A cellular pathway controlling functional plasma membrane incorporation of the cold sensor TRPM8

The transient receptor potential melastatin 8 (TRPM8) plays a crucial part in cold detection by the somatosensory system. In heterologous expression systems, TRPM8 activity steeply increases upon cooling and in the presence of substances that are known to produce a cooling sensation, including menthol, and the ‘super-cooling agent’ icilin. TRPM8-deficient mice exhibited a striking deficit in avoiding cool temperatures (18–30 C). Moreover, whereas mild cooling can evoke analgesia in wild-type mice, cooling-induced analgesia was absent in TRPM8-deficient mice. Importantly, increased functional expression of TRPM8 contributes to pathological cold hypersensitivity and cold allodynia in various animal models of neuropathic and inflammatory pain. In recent years, important advances have been made in our knowledge about the biophysical properties of TRPM8. However, the knowledge about the trafficking mechanism that determine the abundance of TRPM8 at the plasma membrane is very sparse. Nevertheless, modulation of the number of active cold sensitive TRPM8 channels at the plasma membrane represents an important regulatory mechanism under normal and pathophysiological conditions. In this article we discuss our recent findings published in the article ’VAMP7 regulates constitutive membrane incorporation of the cold-activated channel’ in which we have uncovered a cellular pathway that controls functional plasma membrane incorporation of TRPM8, and thus regulates thermo-sensitivity in vivo. By the use of Total internal reflection fluorescence (TIRF) microscopy, in which only a thin layer of illumination above the interface is created and only fluorophores within this thin layer (»100–300 nm) in the sample are excited, we revealed that fluorescently tagged TRPM8 channels are located in a population of highly dynamic vesicular and tubular structures. By treatment of TRPM8-mCherry expressing cells with microtubuleor actindepolymerizing agents and additional TIRF Recovery after Photobleaching (TIRF-FRAP) experiments, we were able to show that TRPM8-positive structures use microtubules as principal track for rapid near-membrane intracellular movement. Further characterization of the mobile TRPM8-positive structures was done by co-expression of TRPM8-mCherry along with known markers of various cellular compartments tagged with GFP, and quantified by dual-color TIRFM to simultaneously monitor the movement of TRPM8-mCherry along with GFP-tagged marker proteins. These results showed strong dynamic co-localization of TRPM8 and the Lysosomal associated membrane protein 1 (LAMP1), which was also observed in neurites of TGN co-expressing TRPM8-mCherry and LAMP1-GFP (Fig. 1A). Although LAMP1 is typically associated with endo-lysosomal structures, additional TIR-FRAP experiments indicated that TRPM8and LAMP1-positive mobile vesicles transport TRPM8 from the cell center toward the plasma membrane via microtubules. The pool of mobile TRPM8-positive vesicles is a stable compartment rather than a lysosomal structure targeted for degradation. This was further supported by the fact that the large majority of TRPM8– or LAMP1-positive structures observed in the near-membrane zone were not stained by lysotracker red or pHrodo red dextran, fluorescent dyes that selectively stain acidic lysosomal compartments, indicating that the luminal pH of these structures is higher than that of classical lysosomes (pH < 6). Further co-localization studies showed association between TRPM8 and the vesicular SNARE protein VAMP7. VAMP7 is present in LAMP1-positive structures and mediates their fusion with the plasma membrane. TIR-FRAP experiments showed dynamic co-localization of VAMP7-GFP and TRPM8-mCherry in mobile