The main molecular entity involved in innocuous cold detection in mammals is TRPM8. This polymodal TRP channel is activated by cold, cooling compounds such as menthol, voltage, and rises in osmolality. Basal kinase activity phosphorylates TRPM8 and modulates its function under resting conditions. However, which specific residues, how this post-translational modification modulates TRPM8 activity, and its influence on cold sensing are still poorly understood. We identified four serine residues within the N-terminal domain constitutively phosphorylated in the mouse ortholog by mass spectrometry. TRPM8 function was assessed by Ca2+-imaging and patch-clamp recordings, revealing that treatment with staurosporine, a kinase inhibitor, increased cold- and mentholevoked responses of the channel. S29A mutation is sufficient to enhance TRPM8 activity, suggesting that phosphorylation of this residue is a critical molecular determinant of this negative regulation. Biophysical and TIRF-based analysis revealed a dual mechanism in the potentiated responses of unphosphorylated TRPM8: an increase in the number of active channels at the plasma membrane and a shift in the voltage activation curve towards more negative potentials. Notably, basal kinase activity downregulates TRPM8 function at cold thermoreceptor neurons, an observation accounted for by mathematical modeling. Overall, our findings suggest that cold temperature detection could be rapidly and reversibly fine-tuned by controlling the TRPM8 basal phosphorylation state, a mechanism that acts as a dynamic molecular brake of this thermo-TRP channel function in primary sensory neurons.

Supported by Grants Millennium Nucleus for the Study of Pain (MiNuSPain) (RM, MP), Millennium Nucleus of Ion Channel-Associated Diseases (MiNICAD) (RM, MP), DICYT VRIDeI-USACH 022143PP (MP, RM) and by VRIDeI-USACH 021843MM (RM).

Resumen del trabajo presentado al VIII Congreso Red Española de Canales Iónicos, celebrado en Alicante del 24 al 27 de mayo de 2022.

Peer reviewed