Direct inhibition of the cold-activated TRPM8 ion channel by Gα(q).

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 Gα_q-coupled receptors that, when bound by ligand, catalyze the exchange of GDP for GTP in the guanine nucleotide-binding site of Gα_q, causing Gα_q activation. Gα_q 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 Gα_q 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 (3Gα_qiq), in which the PLCβ-binding domain of Gα_q was replaced with the analogous region from Gα_i, which does not interact with PLCβ, was able to potently inhibit TRPM8 activity. Activated Gα_i alone had no effect on TRPM8, suggesting that signaling through Gα_i 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 Gα_i 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 Gα_i on TRPM8 activity is unclear (perhaps differences in experimental design?) and surely warrants further study.

The authors go on to show that activated Gα_q 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 Gα_q-mediated suppression of TRPM8. Importantly, the authors found that Gα_q binds directly to TRPM8 and that the interaction between the two proteins is not affected by the activity state of Gα_q. Indeed, the presence of BK and its receptor, or histamine and its receptor, had no effect on the amount of Gα_q bound to TRPM8. It appears as if Gα_q is constitutively bound to TRPM8. If Gα_q is always bound to TRPM8, how is it regulating TRPM8 activity? The authors hypothesize that, since activated Gα_q can inhibit TRPM8 in excised patches where it is presumably bound by inactive Gα_q, it is likely that activated Gα_q binds to TRPM8 at a different interface than inactive Gα_q, facilitating inhibition of TRPM8 by inducing a conformational change in the channel. This would also suggest that inactive Gα_q may have a role in modulating TRPM8 activity. Indeed, TRPM8-expressing cultured mouse embryonic fibroblasts (MEFs) null for Gα_q were more potently activated by menthol than TRPM8-expressing wildtype MEFs that do express Gα_q. Although using a different experimental protocol, forcing Gα_q into the inactive state did not potentiate TRPM8 activity, suggesting that it is not low levels of activated Gα_q that are responsible for the differences seen between wildtype and Gα_q-null MEFs, but rather that inactive Gα_q binding to TRPM8 may also inhibit TRPM8 activity. Of note is that Gα_q is able to interact with both the N- and C-terminal domains of TRPM8. What role these multiple interaction domains may play in Gα_q-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 Gα_q 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 Gα_q-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 Gα_q-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 Gα_q 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 Gα_q-coupled receptors within these subpopulations in both normal and inflammatory/injury conditions.

References:

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.

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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.

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