Box 1. Activation of the Pain System Heterologous expression studies have identified a number of sensory neuron–associated genes that have been invoked as sensors for different sorts of damaging stimuli. Cold Pain Several mechanisms have been proposed for the transduction of noxious cold stimuli, including direct activation of specific TRP channels, inhibition of K+ currents, and reduction of electrogenic pump activity. Recent work suggests that TRPM8 and TRPA1 may play important roles in the detection of noxious cold, and that the inhibition of an as-yet-unidentified background K+ channel may be important. In addition to current research focusing on transduction by primary sensory neurons, recent reports have suggested that epidermal cells, particularly keratinocytes, may be responsible for the transduction of certain stimuli, synapsing with primary sensory neurons (see recent review from [74]). Other studies have proposed a role for vascular nociceptors in cold pain, suggesting that cold-induced vasoconstriction may begin this process [43]. It seems clear that our understanding of cold transduction is still incomplete. The discovery that the voltage-gated sodium channel NaV1.8 (SCN10A) is not inactivated by cooling, in contrast to all other voltage-gated sodium channels, provides an insight into the mechanisms behind the detection of noxious cold [10]. It provides a clear explanation for the observation that while non-nociceptive sensory neurons become inactive upon cooling, resulting in numbness, nociceptors are sensitised. The evolutionary conserva- tion of NaV1.8 reflects the vital importance of the ability to detect noxious stimuli in cold environments, particularly relevant to cold- blooded animals. A more detailed treatment of the mechanisms involved in cold pain is given in [75]. Heat Pain The identification of a cation selective ion channel, TRPV1, expressed in sensory neurons and activated by both heat and red hot chili peppers (the capsaicin receptor), caught the public imagination and suggested that the mechanism for noxious thermosensation had been identified [76]. Despite strong evidence of a role for TRPV1 in altering inflammatory pain thresholds, the evidence that noxious heat is transduced solely or principally by TRPV1 is weak [77,78]. Subsets of thermosensitive neurons defined by isolectin binding (IB4) have been shown not to express TRPV1 or TRPV2. TRPV1 knock-outs have fairly normal patterns of responses to acute noxious heat [77]. TRPV4 seems to have a more significant role in mechanosensation rather than thermosensation [63], but the (perhaps inappropriate) epithet thermoTRPs has been attached to these pleiotropic sensors that are involved in many aspects of sensation. It seems clear that other transducers of noxious heat, either in the skin or in neurons, remain to be identified. Touch and Noxious Mechanosensation Sensory neurons express ion channels that can be directly activated by mechanical stimuli, and evidence has been presented that such channels underlie noxious mechanosensation [79]. The genes encoding such transducing channels, however, have not been identified. Although some electrical deficits in neuronal responses to mechanical stimuli have been observed in mouse knock-outs (e.g., ACCN3 encoding ASIC 3, as described by [80]), there are no known deficits in potential transducing channels, apart from TRPV4 [32], that show effects on behavioural responses to noxious mechanical stimuli. Loss of sensory neurons, or downstream sodium channels characteristically expressed by these neurons (e.g, SCN9A and SCN10A), do have a mechano- insensitive phenotype [6,11], implying that these neurons are responsible for mechanosensation. Box 2. Pain-Linked SNPs in COMT (catechol-O- methyltransferase) Many SNPs affecting pain perception have been identified in recent years. Possibly the best-characterised of these are the SNPs present in COMT, the gene coding for catechol-O-methyltransfer- ase (COMT). COMT mediates the inactivation of catecholamine neurotransmitters, including dopamine, adrenaline, and noradren- aline, and reduced COMT enzymatic activity appears to result in increased pain sensitivity and temporal summation of pain [26]. The precise mechanism by which this occurs has yet to be defined, although Zubieta et al. [65] propose a depletion of enkephalins due to elevated dopamine levels, leading to an upregulation of m- opioid receptors and increased sensitivity to temporally integrated noxious stimulation. Alternatively, or perhaps additionally, de- creased adrenaline metabolism (due to decreased COMT activity) may increase pain through the stimulation of b2/3-adrenergic receptors, since the pain induced by pharmacological COMT inhibition is blocked by combined b2/3-adrenergic receptor antagonists [27]. Zubieta et al. [65] investigated the effect on pain sensitivity of an abundant, nonsynonymous SNP in COMT. This SNP results in the substitution of valine by methionine at position 158 (val158met), and is associated with reduced thermo- stability and a 3–4-fold reduction in COMT activity. Humans homozygous for the met158 allele reported very slightly increased sensory and affective ratings of pain in response to muscular infusion of hypertonic saline compared to heterozygotes, while ratings for val158 homozygotes were lower than heterozygotes. Corresponding variations in regional, pain-related m-opioid responses were observed using positron emission tomography, and were taken to explain the differences in pain perception [65]. A broader investigation of the influence of SNPs in the COMT gene on pain sensitivity provided a more comprehensive picture. Diatchenko et al. [26] selected six SNPs with high polymorphism frequency: two synonymous and one nonsynonymous (val158met) in the coding region, two in the promoter region, and one at the end of the 39 UTR; no other SNPs exist in the coding region at frequency .0.15%. Two of these (not including val158met) were found to be associated with altered pain sensitivity, present in the same haploblock. The haplotype at this haploblock was found to have a significant effect on pain perception, with haplotypes designated as high, low, or intermediate pain sensitivity (population frequencies 10.7%, 36.5%, and 48.7%, respectively). This was found to correlate inversely with levels of COMT activity. Different combinations of these haplotypes were strongly associated with variations in experimental pain sensitivity. Additionally, a reduction in risk of a common painful musculoskeletal condition, temporomandibular joint disorder, was observed in the presence of even a single low- sensitivity haplotype. The mechanism by which these COMT haplotypes influence activity was recently uncovered using in silico modelling of mRNA secondary structures. It was found that the haplotypes differed with respect to mRNA local stem-loop structures, and that the most stable structure resulted in lower protein levels and thus activity. Further evidence was provided by site-directed mutagenesis, restoring normal levels of activity by eliminating the stable structure [81]. This illustrates two points: first, that synonymous polymorphisms may have large influences on protein activity; and second, the potential for interaction of multiple SNPs and therefore the importance of investigating haplotypes over single SNPs. relevant to the study of pain [4]. This database, regularly updated and currently containing more than 230 genes, is a valuable resource to the research community. Gaveriaux-Ruff and Kieffer [33] have also catalogued neuronal Cre lines that are relevant to genetic studies of pain pathways. PLoS Genetics | www.plosgenetics.org 4 July 2008 | Volume 4 | Issue 7 | e1000086