INTRODUCTION

Our sensory systems are able to detect subtle changes in ambient temperature, due to the coordinated efforts of thermosensory neurons. At the level of the primary afferent nerve, the site at which thermal stimuli are converted into neuronal activity, temperature-sensitive members of the TRP channel family are found. Remarkably, the range of temperatures that these channels respond to covers the entire perceived temperature spectrum, from warm to painfully hot, from pleasingly cool to excruciatingly cold [1]. Moreover, many of these channels are receptors for ligands that elicit distinct psychophysical sensations, such as the heat associated with capsaicin and the cold felt with menthol. The latter of these was influential in the discovery of the first TRP channel shown to be responsive to temperatures in the cold range (<30°C), TRPM8, a member of the melastatin TRP channel subfamily [2,3]. This chapter focuses on TRPM8, describing what was known about cold signaling before the channel was cloned, how TRPM8 was identified as a cold sensor, and what advances have been made in our understanding of the molecular logic for cold sensation since its identification.

COLD SENSING AND MENTHOL

The perception of nonpainful, cool temperatures is reported to occur when the skin is cooled as little as 1°C from normal body temperature [4]. However, once temperatures approach 15°C, the perception of cold pain is felt, with qualities described as burning, aching, and prickling [5]. In the early to mid-twentieth century, a number of laboratories began to observe cold-induced electrical impulses when recording from mammalian sensory nerves. These peripheral cold receptors, both Aδ- and C-fibers, have thermal thresholds (i.e., the temperature at which nerve impulses are generated) for cold activation between 30–20°C, temperatures considered to be innocuously cool [4,6]. Further cooling to temperatures below what is considered noxious (<15°C) was also shown to excite a small percentage of nociceptors (20–30 percent), while cooling to <0°C was reported to activate all fibers [7,8] (for review see reference 9). Thus, there is significant diversity in the types of neurons that respond to cold, as well as an expansive range of cold activation thresholds. Moreover, no defined mechanism for cold sensing was described.

Most cold-sensitive neurons are also sensitive to the ubiquitous cooling compound menthol, a cyclic terpene alcohol found in mint leaves [10]. It is well known that moderate concentrations of menthol induce a pleasant cool sensation, such as that felt when using menthol-containing products such as candy and vapo-rubs. However, when present at higher doses menthol can be noxious, causing burning, irritation, and pain [10–12]. In seminal studies conducted by Hensel and Zotterman in the 1950s, menthol elicited its “cool” sensation by increasing the threshold temperature for activation of cold receptors [13]. Indeed, the researchers hypothesized that menthol exerted its actions on “an enzyme” that was involved in the activation of these nerves [13]. Surprisingly, it took more than 50 years for Hensel and Zotterman’s hypothesis to be validated.

THE HUNT FOR A MENTHOL RECEPTOR

As described previously, studies into cold-sensitive fibers were elusive in defining the biological basis for cold signaling. However, in the mid- to late 1990s, a number of laboratories interested in cold transduction began to use primary cultures of either dorsal root (DRG) or trigeminal (TG) ganglia neurons as in vitro models of sensory afferents. Approximately 10–20 percent of ganglia neurons respond to cold temperatures, with thresholds for activation below 30°C [2,14–16]. Suto and Gotoh showed that cold stimuli (~20°C) evoke a robust influx of calcium in a small percentage (~10 percent) of cultured DRG neurons [15]. Similarly, Kobayashi and colleagues obtained comparable results using menthol as the stimulus, demonstrating that the cooling compound promoted membrane depolarization and generated nerve impulses [17].

