Journey of a cold sensor – VAMP7-dependent transport of TRPM8

TRPM8, a member of the transient receptor potential (TRP) superfamily of cation channels, is expressed in a subset of somatosensory neurons, where it plays a central role in thermosensation, as the principal sensor of innocuous cold temperatures and cooling agents such as menthol or icilin. Pharmacological inhibition or genetic deletion of TRPM8 causes strong deficits in the sensation of innocuously cold temperatures, whereas hyperactivation causes pathological cold hypersensitivity. The (patho)physiological functions of TRPM8 depend on the total ionic current through active channels in the plasma membrane, which in turn is governed by its gating mechanisms, ion permeability and the total number of channels at the site of action. Earlier work has greatly advanced our knowledge about the gating and ion permeability properties of TRPM8, including its regulation by cellular signaling pathways. However, very little insight was available about the intracellular trafficking mechanism of TRPM8 to the plasma membrane (PM), as is true for most other TRP channels. Nevertheless, modulation of the number of active TRP channels at the cell surface represents an important regulatory mechanism under normal and pathophysiological conditions. Earlier studies had provided evidence that TRPM8 may reside in lipid rafts at the PM, indicated that TRPM8 may exhibit various modes of near membrane trafficking, and suggested that channel stimulation may stimulate incorporation of additional channels at the PM via exocytosis. However, in these earlier studies, information regarding cellular structures and molecular determinants that governed the intracellular trafficking of TRPM8 to the cell surface were not elucidated. In the article “VAMP7 regulates constitutive membrane incorporation of the cold-activated channel TRPM8” we have elucidated important aspects of the pathway carrying TRPM8 channels to the PM. In the first part of the work, we used Total-Internal-Reflection-Fluorescence–microscopy (TIRFM) to observe near membrane events in live cells. Using this technique, we showed that fluorescently tagged TRPM8 resides in very dynamic vesicles (speed 0.58 mm/s), which exhibit constitutive movement, mainly via microtubules. However, we were unable to measure an increased TRPM8 trafficking to the PM following agonist treatment, in contrast to a previous study. Next, we characterized the nature of these TRPM8-transporting vesicles, by determining which of a list of known markers of intracellular compartments are co-transported within these vesicles. To achieve this, we used dual color TIRFM to simultaneously monitor the movement of TRPM8-mCherry along with GFP-tagged marker proteins. Thus, TRPM8 was considered to dynamically colocalize with specific marker proteins when we observed a vesicular structure exhibiting both GFP and mCherry fluorescence moving alongside. Using this approach, we observed that TRPM8 is primarily found in LAMP1and Rab7carrying vesicles. Although Rab7 and LAMP1 are classically known to as marker of endo-lysosomes, we observed that the TRPM8-containing vesicles were non-acidic in nature, and did not function as protein breakdown organelles.