ATP hydrolysis by a CFTR domain: pharmacology and effects of G551D mutation.
Residues 417-830 of the cystic fibrosis transmembrane conductance regulator (CFTR) were expressed as a glutathione-S-transferase fusion protein. This fusion protein, NBD1/R/GST, contains the regulatory and first nucleotide binding domains of CFTR. NBD1/R/GST hydrolyzed ATP with a K(M) (60 microM) 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 the NBD1 ATPase catalytic mechanism. ATP hydrolysis by NBD1/R/GST was unaffected by genistein, glybenclamide, and other agents known to affect CFTR's chloride channel function, suggesting that these agents do not act by directly influencing the ATPase function of NBD1. The disease-causing mutation, G551D, reduced ATP hydrolysis by NBD1/R/GST by increasing the K(M) for ATP fourfold. This suggests that when G551D occurs in patients with cystic fibrosis, it affects CFTR function by reducing the affinity of NBD1 for ATP.
Howell
L D
LD
Department of Medicine, Duke University and VA Medical Centers, Durham, North Carolina, 27710-3378, USA.
Borchardt
R
R
Cohn
J A
JA
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D002384:Catalysis; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D044948:Flavonols; D005982:Glutathione Transferase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D010957:Plasmids; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology; D014970:Xanthines / Q000494:pharmacology
D005419:Flavonoids; D044948:Flavonols; D011993:Recombinant Fusion Proteins; D014970:Xanthines; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D014638:Vanadates; D000255:Adenosine Triphosphate; C051360:1,3-dipropyl-8-cyclopentylxanthine; D005982:Glutathione Transferase; D000251:Adenosine Triphosphatases