Expression and functional profile of TRPV1-exCellHalo. (A) Reconstructed model of the extracellular region on a TRPV1 subunit (PDB accession no. 7L2H). The loop between TM S1 and S2 used for ncAA insertion is shown in grey. Yellow termini demarcate the disordered region between TM segment 5 and the pore helix where circularly permutated HaloTag is inserted. (B) Ca²⁺ imaging experiment in HEK293T/17 cells comparing responses of wild-type TRPV1 against TRPV1-exCellHalo when activated by 500 nM capsaicin. Statistical comparison determined by Mann–Whitney U-test. (C) Dose response experiment of wild-type TRPV1 (n = 4; K_1/2 = 0.53 ± 0.28) and TRPV1-exCellHalo (n = 7; K_1/2 = 0.34 ± 0.19). These K_1/2 values are not significantly different as determined by Student’s two-tailed t test (P = 0.66). (D) Left: DIC image (10× magnification) of HEK293T/17 cells transfected with TRPV1-exCellHalo. Right: Fluorescence Ca²⁺-imaging experiment of TRPV1-exCellHalo expressing cells shown in the left panel with Fluo4 following activation with 500 nM capsaicin (cyan). After Ca²⁺ imaging experiment, surface labeling is accomplished with the incubation of HaloTag ligand Alexa660 (magenta) for 3 min. (E) Western blot of immune precipitation with anti-HaloTag antibody (Chemtek). Cell lysate is incubated with anti-HaloTag resin and the blot is probed with anti-GFP antibody. Two conditions tested are co-expression of TRPV1-exCellHalo/TRPV1-cGFP or TRPV1-cGFP alone (IN: input; FT: flow through; IP: immunoprecipitated). Source data are available for this figure: SourceData F4.