Characterization of a CFTR construct with a C‐terminal tetracysteine sequence and its use in the visualization of trafficking pathways

Abstract

Membrane permeable bi‐arsenical dyes, such as fluorescein arsenical hairpin (FlAsH), bind tetracysteine‐tagged‐proteins emitting fluorescence in‐vivo. This fluorescence method (developed by R. Tsien) provides a tool to evaluate trafficking pathways in living cells. We generated a tetracysteine‐tagged Cystic Fibrosis Transmembrane Regulator (CFTR) protein and studied its expression with the long‐term goal of using this protein to study its trafficking during endocytosis. The tetracysteine‐tagged CFTR (CFTR‐TC) was predominantly expressed in both BHK and in Cos‐7 cells as the immature core‐glycosylated form (band B) rather than the mature glycosylated band C. Iodide efflux assays of CFTR‐TC's function as a chloride channel at the cell surface revealed an insignificant response after cAMP stimulation, likely reflecting its low band C expression. Interestingly, 1 hour FlAsH pre‐treatment evoked a mean response difference of −16.0 mV versus untreated (one‐way ANOVA p<0.05), suggesting increased band C expression. Our results suggest that FlAsH rescues the surface expression of CFTR‐TC and studies are ongoing to visualize its localization and the regulation of its trafficking by proteins involved in the endocytic pathway. The work is supported by the Natural Sciences and Engineering Research Council (postgraduate scholarship) and the Canadian Cystic Fibrosis Foundation BREATHE Program.