Using a similar approach, Reid and Flonta recorded membrane currents from cultured DRG neurons that they selected based on the ability of cold to elicit an increase in cytoplasmic calcium [16]. Cooling generated an inward current in these neurons, when the cells were held at negative membrane potentials, with an average temperature threshold near 29°C. This threshold shifted to warmer temperatures when the recordings were conducted in the presence of menthol, as was predicted from Hensel and Zotterman’s original hypothesis [13]. Similar responses were observed in cultured TG neurons by Julius and colleagues, who went on to demonstrate that both menthol and cold evoke rapidly activating, nonselective cation conductances that were characterized by strong outward rectification (Figure 13.1A, B) [2]. Both studies showed that the effects of menthol were temperature dependent and that warming neurons to >37°C could strongly reduce menthol’s effects [2,16].

FIGURE 13.1

Menthol- and cold-evoked responses in sensory neurons and cells heterologously expressing TRPM8. (A) Whole-cell voltage clamp recordings from dissociated primary cultures of trigeminal neurons. Menthol (100 μM) and cold (see temperature plot) (more...)

Both menthol- and cold-evoked currents also adapt to prolonged stimulation at a rate that is similar to what is observed in primates and humans [9]. Thus, this in vitro data supported the hypotheses of Hensel and Zotterman in that it seemed likely that cold and menthol work through a similar mechanism, leading to the search for their common molecular site of action.

THE CLONING OF A COLD AND MENTHOL RECEPTOR

The long-sought confirmation of Hensel and Zotterman’s original hypothesis for the action of menthol finally came in 2002. Two groups working independently and using different experimental approaches concurrently cloned a cold- and menthol-sensitive ion channel from sensory neurons [2,3]. The first group used menthol to expression clone a complementary DNA (cDNA; a synthesized copy of an RNA transcript) from rat TG neurons that could confer menthol sensitivity to cells that were normally insensitive to temperature [2]. The second group searched for TRP channel–like sequences in mouse DRG neurons and tested these channels for temperature sensitivity [3]. With these divergent approaches, both groups simultaneously identified TRPM8 (also referred to as trp-p8 or CMR1), a member of the melastatin or long-TRP channel subfamily [18]. Surprisingly, TRPM8 had been identified prior to these neuronal studies as a transcriptional marker of prostate epithelia, but was not detected in sensory tissue at the time (see below) [19]. Nonetheless, TRPM8 was the first cold-activated ion channel to be identified, and it established the general role for TRP ion channels in thermosensation [1].

TRPM8 HAS PROPERTIES SIMILAR TO THOSE OF COLD RECEPTORS

In both TG and DRG, TRPM8 is expressed in <15 percent of small-diameter (~20 μm) sensory neurons, consistent with the proportion of neurons shown to be cold- and menthol-sensitive in neuronal cultures [2,14,20,21]. In initial characterizations of the molecular and neurochemical phenotypes of sensory neurons that express TRPM8, it was found that RNA transcripts for the channel were not coexpressed with the heat-activated TRP channel TRPV1, calcitonin gene-related peptide (CGPR), neurofilament, or isolectin B4 binding [3]. Later studies confirmed these results, further demonstrating that TRPM8 transcripts are in both Aδ- and C-fibers and that it coexpresses with the receptor tyrosine kinase TrkA, but not TrkB or TrkC or another putative temperature-sensitive channel, TRPA1 (see Chapter 11) [22]. Transcripts for TRPM8 are also more abundant in trigeminal versus dorsal root ganglia [2], particularly in the mandibular region that innervates the tongue [22]. TRPM8 was also found to be expressed in lingual nerve fibers that project in to the fungiform papillae of the tongue [23]. Interestingly, TRPM8 fibers were in close proximity to taste buds, but did not innervate these structures. Thus, TRPM8 defines a small and discrete population of sensory afferents that innervate tissues known to be highly sensitive to cold and nociceptive stimuli.

When expressed in heterologous expression systems, such as Xenopus oocytes or mammalian cell lines, TRPM8-mediated currents are activated by a number of cooling compounds in addition to menthol, such as eucalyptol (the active ingredient in eucalyptus oil) and the super-cooling AG-3-5 (Figure 13.1C) [2]. Biophysically, TRPM8 has surprisingly similar properties to those recorded in both cultured DRG and TG using similar experimental paradigms (see Table 13.1). These include selectivity for ions, potency of menthol in activating currents, and voltage dependence of membrane currents induced by either cold or menthol [2,3,16,24]. Like almost all TRP channels, TRPM8 is a nonselective cation channel that displays strong outward rectification (Figure 13.1D). Like native menthol-evoked currents, TRPM8 showed relatively high selectivity for calcium and little selectivity among monovalent cations [2]. Menthol-evoked single-channel currents are also characterized by strong outward rectification and have a slope conductance of 83 pS [2].

TABLE 13.1

Comparison of the Biophysical Properties of Native Cold/Menthol Currents and Heterologously Expressed TRPM8

More remarkably, TRPM8 currents are also evoked by temperature decreases with an activation temperature threshold of ~26°C, with activity increasing in magnitude down to 8°C (Figure 13.2A, B) [2,3]. Interestingly, this broad range spans what are considered both innocuous cool (~30–15°C) and noxious cold temperatures (<15°C). Moreover, neither the rate nor direction of temperature change altered the response profile of TRPM8 currents (Figure 13.2B) [2]. Therefore, the channel’s response is directly proportional to the temperature presented in these restricted in vitro systems. When menthol is applied, the threshold for activation shifts to warmer temperatures (Figure 13.2C) [2], and increasing concentrations of menthol shift this curve even more, suggesting that menthol mimics the endogenous mechanism for thermal activation of TRPM8 (Figure 13.2D; D. McKemy, unpublished observations).

FIGURE 13.2

Cold activation of TRPM8. (A) Reduction in perfusate temperature (from ~32 to 10°C) evokes robust inward currents in TRPM8-expressing Xenopus oocytes (−60 mV hp.). Two rates of temperature change are shown (0.2°C/sec.; 1°C/sec.). (more...)

OTHER COOLING COMPOUNDS ACTIVATE TRPM8

In addition to menthol and AG-3-5, a number of cooling agents, including Cool-actP, Cooling Agent 10, FrescolatMGA, FrescolatML, geraniol, hydroxycitronellal, linalool, PMD38, WS-3, and WS-23 activate TRPM8 in vitro [2,25,26]. Of these AG-3-5 (also known as icilin) was first identified as a super-cooling agent in the early 1980s and bears little resemblance to menthol structurally [27]. AG-3-5 is more potent and effective than menthol in activating TRPM8, and when given intravenously, it will induce characteristic shivering or “wet dog” shakes [2,27]. Interestingly, the mechanism whereby AG-3-5 activates TRPM8 is different than that of menthol or cold [2]. AG-3-5 requires a coincident rise in cytoplasmic calcium, either via permeation through the channel or by release from intracellular stores, in order to evoke TRPM8 currents [28]. This requirement of a calcium rise for TRPM8 activity is not needed for cold- or menthol-induced channel activity, suggesting the channel can be activated by multiple mechanisms. Additionally, a critical amino acid was identified: when mutated, it rendered AG-3-5 incapable of activating TRPM8. This residue was located between the second and third trans-membrane domains of the channel, a region known to be important for capsaicin sensitivity of TRPV1 [28,29].

While various compounds activate TRPM8, a more relevant class of molecules that may be of use clinically is of those that antagonize or block the channel. A number of antagonists have been identified, including BCTC, thio-BCTC, CTPC, and capsazepine [26,30]. Surprisingly, many of these compounds also antagonize the heat-gated channel TRPV1. Thus, there is significant overlap pharmacologically between the two channels. Along with the analogous positions critical for capsaicin and AG-3-5 activation of TRPV1 and TRPM8, respectively, these results suggest a conserved mechanism for ligand activation of these thermosensitive TRP channels. While of interest pharmacologically, these results complicate the search for selective agents for these channels that are such good targets for drug discovery.

