Nucleotide occlusion in the human cystic fibrosis transmembrane conductance regulator. Different patterns in the two nucleotide binding domains.
The function of the human cystic fibrosis transmembrane conductance regulator (CFTR) protein as a chloride channel or transport regulator involves cellular ATP binding and cleavage. Here we describe that human CFTR expressed in insect (Sf9) cell membranes shows specific, Mg2+-dependent nucleotide occlusion, detected by covalent labeling with 8-azido-[alpha-32P]ATP. Nucleotide occlusion in CFTR requires incubation at 37 degrees C, and the occluded nucleotide can not be removed by repeated washings of the membranes with cold MgATP-containing medium. By using limited tryptic digestion of the labeled CFTR protein we found that the adenine nucleotide occlusion preferentially occurred in the N-terminal nucleotide binding domain (NBD). Addition of the ATPase inhibitor vanadate, which stabilizes an open state of the CFTR chloride channel, produced an increased nucleotide occlusion and resulted in the labeling of both the N-terminal and C-terminal NBDs. Protein modification with N-ethylmaleimide prevented both vanadate-dependent and -independent nucleotide occlusion in CFTR. The pattern of nucleotide occlusion indicates significant differences in the ATP hydrolyzing activities of the two NBDs, which may explain their different roles in the CFTR channel regulation.
Szabó
K
K
National Institute of Haematology and Immunology, Membrane Research Group of the Hungarian Academy of Sciences, Hungarian Academy of Sciences, H-1113 Budapest, Hungary.
Szakács
G
G
Hegeds
T
T
Sarkadi
B
B
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; C505032:CFTR protein, human; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate