Constitutive phosphorylation as a key regulator of TRPM8 channel function

In mammals, environmental cold sensing conducted by peripheral cold thermoreceptor neurons mostly depends on TRPM8, an ion channel that has evolved to become the main molecular cold transducer. This TRP channel is activated by cold, cooling compounds, such as menthol, voltage, and rises in osmolality. TRPM8 function is regulated by kinase activity that phosphorylates the channel 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. By mass spectrometry, we identified four serine residues within the N-terminus (S26, S29, S541, and S542) constitutively phosphorylated in the mouse ortholog. TRPM8 function was examined by Ca2+ imaging and patch-clamp recordings, revealing that treatment with staurosporine, a kinase inhibitor, augmented its cold- and menthol-evoked responses. S29A mutation is sufficient to increase TRPM8 activity, suggesting that phosphorylation of this residue is a central molecular determinant of this negative regulation. Biophysical and total internal reflection fluorescence-based analysis revealed a dual mechanism in the potentiated responses of unphosphorylated TRPM8: a shift in the voltage activation curve toward more negative potentials and an increase in the number of active channels at the plasma membrane. Importantly, basal kinase activity negatively modulates TRPM8 function at cold thermoreceptors from male and female mice, 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.This work was supported by Grants DICYT VRIDeI-USACH 022143PP to M.P. and R.M.; FONDECYT 1161733 to R.M. and M.P.; FONDECYT 1181076 to P.O.; Centro Basal AC3E FB0008 to P.O.; VRIDeI-USACH AP-539MM to R.M.; National Research Foundation of Korea NRF-2019R1A2C2003642 to K.-S.P.; MINECO PID2019-108194RB and the Severo Ochoa Program for Centers of Excellence in R&D SEV-2017-0723 to F.V.; the Millennium Nucleus of Ion Channel-Associated Diseases (MiNICAD) to R.M. and M.P.; and the Millennium Nucleus for the Study of Pain (MiNuSPain) to R.M. and M.P. R.M. also thanks VRIDeI-USACH for postdoctoral support to B.R. (02183MM). B.R. (21161660) and B.L. (21181611) thank Comisión Nacional de Investigación Científica y Tecnológica de Chile PhD fellowships. MiNICAD is a Millennium Nucleus supported by the Millennium Science Initiative of the Ministry of Science, Technology, Knowledge and Innovation (Chile). MiNuSPain is a Millennium Nucleus supported by the Millennium Science Initiative of the Ministry of Science, Technology, Knowledge and Innovation (Chile). The CINV is a Millennium Science Institute funded by Agencia Nacional de Investigación y Desarrollo de Chile Grant ICN09_022 of the Ministry of Science, Technology, Knowledge and Innovation (Chile).Peer reviewe