Differential interactions of nucleotides at the two nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator.
After phosphorylation by protein kinase A, gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by the interaction of ATP with its nucleotide binding domains (NBDs). Models of this gating regulation have proposed that ATP hydrolysis at NBD1 and NBD2 may drive channel opening and closing, respectively (reviewed in Nagel, G. (1999) Biochim. Biophys. Acta 1461, 263-274). However, as yet there has been little biochemical confirmation of the predictions of these models. We have employed photoaffinity labeling with 8-azido-ATP, which supports channel gating as effectively as ATP to evaluate interactions with each NBD in intact membrane-bound CFTR. Mutagenesis of Walker A lysine residues crucial for azido-ATP hydrolysis to generate the azido-ADP that is trapped by vanadate indicated a greater role of NBD1 than NBD2. Separation of the domains by limited trypsin digestion and enrichment by immunoprecipitation confirmed greater and more stable nucleotide trapping at NBD1. This asymmetry of the two domains in interactions with nucleotides was reflected most emphatically in the response to the nonhydrolyzable ATP analogue, 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP), which in the gating models was proposed to bind with high affinity to NBD2 causing inhibition of ATP hydrolysis there postulated to drive channel closing. Instead we found a strong competitive inhibition of nucleotide hydrolysis and trapping at NBD1 and a simultaneous enhancement at NBD2. This argues strongly that AMP-PNP does not inhibit ATP hydrolysis at NBD2 and thereby questions the relevance of hydrolysis at that domain to channel closing.
Aleksandrov
L
L
S. C. Johnson Medical Research Center, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259, USA.
Mengos
A
A
Chang
X
X
Aleksandrov
A
A
Riordan
J R
JR
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000493:pharmacokinetics*; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D001386:Azides / Q000493:pharmacokinetics*; D001665:Binding Sites; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019920:Photoaffinity Labels; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014357:Trypsin; D014638:Vanadates / Q000493:pharmacokinetics
D001386:Azides; C505032:CFTR protein, human; D019920:Photoaffinity Labels; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000266:Adenylyl Imidodiphosphate; D014638:Vanadates; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D008239:Lysine