8 in non-sensory normal and neo-plasmic tissue.

ACKNOWLEDGEMENT

Work in the authors’ laboratory is supported by grant PN2 Idei 164/2007 from the Romanian Research Council (CNCSIS).

ABBREVIATIONS

PNS = Peripheral nervous system

CNS = Central nervous system

HEK = Human embryonic kidney

BCTC = N-(4-Tertiarybutylphenyl)-4-(3-chloropyridin-2-l)tetrahydropyrazine-1(2H)-carbox-amide

NGF = Nerve growth factor

trkA = Neurotrophic tyrosine kinase receptor type 1

CPS-369 = (R)-2-[((1R,2S,5R)-2-isopropyl-5-methyl-cyclohexanecarbonyl)-amino]-propionic acid ethyl ester

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Received: ???????????????? Revised: ???????????????? Accepted: ????????????????

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