8-azidoadenosine-5--triphosphate and Cystic-Fibrosis

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels appear to be regulated by hydrolysis of ATP and are inhibited by a product of hydrolysis, ADP. We assessed the effect of the other product of hydrolysis, inorganic phosphate (P(i)), on CFTR Cl- channel activity using the excised inside-out configuration of the patch-clamp technique. Millimolar concentrations of P(i) caused a dose-dependent stimulation of CFTR Cl- channel activity. Single-channel analysis demonstrated that the increase in macroscopic current was due to an increase in single-channel open-state probability (po) and not single-channel conductance. Kinetic modeling of the effect of P(i) using a linear three-state model indicated that the effect on po was predominantly the result of an increase in the rate at which the channel passed from the long closed state to the bursting state. P(i) also potentiated activity of channels studied in the presence of 10 mM ATP and stimulated Cl- currents in CFTR mutants lacking much of the R domain. Binding studies with a photoactivatable ATP analog indicated that Pi decreased the amount of bound nucleotide. These results suggest that P(i) increased CFTR Cl- channel activity by stimulating a rate-limiting step in channel opening that may occur by an interaction of P(i) at one or both nucleotide-binding domains.

Topics: 3T3 Cells; Adenosine Triphosphate; Animals; Azides; Biophysical Phenomena; Biophysics; Cell Line; Chloride Channels; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; HeLa Cells; Humans; In Vitro Techniques; Membrane Proteins; Mice; Models, Biological; Mutation; Phosphates; Sulfates; Transfection

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by cytosolic nucleotides and by cAMP-dependent phosphorylation. In excised membrane patches, CFTR Cl- channel activity requires hydrolyzable nucleotides and Mg2+, and is inhibited by ADP. We examined the interactions between CFTR and nucleotides using 8-azidoadenosine 5'-triphosphate (8-N3-ATP), a photoactivatable ATP analog. Because CFTR functions as a membrane ion channel, we studied CFTR in membranes of Sf9 insect cells. We found that [alpha-32P]8-N3ATP specifically photolabeled CFTR, with half-maximal labeling at 10 microM 8-N3ATP in the presence of Mg2+ and 100 microM in the absence of Mg2+. The 8-N3ATP also substituted for ATP in activating CFTR Cl- channels, indicating that it interacts with the active site(s). Both ATP and GTP prevented photolabeling with half-maximal inhibition at 1 mM. ADP and adenyl-5'-yl imidodiphosphate (AMP-PNP) prevented photolabeling but at much higher concentrations, whereas AMP did not inhibit photolabeling at concentrations of up to 100 mM. Phosphorylation of CFTR was not a prerequisite for nucleotide binding. These results demonstrate that CFTR interacts directly with nucleotides at concentrations that regulate CFTR Cl- channel activity.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Affinity Labels; Animals; Azides; Baculoviridae; Binding Sites; Cells, Cultured; Chloride Channels; Cloning, Molecular; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; Magnesium; Membrane Proteins; Moths; Phosphorylation; Protein Kinases

The majority of mutations which lead to clinical cystic fibrosis are located within the two predicted nucleotide binding domains of the cystic fibrosis gene product. We have used a prokaryotic expression system to synthesize and purify the first nucleotide binding domain (NBD-1, amino acids 426-588) with and without the most common mutation associated with the disease (the deletion of phenylalanine at position 508, delta F508). Both wild type and delta F508 NBD-1 bind ATP-agarose in a quantitatively comparable manner; this binding was inhibited by excess Na2ATP, trinitrophenol-ATP, or 8-azido-ATP. Irreversible NBD-1 labeling by an ATP analog was demonstrated using [32P]8-azido-ATP. This covalent labeling was inhibited by preincubation with Na2ATP, with half-maximal inhibition for Na2ATP occurring at approximately 5 mM for both the wild type and delta F508 nucleotide binding domain. These experiments are among the first to confirm the expectation that the cystic fibrosis transmembrane conductance regulator NBD-1 binds nucleotide. Since, under the conditions used in our study, NBD-1 without phenylalanine 508 displays very similar nucleotide binding characteristics to the wild type protein, our results support previous structural models which predict that the delta F508 mutation should not cause an alteration in ATP binding.

Topics: Adenosine Triphosphate; Affinity Labels; Azides; Base Sequence; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA; Electrophoresis, Polyacrylamide Gel; Humans; Membrane Proteins; Molecular Sequence Data; Mutation; Nucleotides; Phenylalanine