REGULATION OF TRPM8 CURRENTS

Experience tells us that temperature sensation is a dynamic process. For instance, we can easily adapt to cold temperatures, a process observed in both psychophysical and cellular assays [6,16,24,31]. Similarly, cold- or menthol-induced TRPM8 currents will adapt or desensitize in a calcium-dependent manner during prolonged stimulation [2]. Increased intracellular calcium is known to lead to the breakdown of a membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP₂), via activation of phospholipase C (PLC) [32]. Thus, it has been proposed that calcium influx, via TRPM8, activates PLC to cleave PIP₂, thereby altering the concentration of this phospholipid in the plasma membrane [33,34]. Thus, adaptation is thought to be due to cellular changes in the levels of PIP₂, which modulates the ability of TRPM8 to respond to cold or menthol. At the molecular level, a number of amino acid residues have been identified in the carboxy-terminal domain of the channel, adjacent to the sixth transmembrane domain, that appear to be involved in PIP₂’s effects on TRPM8 [34]. Interestingly, these residues are near the highly conserved TRP box of the channel and are found in other PIP₂-sensitive TRPM channels, including TRPM4 and TRPM5 [35,36].

In addition to calcium, many types of pathological conditions, such as peripheral inflammation, can lead to altered membrane levels of PIP₂ [32]. Many cell-surface receptors, such as TrkA, the receptor for nerve growth factor, and the bradykinin receptor activate PLC, thereby cleaving PIP₂ in the membrane. A consequence of PIP₂ breakdown is the generation of diacylglycerol (DAG) and inositol-trisphosphate (IP₃). DAG, along with increased intracellular calcium, activates protein kinase C (PKC), leading to phosphorylation of several cellular substrates including many ion channels. Phosphorylation is a common mechanism whereby channel activity is modulated, and increased PKC activity causes decreased TRPM8 membrane currents [37]. Interestingly, PKC activation does not lead to increased incorporation of phosphate on TRPM8, but rather a decrease in TRPM8 phosphorylation. This was blocked by treatment with phosphatase inhibitors, suggesting that the PKC-mediated effects are not due to direct phosphorylation of TRPM8, but that PKC plays a role upstream of channel phosphorylation.

Intracellular pH also regulates TRPM8. When pH is increased to above physiological levels, TRPM8 activity is inhibited [26,30]. These effects of pH are thought to be mediated intracellularly [30], but there is disagreement on the effects of pH on cold-, menthol-, and AG-3-5–evoked currents. Andersson et al. reported that menthol’s ability to activate TRPM8 is unaffected by pH, but that cold and AG-3-5 responses are inhibited [30]. Behrendt et al. also found that AG-3-5 was less effective in activating TRPM8 at high pH, but in contrast, menthol-evoked responses were also suppressed [26]. Cold was not tested in the latter study. Thus, it seems likely that either cell-to-cell variation in temperature thresholds for cold, or altered sensitivity due to experience and pathological state of the neuron, may be a result of TRPM8 regulation via cellular levels of PIP₂, protons, or kinase activity. This level of channel modulation may also account for the complexity and variability in cold-evoked temperature responses observed both in vivo and in vitro.

MECHANISM OF COLD ACTIVATION

Ever since the identification of thermosensitive TRP ion channels, the basis of the precipitous temperature sensitivity of these proteins has been of keen interest, but a physiological or molecular mechanism has remained elusive. Several plausible mechanisms have been proposed, including temperature-dependent structural reorganization of the channels, production of endogenous channel-activating ligands by a change in temperature, or that the channels respond to temperature-dependent changes in membrane fluidity [38].

