ATP hydrolysis by a CFTR domain: Pharmacology and effects of G551D mutation

Residues 417-830 of the cystic: fibrosis transmembrane conductance regulato r (CFTR) were expressed as a glutathione-S-transferase fusion protein. This fusion protein, NBD1/R/GST, contains the regulatory and first nucleotide b inding domains of CFTR. NBD1/R/GST hydrolyzed ATP with a K-M (60 mu M) and V-max (330 nmol/min/mg) that differed from those reported for CFTR and for a peptide containing CFTR residues 433-589. The ATPase inhibitor profile of NBD1/R/GST indicates that CFTR resembles P-glycoprotein with respect to th e NBD1 ATPase catalytic mechanism. ATP hydrolysis by NBD1/R/GST was unaffec ted by genistein, glybenclamide, and other agents known to affect CFTR's ch loride channel function, suggesting that these agents do not act by directl y influencing the ATPase function of NBD1. The disease-causing mutation, G5 51D, reduced ATP hydrolysis by NBD1/R/GST by increasing the K-M for ATP fou rfold. This suggests that when G551D occurs in patients with cystic fibrosi s, it affects CFTR function by reducing the affinity of NBD1 for ATP. (C) 2 000 Academic Press.