The TRPV3 channel is crucial for skin barrier formation and hair growth, and its dysregulation is linked to itch, atopic dermatitis, rosacea, and genetic disorders like Olmsted syndrome. Heat and voltage can directly activate TRPV3 channels to conduct cations through their pore. In addition, direct interactions between TRPV3 and the transmembrane protein TMEM79 influence channel trafficking rather than pore opening. Otherwise, little is known about how the TRPV3 channel is endogenously controlled. Importantly, we lack the experimental tools to reliably quantify TRPV3 channel cell surface expression under diverse experimental conditions, including live cells. To address this gap, we successfully fused a cpHalo-tag to the extracellular face of the mouse TRPV3 channel while retaining its sensitivity to heat and agonists. We show that we can differentially detect surface-expressed and intracellularly localized channels in HEK293 cells by sequentially labeling with spectrally separable membrane-permeable and impermeable Halo-tag dyes. Using this tool, we detect robust changes in TRPV3 channel localization on the cell surface caused by truncation of a portion of the channel or by its co-expression with TMEM79. Notably, we show that similar results are obtained with epifluorescence or fluorescence-activated cell sorting as can be obtained by using confocal microscopy, making our tool ideal for high-throughput screening applications. Furthermore, we co-express Halo-TRPV3 channels together with a fluorescent Ca2+-reporter and show that this system can be used to assess pore gating in response to activators. Finally, we show that it is possible to detect channel fluorescence after denaturing gel electrophoresis using a cell sample size that is compatible with medium-throughput biochemistry. This work thus establishes a flexible and highly sensitive new tool to elucidate the molecular and cellular mechanisms of TRPV3 channel function.
Holloway et al. (Sun,) studied this question.