Epitope tagging permits cell surface detection of functional CFTR

Area of Science:

• Molecular Biology
• Cell Biology
• Biophysics

Background:

• The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial chloride channel involved in epithelial cell function.
• Accurate detection of CFTR localization is vital for understanding its role and developing therapies.

Purpose of the Study:

• To optimize detection methods for CFTR membrane localization studies.
• To develop a reliable reporter for CFTR trafficking to the cell surface.

Main Methods:

• CFTR was tagged with epitope sequences (M2 or HA) at the carboxy terminus or fourth external loop.
• N-linked glycosylation sites in the fourth loop were either preserved or mutated to create deglycosylated CFTR (dgCFTR).
• Tagged CFTRs were expressed in HeLa cells and Xenopus oocytes; cAMP-sensitive chloride permeability was assayed using SPQ fluorophore.

Main Results:

• M2-tagged CFTR constructs demonstrated cAMP-sensitive halide permeability, unlike HA-tagged CFTR.
• Deglycosylated CFTR (dgCFTR) showed reduced chloride conductance, while M2-tagged CFTRs retaining glycosylation sites responded like wild-type.
• External M2-tagged CFTR was successfully detected on the surface of nonpermeabilized cells, correlating with proper mutant processing.

Conclusions:

• The M2 epitope tag is effective for detecting CFTR localization at the cell surface.
• M2-901/CFTR serves as a valuable reporter for monitoring the trafficking of both wild-type and mutant CFTR proteins.
• Glycosylation plays a role in CFTR function and surface expression.