Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia

Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242 The regulatory domain of cystic fibrosis transmembrane conductance regulator (CFTR) regulates channel activity when several serines are phosphorylated by cAMP-dependent protein kinase. To further define the functional role of individual phosphoserines, we studied CFTR containing previously studied and new serine to alanine mutations. We expressed these constructs in Fischer rat thyroid epithelia and measured transepithelial Cl current. Mutation of four in vivo phosphorylation sites, Ser 660 , Ser 737 , Ser 795 , and Ser 813 (S-Quad-A), substantially decreased cAMP-stimulated current, suggesting that these four sites account for most of the phosphorylation-dependent response. Mutation of either Ser 660 or Ser 813 alone significantly decreased current, indicating that these residues play a key role in phosphorylation-dependent stimulation. However, neither Ser 660 nor Ser 813 alone increased current to wild-type levels; both residues were required. Changing Ser 737 to alanine increased current above wild-type levels, suggesting that phosphorylation of Ser 737 may inhibit current in wild-type CFTR. These data help define the functional role of regulatory domain phosphoserines and suggest interactions between individual phosphoserines. Cl channel; cystic fibrosis transmembrane conductance regulator; regulatory domain; cystic fibrosis