Ajay Dhaka, University of Washington School of Medicine
A compelling new study by Zhang and colleagues proposes a novel mechanism for the inhibition of the cold/menthol/icilin-activated ion channel TRPM8 by inflammatory mediators (1). TRPM8, which is expressed primarily in somatosensory neurons, appears to be the principle transducer of cool and cold temperature (2). TRPM8 is required for normal environmental cool temperature detection facilitating behavioral thermal homeostasis and it appears to mediate some of the analgesic effects of cool temperature following injury (3–6). Perhaps paradoxically, TRPM8 is also required for normal nocifensive responses to painful cold stimuli and TRPM8-null mice have clear deficits in the development of cold allodynia in response to inflammation and neuropathic injury (3-5;7;8). How TRPM8 is able to regulate these different aspects of cold perception is still an open question.
Previous studies have shown that inflammatory mediators such as bradykinin (BK) and histamine can enhance pain by sensitizing the heat/capsaicin receptor TRPV1 and while also potently inhibiting TRPM8 activity (9-12). BK and histamine signal through Gαq-coupled receptors that, when bound by ligand, catalyze the exchange of GDP for GTP in the guanine nucleotide-binding site of Gαq, causing Gαq activation. Gαq then activates phospholipase Cβ (PLCβ), leading to the depletion of membrane phosphatidylinositol-4,5-bisphosphate (PIP2) and the activation of protein kinase C (PKC). TRPM8 activity is regulated by membrane PIP2 and PIP2 depletion leads to the inhibition of TRPM8 activity (11;13). Furthermore, activation of PKC has also been shown to depress TRPM8 activity in response to BK in studies using both in vitro expression model systems and cultured DRG neurons (9;10).
The present study, however, has not been able to replicate these findings. Using pharmacological agents, the authors found no evidence supporting the idea that PLC-mediated hydrolysis of PIP2 or PKC activation mediate the effects of BK-induced inhibition of TRPM8. Instead, the authors found in a series of well thought-out experiments that Gαq appears to directly inhibit TRPM8 activity. They provided evidence that BK inhibition of TRPM8 but not sensitization of TRPV1 is membrane-delimited, suggesting that diffusible signaling messengers are not required to mediate the effect of BK on TRPM8. Furthermore, they found that a constitutively activated chimera (3Gαqiq), in which the PLCβ-binding domain of Gαq was replaced with the analogous region from Gαi, which does not interact with PLCβ, was able to potently inhibit TRPM8 activity. Activated Gαi alone had no effect on TRPM8, suggesting that signaling through Gαi was not contributing to inhibition of TRPM8. This data further supported the contention that signaling through PLC is not required for BK-mediated inhibition of TRPM8. To add to the confusion, another study has shown that activated Gαi via protein kinase A inhibition could potently suppress TRPM8 activity (14). Why there have been disparate findings between the current study and others investigating BK-mediated inhibition of TRPM8 as well as the effects of Gαi on TRPM8 activity is unclear (perhaps differences in experimental design?) and surely warrants further study.
The authors go on to show that activated Gαq inhibited TRPM8 in excised patches even in the presence of excess PIP2, again strongly supporting the suggestion that neither PIP2 depletion nor downstream signaling pathways are required for Gαq-mediated suppression of TRPM8. Importantly, the authors found that Gαq binds directly to TRPM8 and that the interaction between the two proteins is not affected by the activity state of Gαq. Indeed, the presence of BK and its receptor, or histamine and its receptor, had no effect on the amount of Gαq bound to TRPM8. It appears as if Gαq is constitutively bound to TRPM8. If Gαq is always bound to TRPM8, how is it regulating TRPM8 activity? The authors hypothesize that, since activated Gαq can inhibit TRPM8 in excised patches where it is presumably bound by inactive Gαq, it is likely that activated Gαq binds to TRPM8 at a different interface than inactive Gαq, facilitating inhibition of TRPM8 by inducing a conformational change in the channel. This would also suggest that inactive Gαq may have a role in modulating TRPM8 activity. Indeed, TRPM8-expressing cultured mouse embryonic fibroblasts (MEFs) null for Gαq were more potently activated by menthol than TRPM8-expressing wildtype MEFs that do express Gαq. Although using a different experimental protocol, forcing Gαq into the inactive state did not potentiate TRPM8 activity, suggesting that it is not low levels of activated Gαq that are responsible for the differences seen between wildtype and Gαq-null MEFs, but rather that inactive Gαq binding to TRPM8 may also inhibit TRPM8 activity. Of note is that Gαq is able to interact with both the N- and C-terminal domains of TRPM8. What role these multiple interaction domains may play in Gαq-mediated regulation of TRPM8 will have to be determined in future experiments. Also, since PIP2 is clearly required for normal TRPM8 activity, could PIP2 depletion have any role in BK-mediated inhibition of TRPM8? Perhaps under physiological conditions depletion of PIP2 could lead to a longer suppression of TRPM8 activity than that caused by activated Gαq alone. Future experiments could also address this possibility.
