Identification and partial characterization of a domain in CFTR that may bind cyclic nucleotides directly.
BACKGROUND
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is activated by cAMP-dependent phosphorylation. CFTR channel activity is also stimulated by cGMP-dependent protein kinase and protein kinase C.


RESULTS
Here, we show that CFTR channel activation by cGMP may also occur directly. In oocytes from one-third of Xenopus donors, the activation of CFTR by cGMP averaged 87% of the level achieved by cAMP. The currents activated by either cyclic nucleotide displayed similar current-voltage relationships, kinetics, pharmacology and halide selectivity. Sequential stimulation by cAMP and cGMP was not additive, suggesting that both cyclic nucleotides activate the same channel; cGMP was one order of magnitude more potent than cAMP, and its action was insensitive to protein kinase inhibitors. Analysis of the amino-acid sequence of CFTR revealed a domain in the amino-terminal portion of the third cytoplasmic loop that resembles a class of cyclic-nucleotide-binding domains related to that of the catabolite-gene activator protein, CAP. Two CFTR residues in this domain--Val397 and Lys420--were identified which, when changed to alanine, altered the response to cGMP independently of the response to cAMP.


CONCLUSIONS
We conclude that direct cyclic nucleotide binding may play a role in channel gating of CFTR. The cGMP-binding domain may provide a useful target for pharmacologic intervention in cystic fibrosis.
Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
S K
SK
Sullivan
L B
LB
Agellon
R
R
Schick
D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions; D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry; D006152:Cyclic GMP / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D003600:Cytosol; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D008969:Molecular Sequence Data; D009865:Oocytes; D011994:Recombinant Proteins; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D000838:Anions; C505032:CFTR protein, human; D002373:Cyclic AMP Receptor Protein; D007546:Isoquinolines; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C053177:N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide; D000242:Cyclic AMP; D006152:Cyclic GMP