The cold-activated trpm8 channelagonism by macrolide immunosuppressants and modulation by gq protein-coupled receptors signaling pathways

Resumen

TRPM8 is a polymodal, non-selective cation channel activated by cold temperature and cooling compounds (i.e. menthol) which is mainly expressed in a small subpopulation of cold-sensitive peripheral sensory neurons. TRPM8 is the principal physiological sensor of environmental cold temperatures and is also involved in different pathophysiological conditions such as cold allodynia or dye eye disease. At the same time, activation of TRPM8-expressing fibers, for example by cold or menthol, has analgesic and antipruritic effects. The dual role of TRPM8 in pain transmission has led to a strong interest in the pursuit of novel modulators of TRPM8 channels. In this thesis, different aspects of TRPM8 channel modulation have been studied. First, I show that TRPM8 is a pharmacological target of two natural macrolide molecules; tacrolimus (FK506), a calcineurin inhibitor, and rapamycin (Sirolimus), an mTOR inhibitor. These two molecules share immunosuppressant properties and are widely used in the clinic, mainly for the treatment of organ rejection following transplants. Additionally, tacrolimus is also used in topical formulations for the treatment of atopic dermatitis and dry eye disease, and its use is often accompanied by adverse sensory side effects such as burning pain. I demonstrate by calcium imaging and patch clamp experiments that tacrolimus and rapamycin activate heterologously expressed TRPM8 channels in different species, including humans, and sensitize their response to cold temperatures by inducing a leftward shift in the voltage-dependent activation curve. The effect of tacrolimus on TRPM8 channels is direct and independent of the already known binding sites for other TRPM8 agonists (e.g. menthol or icilin).In cultured mouse DRG neurons, tacrolimus and rapamycin activate almost exclusively TRPM8-expressing cold-sensitive neurons, and these responses were drastically blunted in TRPM8 KO mice or after the application of TRPM8 antagonists. Patch-clamp recordings of cold-sensitive neurons confirm that both compounds activate inward currents and strongly potentiate the inward current evoked by cold. Behavioral experiments demonstrated that tacrolimus triggers eye blinking although this effect remains in TRPM8 KO mice, suggesting additional molecular targets. Second, the mechanisms involved in TRPM8 modulation by Gq Protein-Coupled Receptors (GqPCR) have been studied. This modulation could be important in the context of inflammation, considering that some GqPCRs for pro-inflammatory mediators (e.g. bradykinin type 2 or histamine H1 receptors) can modulate the excitability of peripheral sensory nerve terminals. TRPM8 channels are inhibited by GqPCRs activation; however, the molecular mechanisms underlying TRPM8 modulation remain unclear. Some studies support the modulation through the canonical signaling pathway, involving PLC activation and reduction in PI(4,5)P2 levels or PKC-mediated phosphorylation of the channel, while others point to a direct interaction of TRPM8 with the Gαq subunit.I studied the GqPCR-mediated inhibition of TRPM8 in heterologous expression systems and the mechanisms underlying this effect. Activation of GqPCRs modulates TRPM8 menthol- and cold-evoked currents in a fast and reversible manner, an effect that is dependent on temperature and voltage. By different strategies I confirmed that PI(4,5)P2 depletion is necessary for TRPM8 modulation by GqPCR. The selective depletion of PI(4,5)P2 by activation of a voltage sensitive lipid 5 phosphatase (Dr-VSP) is enough to recapitulate the effect of GqPCR activation on TRPM8 inward currents. Moreover, I demonstrated that PI(4,5)P2 hydrolysis shows precise temporal correlation with TRPM8 current inhibition in simultaneous patch-clamp and fluorescence-based PI(4,5)P2 level measurements. Importantly, pharmacological blockade of PLC and therefore PI(4,5)P2 hydrolysis prevents the inhibitory effect of GqPCRs activation on TRPM8 channels. In DRG cold-sensitive neurons, cold-evoked responses were not significantly affected by activation of GqPCRs. Altogether, these results identify TRPM8 channels in sensory neurons as molecular targets of the immunosuppressants tacrolimus and rapamycin, two clinically approved drugs. In addition, these findings contribute to the characterization of TRPM8 modulation by GqPCRs, and demonstrate that PLC-mediated PI(4,5)P2 depletion is a necessary event and underlies TRPM8 inhibition evoked by GqPCR activation.