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

The regulatory domain of cystic fibrosis transmembrane conductance regulato r (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 a nd new serine to alanine mutations. We expressed these constructs in Fische r rat thyroid epithelia and measured transepithelial Cl- current. Mutation of four in vivo phosphorylation sites, Ser(660), Ser(737), Ser(795), and Se r(813) (S-Quad-A), substantially decreased cAMP-stimulated current, suggest ing that these four sites account for most of the phosphorylation-dependent response. Mutation of either Ser(660) or Ser(813) alone significantly decr eased current, indicating that these residues play a key role in phosphoryl ation-dependent stimulation. However, neither Ser(660) nor Ser(813) alone i ncreased current to wild-type levels; both residues were required. Changing Ser(737) to alanine increased current above wild-type levels, suggesting t hat phosphorylation of Ser(737) may inhibit current in wild-type CFTR. Thes e data help define the functional role of regulatory domain phosphoserines and suggest interactions between individual phosphoserines.