Constitutive Phosphorylation as a Key Regulator of TRPM8 Channel Function

Abstract

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 Ca 21 imaging and patchclamp 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.

Validation of six commercial antibodies for detection of heterologous and endogenous TRPM8 ion channel expression

“…Only cells that responded to 50 µM Carbachol, a compound that activates endogenous muscarinic receptors in HEK-293 cells [29], were included in the analysis. [30][31][32], both for the EYFP and antibody signal, further confirming antibody specificity (Figure S1). No TRPM8 immunostaining was found in neighboring EYFP-(i.e.…”

Control of TRPM3 Ion Channels by Protein Kinase CK2-Mediated Phosphorylation in Pancreatic β-Cells of the Line INS-1

“…For TRPM4 channels, the Ca 2+ sensitivity has been shown to be regulated by protein kinase C–dependent phosphorylation at S 1152 and S 1145 within the C-terminus of the protein [ 32 ] and that casein kinase 1 (CK1)–mediated phosphorylation of S 839 is responsible for the basolateral localization of this channel in polarized epithelial cells [ 33 ]. Very recently, constitutive phosphorylation of four C-terminal serine residues has been identified as a key regulator of TRPM8 channels [ 34 ]. TRPM3 channels, however, have never been described as targets of phosphorylation.…”

Mutations of TRPM8 channels: Unraveling the molecular basis of activation by cold and ligands

“…This behavior could be attributed to a leftward shift of the voltage dependence of activation, thus, leading to a higher channel opening probability at physiological membrane potentials. In a recent study, the same authors identified S26 as a phosphorylation site, together with S29, S541, and S542, and showed that there is a TRPM8 downregulation by basal phosphorylation 64 . Point mutations to Ala in each of these Ser, showed that S29 is a key residue for TRPM8 modulation, as only the S29A mutation led to a strong potentiation of the response to cold and menthol.…”

“…Based on previous studies on S26 and S27 it was argued that this mutation might influence the stability of the TRPM8 N ‐terminus. The identification of the key role of S29 led to the hypothesis that this R30Q might prevent S29 phosphorylation or the effect of this posttranscriptional modification 64 …”

“…The identification of the key role of S29 led to the hypothesis that this R30Q might prevent S29 phosphorylation or the effect of this posttranscriptional modification. 64 The N-terminus was also explored by Morgan et al 66 that analyzed the effect of nonconserved substitutions located within conserved regions, namely TRPM8 rare variants G150R, K423N, R475C, R485W, and the experimental mutant Y516E. The rare variants were selected from the human genome SNP database, and are rather infrequent in human population.…”