For TRPM8, temperature has been shown to produce two fundamental changes in channel properties: a shift in the voltage dependence of the channel and modification of the maximum probability of channel opening [39,40]. Like other members of the TRPM subfamily, TRPM8 currents are voltage sensitive [2], and an initial study reported that declining temperatures shift the voltage-dependent activation curve of TRPM8 toward negative membrane potentials [40]. Kinetic analyses suggested that temperature sensitivity of the channel was a result of an over ten-fold difference between the activation energies associated with opening and closing the channel (E_a,open < E_a,close). Thus, cold activation of TRPM8 was described with a simple, single-thermodynamic principle based on this difference and was not due to significant changes in the steady-state open probability of the channel. Moreover, menthol was shown to mimic the effect of cold temperatures on voltage dependence of the channel, serving as a gating modifier [40].

A subsequent report also provided evidence for a temperature-dependent shift in the voltage dependence of TRPM8 [39]. However, in contrast to the previous findings, a significant increase in the maximum open probability of the channel was observed upon cooling, leading to the hypothesis that temperature affects not only the voltage dependence of the channel, but also leads to large conformational changes in TRPM8. Thus, a more complex allosteric model for channel activation was proposed, one that suggests that TRPM8 has two autonomous sensors, one for voltage and one for temperature, that are activated independently and interact to promote channel opening [39]. To date, the structural determinants of voltage and temperature sensitivity of TRPM8 have not been identified. However, the similarity of the TRP channel family to classic voltage-gated channels suggests that a putative voltage sensor may reside in the fourth transmembrane domain [41]. Whether the temperature sensor exists in this or other regions of the channel remains to be determined.

A ROLE FOR TRPM8 OUTSIDE THE NERVOUS SYSTEM

Although TRPM8 is undoubtedly critical for transduction of thermal stimuli in the peripheral nervous system, its expression in other tissues suggests it serves other biological roles in addition to neuronal thermal sensing. Along with expression in the prostate, TRPM8 has also been found in the bladder and male genital tract [42]. It is also observed that TRPM8 expression increases dramatically in cancers of the prostate, as well as other nonprostatic tumors such as breast, colon, lung, and skin [19]. In the prostate, low levels of TRPM8 transcripts have been identified [19]. However, TRPM8 expression greatly increases in transformed prostate epithelia. A role for TRPM8 in either normal or cancerous prostate tissues is still unclear, and studies using models of prostate cancer, such as LNCaP cells, have shown that the channel does respond to cold and menthol in these cells [43]. However, the channel is not present on the cell surface but is trapped in intracellular membrane compartments such as the endoplasmic reticulum. This expression is androgen dependent, suggesting that TRPM8 may play a role in differentiating these cells [44,45].

In addition to prostate and cancerous tissues and cells, TRPM8 transcripts have been found in the gastric fundus [46]. Cooling that takes place following the consumption of cold foods induces contraction of gastrointestinal smooth muscles, resulting in a short-lived gastric voiding [47]. When a putative TRPM8 inhibitor capsazepine was applied, these cooling-evoked contractions were reduced, suggesting that TRPM8 may be involved in this process in a nonneuronal capacity [46]. However, capsazepine is also a potent antagonist for TRPV1, thus complicating these results. Nonetheless, these and other data strongly suggest that TRPM8 may have diverse biological functions outside of the peripheral nervous system.

CONCLUSIONS

The elucidation of TRP channels as molecular detectors of thermal stimuli addressed a fundamental issue in sensory transduction: how are thermal stimuli converted into neuronal activity? The identification of TRPM8 and the subsequent studies characterizing its properties have shed light on the molecular mechanisms of cold sensation. Future studies will undoubtedly continue to elucidate the biological importance of this ion channel in mediating sensory signaling, as well as the role of TRPM8 in nonneuronal tissues. Nonetheless, cloning TRPM8 established the first molecular detector of cold stimuli, and its in vitro properties are consistent with this role in vivo. Moreover, TRPM8 confirmed Hensel and Zotterman’s half-century-old hypothesis [13] and established that TRP channels can confer thermal stimuli over broad ranges of temperature.

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