Although it is not clear what the physiological relevance of BK-mediated TRPM8 inhibition is, it has been hypothesized that the inhibition of TRPM8 by inflammatory mediators such as BK serves to enhance inflammatory pain by inhibiting the analgesic properties of TRPM8 (9;10). A number of reports suggest that TRPM8 is expressed in more than one type of somatosensory neuron, potentially consisting of a nociceptive population that co-expresses TRPV1 and/or other nociceptor markers, and an innocuous cool-sensing population that does not co-express nociceptive markers (2;15-17). In the present study the authors found, in agreement with earlier studies, that only a fraction (11/33) of cultured DRG neurons showed BK-mediated inhibition of TRPM8-dependent responses, suggesting that only a subpopulation of TRPM8+ neurons express BK receptors (9). In the one example of a BK-inhibited TRPM8+ neuron shown in the study, the neuron also co-expressed TRPV1. What if BK receptors or other inflammatory mediator Gαq-coupled receptors were only co-expressed with TRPM8 in TRPV1+ neurons, which would likely be nociceptive neurons? It is known that BK receptors are widely co-expressed with TRPV1 in somatosensory neurons (18). What then would be the functional implication of inhibiting TRPM8 in these neurons? By what mechanism would inhibition of TRPM8 expressed in nociceptors facilitate inflammatory pain? How would one reconcile this with the requirement of TRPM8 in the development of cold allodynia? If, however, inflammatory mediator Gαq-coupled receptors such as BK receptors were only expressed in innocuous cool-sensing TRPM8+ neurons and not in TRPM8+ nociceptors, which does not appear to be the case, then the model could be easily explained. BK would suppress the activation of potentially analgesic, innocuous cool-sensing neurons while not affecting TRPM8+ nociceptors and therefore promoting inflammatory pain, including cold pain. These are but two, likely overly simplistic, possibilities and both assume that different subpopulations of TRPM8-expressing neurons act in labeled lines for modality-specific sensation, which may not be the case. Understanding the consequences of inflammation-mediated Gαq activation on TRPM8-dependent pain modulation requires a greater knowledge of the function of different subpopulations of TRPM8-expressing neurons and of the in vivo expression of inflammatory mediator Gαq-coupled receptors within these subpopulations in both normal and inflammatory/injury conditions.
1. Zhang X, Mak S, Li L, Parra A, Denlinger B, Belmonte C, McNaughton PA. Direct inhibition of the cold-activated TRPM8 ion channel by Gαq. Nat Cell Biol. 2012 Jul 1. [Epub ahead of print]
2. McCoy DD, Knowlton WM, McKemy DD. Scraping through the ice: uncovering the role of TRPM8 in cold transduction. Am J Physiol Regul Integr Comp Physiol. 2011 Jun;300(6):R1278-87.
3. Bautista DM, Siemens J, Glazer JM, Tsuruda PR, Basbaum AI, Stucky CL, Jordt SE, Julius D. The menthol receptor TRPM8 is the principal detector of environmental cold. Nature. 2007 Jul 12;448(7150):204-8.
