HaloTag-based approach to quantify subcellular localization of TRPV3 channels.

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 directly activate TRPV3 channels to conduct cations through their pore. In addition, interactions between TRPV3 and the transmembrane protein TMEM79 influence channel trafficking rather than pore gating. 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 in knowledge, we successfully fused a cpHaloTag to the extracellular face of the mouse TRPV3 channel while retaining its sensitivity to voltage, 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 HaloTag dyes. Using this tool and confocal microscopy, we detect robust changes in TRPV3 channel localization on the cell surface of live and fixed cells caused by truncation of an N-terminal portion of the channel or by its co-expression with TMEM79. Notably, we show that these changes in TRPV3 channel subcellular localization can be robustly quantified by epifluorescence microscopy at low magnification and flow cytometry, making our tool ideal for high-throughput screening applications. Furthermore, we co-express Halo-TRPV3 channels together with a fluorescent Ca