Regulation of TRPM8 channel activity by Src‐mediated tyrosine phosphorylation

• Published in: Journal of cellular physiology 2020-06, Vol.235 (6), p.5192-5203
• Main Authors: Manolache, Alexandra, Selescu, Tudor, Maier, G. Larisa, Mentel, Mihaela, Ionescu, Aura Elena, Neacsu, Cristian, Babes, Alexandru, Szedlacsek, Stefan Eugen
• Format: Article
• Language: English
• Subjects: Activation; Animals; Biological Transport - genetics; Cancer; channel activity; Channel gating; Cold; Cold Temperature; cultured rat dorsal root ganglion neurons; Dorsal root ganglia; Enzyme inhibitors; Ganglia, Spinal - metabolism; Ganglia, Spinal - pathology; HEK293 Cells; Humans; Inflammation; Inflammation - drug therapy; Inflammation - genetics; Inflammation - pathology; Inhibitors; Intracellular signalling; Kinases; Metastases; Modulation; Neoplasms - drug therapy; Neoplasms - genetics; Neoplasms - pathology; Neurons; Neurons, Afferent - metabolism; Neurons, Afferent - pathology; Pain - drug therapy; Pain - genetics; Pervanadate; Phosphorylation; Phosphorylation - genetics; Protein Kinase Inhibitors - pharmacology; Protein-tyrosine-phosphatase; Pyrimidines - pharmacology; Rats; RNA-mediated interference; Sensory neurons; Signaling; Src protein; src-Family Kinases - antagonists & inhibitors; src-Family Kinases - genetics; Src‐mediated tyrosine phosphorylation; Transient receptor potential proteins; TRPM Cation Channels - genetics; TRPM8; Tumors; Tyrosine; Tyrosine - metabolism
• ISSN: 0021-9541; 1097-4652
• DOI: 10.1002/jcp.29397

The transient receptor potential melastatin type 8 (TRPM8) receptor channel is expressed in primary afferent neurons where it is the main transducer of innocuous cold temperatures and also in a variety of tumors, where it is involved in progression and metastasis. Modulation of this channel by intracellular signaling pathways has therefore important clinical implications. We investigated the modulation of recombinant and natively expressed TRPM8 by the Src kinase, which is known to be involved in cancer pathophysiology and inflammation. Human TRPM8 expressed in HEK293T cells is constitutively tyrosine phosphorylated by Src which is expressed either heterologously or endogenously. Src action on TRPM8 potentiates its activity, as treatment with PP2, a selective Src kinase inhibitor, reduces both TRPM8 tyrosine phosphorylation and cold‐induced channel activation. RNA interference directed against the Src kinase diminished the extent of PP2‐induced functional downregulation of TRPM8, confirming that PP2 acts mainly through Src inhibition. Finally, the effect of PP2 on TRPM8 cold activation was reproduced in cultured rat dorsal root ganglion neurons, and this action was antagonized by the protein tyrosine phosphatase inhibitor pervanadate, confirming that TRPM8 activity is sensitive to the cellular balance between tyrosine kinases and phosphatases. This positive modulation of TRPM8 by Src kinase may be relevant for inflammatory pain and cancer signaling. Transient receptor potential melastatin type 8 (TRPM8) receptor channel is the main transducer of innocuous cold temperatures and is also involved in cancer progression and metastasis. Here we investigate the modulation of TRPM8 by the Src protein tyrosine kinase (PTK), known to be involved in cancer pathophysiology and inflammation. hTRPM8 expressed in HEK293T cells is constitutively tyrosine phosphorylated by heterologously and endogenously expressed Src. Phosphorylation potentiates TRPM8 activity, as treatment with PP2, a selective Src inhibitor, reduces both TRPM8 tyrosine phosphorylation and cold‐induced channel activation. Src RNA interference diminished the PP2‐induced downregulation of TRPM8, confirming that PP2 acts mainly through Src inhibition. Finally, the effect of PP2 on TRPM8 cold activation was reproduced in cultured dorsal root ganglion (DRG) neurons, and this action was antagonized by the protein tyrosine phosphatase (PTP) inhibitor pervanadate, confirming that TRPM8 activity is sensitive to b