4. Colburn RW, Lubin ML, Stone DJ Jr, Wang Y, Lawrence D, D'Andrea MR, Brandt MR, Liu Y, Flores CM, Qin N. Attenuated cold sensitivity in TRPM8 null mice. Neuron. 2007 May 3;54(3):379-86.
5. Dhaka A, Murray AN, Mathur J, Earley TJ, Petrus MJ, Patapoutian A. TRPM8 is required for cold sensation in mice. Neuron. 2007 May 3;54(3):371-8.
6. Proudfoot CJ, Garry EM, Cottrell DF, Rosie R, Anderson H, Robertson DC, Fleetwood-Walker SM, Mitchell R. Analgesia mediated by the TRPM8 cold receptor in chronic neuropathic pain. Curr Biol. 2006 Aug 22;16(16):1591-605.
7. Knowlton WM, Bifolck-Fisher A, Bautista DM, McKemy DD. TRPM8, but not TRPA1, is required for neural and behavioral responses to acute noxious cold temperatures and cold-mimetics in vivo. Pain. 2010 Aug;150(2):340-50.
8. Knowlton WM, Daniels RL, Palkar R, McCoy DD, McKemy DD. Pharmacological blockade of TRPM8 ion channels alters cold and cold pain responses in mice. PLoS One. 2011;6(9):e25894.
9. Linte RM, Ciobanu C, Reid G, Babes A. Desensitization of cold- and menthol-sensitive rat dorsal root ganglion neurones by inflammatory mediators. Exp Brain Res. 2007 Mar;178(1):89-98.
10. Premkumar LS, Raisinghani M, Pingle SC, Long C, Pimentel F. Downregulation of transient receptor potential melastatin 8 by protein kinase C-mediated dephosphorylation. J Neurosci. 2005 Dec 7;25(49):11322-9.
11. Liu B, Qin F. Functional control of cold- and menthol-sensitive TRPM8 ion channels by phosphatidylinositol 4,5-bisphosphate. J Neurosci. 2005 Feb 16;25(7):1674-81.
12. Zhang X, Li L, McNaughton PA. Proinflammatory mediators modulate the heat-activated ion channel TRPV1 via the scaffolding protein AKAP79/150. Neuron. 2008 Aug 14;59(3):450-61.
13. Rohács T, Lopes CM, Michailidis I, Logothetis DE. PI(4,5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain. Nat Neurosci. 2005 May;8(5):626-34.
14. Bavencoffe A, Gkika D, Kondratskyi A, Beck B, Borowiec AS, Bidaux G, Busserolles J, Eschalier A, Shuba Y, Skryma R, Prevarskaya N. The transient receptor potential channel TRPM8 is inhibited via the alpha 2A adrenoreceptor signaling pathway. J Biol Chem. 2010 Mar 26;285(13):9410-9.
15. Dhaka A, Earley TJ, Watson J, Patapoutian A. Visualizing cold spots: TRPM8-expressing sensory neurons and their projections. J Neurosci. 2008 Jan 16;28(3):566-75.
16. Madrid R, de la Peña E, Donovan-Rodriguez T, Belmonte C, Viana F. Variable threshold of trigeminal cold-thermosensitive neurons is determined by a balance between TRPM8 and Kv1 potassium channels. J Neurosci. 2009 Mar 11;29(10):3120-31.
17. Takashima Y, Daniels RL, Knowlton W, Teng J, Liman ER, McKemy DD. Diversity in the neural circuitry of cold sensing revealed by genetic axonal labeling of transient receptor potential melastatin 8 neurons. J Neurosci. 2007 Dec 19;27(51):14147-57.
18. Vellani V, Zachrisson O, McNaughton PA. Functional bradykinin B1 receptors are expressed in nociceptive neurones and are upregulated by the neurotrophin GDNF. J Physiol. 2004 Oct 15;560(Pt 2):391-401.
How does PRF find papers of the week? See our PubMed search string and complete search results by clicking here.
What are you reading? Please share your favorite topical search string and we'll post it here.
Copyright © 2013 Pain Research Forum
All site content, except where otherwise noted, is licensed under a Creative Commons BY-NC-ND License. Additional permissions for appropriate reuse available on request.