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 regulat or (CFTR) protein as a chloride channel or transport regulator involves cel lular ATP binding and cleavage. Here we describe that human CFTR expressed in insect (Sf9) cell membranes shows specific, Mg2+-dependent nucleotide oc clusion, detected by covalent labeling with 8-azido-[alpha-P-32]ATP. Nucleo tide occlusion in CFTR requires incubation at 37 degrees C, and the occlude d nucleotide can not be removed by repeated washings of the membranes with cold MgATP-containing medium. By using limited tryptic digestion of the lab eled CFTR protein we found that the adenine nucleotide occlusion preferenti ally occurred in the N-terminal nucleotide binding domain (NBD). Addition o f 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 modific ation with N-ethylmaleimide prevented both vanadate-dependent and -independ ent nucleotide occlusion in CFTR, The pattern of nucleotide occlusion indic ates significant differences in the ATP hydrolyzing activities of the two N BDs, which may explain their different roles in the CFTR channel regulation .