Abstract
TRPM8 is the main molecular entity responsible for cold sensing. This polymodal ion channel is activated by cold, cooling compounds such as menthol, voltage, and rises in osmolality. In corneal cold thermoreceptor neurons (CTNs), TRPM8 expression determines not only their sensitivity to cold, but also their role as neural detectors of ocular surface wetness. Several reports suggest that Protein Kinase C (PKC) activation impacts on TRPM8 function; however, the molecular bases of this functional modulation are still poorly understood. We explored PKC-dependent regulation of TRPM8 using Phorbol 12-Myristate 13-Acetate to activate this kinase. Consistently, recombinant TRPM8 channels, cultured trigeminal neurons, and free nerve endings of corneal CTNs revealed a robust reduction of TRPM8-dependent responses under PKC activation. In corneal CTNs, PKC activation decreased ongoing activity, a key parameter in the role of TRPM8-expressing neurons as humidity detectors, and also the maximal cold-evoked response, which were validated by mathematical modeling. Biophysical analysis indicated that PKC-dependent downregulation of TRPM8 is mainly due to a decreased maximal conductance value, and complementary noise analysis revealed a reduced number of functional channels at the cell surface, providing important clues to understanding the molecular mechanisms of how PKC activity modulates TRPM8 channels in CTNs.
Highlights
Cold transduction occurs at the free nerve endings of cold thermoreceptor neurons (CTNs), a subpopulation of primary somatosensory neurons of the dorsal root ganglia (DRG) and trigeminal ganglia (TG) that innervate the skin and exposed mucosae
We provide evidence that the Protein Kinase C (PKC) activation by phorbol esters reduces the Transient Receptor Potential Melastatin 8 (TRPM8) response to cold and menthol, mainly due to a decreased expression of functional channels at the plasma membrane
Our functional evaluation of corneal nerve endings of CTNs shows that their electrical activity can be reduced by pharmacological activation of PKC, suggesting that this form of modulation should not be overlooked in studies assessing physiological and physiopathological scenarios, such as those triggered by a local inflammatory process
Summary
Cold transduction occurs at the free nerve endings of cold thermoreceptor neurons (CTNs), a subpopulation of primary somatosensory neurons of the dorsal root ganglia (DRG) and trigeminal ganglia (TG) that innervate the skin and exposed mucosae (reviewed by [1,2,3,4]). TRPM8 is diversely regulated in both physiological and pathological conditions by interacting proteins [24,25], splice variants [26], post-translational modifications [27,28,29,30,31], modulatory regions [32] and G protein-coupled receptor signaling cascades [33,34,35,36,37,38] The latter is relevant during tissue injury and inflammation, where released inflammatory mediators enhance thermal-evoked pain sensation. Most of these receptors, including bradykinin receptor 2 (BR2), are Gαq-coupled receptors that lead to the subsequent activation of phospholipase C (PLC) and PKC
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