Lateral mobility of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and its potential interactions with other proteins were studied by engineering a biotinylation target sequence into an extracellular loop and stably expressing the tagged construct in baby hamster kidney (BHK) cells. The target sequence was specifically biotinylated on intact cells by adding recombinant biotin ligase to the bath solution. Inserting the sequence had little effect on CFTR protein expression according to Western blots, and robust channel activation by cAMP was demonstrated by iodide efflux and patch clamp assays. Confocal microscopy of cells exposed to fluorophore-conjugated streptavidin revealed strong plasma membrane staining of cells expressing biotinylatable wild-type CFTR, but not cells expressing a tagged version of the disease-associated mutant ΔF508. Lateral mobility of CFTR was studied by measuring fluorescence recovery after photobleaching (FRAP) and by image correlation spectroscopy (ICS). FRAP recovery curves were consistent with a randomly diffusing population of CFTR channels with an immobile population of ~20%, and yielded an effective diffusion coefficient D = 1.4 ± 0.6 x 10-10 cm2 s-1 based on simulated diffusive recovery into a strip bleach. By contrast, ICS under the same conditions indicated a diffusion coefficient of 3.8 ± 1.7 x 10-11 cm2 s-1 (mean ± S.E.M., n = 20 cells); i.e. approximately four-fold slower. Computer simulations revealed that FRAP would generate faster diffusion coefficients than ICS when there is a confined, slowly moving population. To investigate possible protein-protein interactions that might account for the immobile pool, tagged CFTR was co-expressed with clathrin-GFP or glycosylphosphoinositol (GPI)-GFP, and visualized by confocal microscopy after biotinylation and binding of fluorophore-conjugated streptavidin. CFTR on the cell surface was partially co-localized with clathrin-GFP but not with GPI-GFP, suggesting that clathrin coated pits, but not lipid rafts, may contribute to the immobile population of CFTR channels.
University of Bristol (2005) J Physiol 567P, PC167
Poster Communications: Lateral diffusion of CFTR on the cell surface measured using an engineered biotinylation target sequence
Bates, Ian R.; Luo, Yishan; Liao, Jie; Bachir, Alexia; Kolin, David L.; Hebert, Benedict; Wiseman, Paul W.; Hanrahan, John W.;
1. Physiology, McGill University, Montreal, QC, Canada. 2. Chemistry, McGill University, Montreal, QC, Canada. 3. Physics, McGill University, Montreal, QC, Canada. 4. Chemistry and Physics, McGill University, Montreal, QC, Canada.
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