Fig. 6. GTTR uptake by TRPV4 is dependent on M680 residue within the channel pore. (A,B) KPT2-derived cell lines that express the M680D TRPV4 mutant were generated and tested for GTTR uptake. Immunofluorescence confirmed that the M680D TRPV4 mutant had normal membrane localization in the cells.(C,D) Immunoblotting showed similar protein expression levels of TRPV4 wild-type and the M680D mutant in KPT2-TRPV4 and the two KPT2-M680D clones (M680D#1 and #2), respectively. After 30 seconds of GTTR treatment, there was no significant enhancement in GTTR uptake by extracellular Ca²⁺ removal in KPT2-M680D cells, and similar results were obtained for the two KPT2-M680D cell lines (D). Fluorescence intensity data are shown as mean ± s.d. ^**P<0.01. Scale bar, 20 µm.