EFFECT OF CYSTIC FIBROSIS-ASSOCIATED MUTATIONS IN THE 4TH INTRACELLULAR LOOP OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR

The cystic fibrosis transmembrane conductance regulator (CFTR) contain s multiple membrane spanning sequences that form a Cl- channel pore an d cytosolic domains that control the opening and closing of the channe l. The fourth intracellular loop (ICL4), which connects the tenth and eleventh transmembrane spans, has a primary sequence that is highly co nserved across species, is the site of a preserved sequence motif in t he ABC transporter family, and contains a relatively large number of m issense mutations associated with cystic fibrosis (CF). To investigate the role of ICL4 in CFTR function and to learn how CF mutations in th is region disrupt function, we studied several CF-associated ICL4 muta nts. We found that most ICL4 mutants disrupted the biosynthetic proces sing of CFTR, although not as severely as the most common Delta F508 m utation. The mutations had no discernible effect on the channel's pore properties; but some altered gating behavior, the response to increas ing concentrations of ATP, and stimulation in response to pyrophosphat e. These effects on activity were similar to those observed with mutat ions in the nucleotide-binding domains, suggesting that ICL4 might hel p couple activity of the nucleotide-binding domains to gating of the C l- channel pore. The data also explain how these mutations cause a los s of CFTR function and suggest that some patients with mutations in IC L4 may have a milder clinical phenotype because they retain partial ac tivity of CFTR at the cell membrane.