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Expression and intracellular processing of chimeric and mutant CFTR molecules

par Pollet, Jean-François ;Van Geffel, Jacqueline ;Van Stevens, E.;Van Geffel, René ;Beauwens, Renaud ;Bollen, Alex ;Jacobs, Paul
Référence Biochimica et biophysica acta. Molecular basis of disease, 1500, 1, page (59-69)
Publication Publié, 2000-01

Article révisé par les pairs

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Résumé :

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride channel comprising two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs) and a unique regulatory (R) domain. The most frequent cystic fibrosis (CF) mutation, a deletion of Phe508 in NBD1, results in the retention of the ΔF508 CFTR in the endoplasmic reticulum, as do many other natural or constructed mutations located within the first NBD. In order to further define the role of NBD1 in CFTR folding and to determine whether the higher frequency of mutations in NBD1 with respect to NBD2 results from its position in the molecule or is related to its primary sequence, we constructed and expressed chimeric CFTRs wherein NBD domains were either exchanged or deleted. Synthesis, maturation and activity of the chimeras were assessed by Western blotting and iodide efflux assay after transient or stable expression in COS-1 or CHO cells respectively. The data showed that deletion of NBD1 prevented transport of CFTR to the cytoplasmic membrane whereas deletion of NBD2 did not impair this process but resulted in an inactive chloride channel. On the other hand, substituting or inverting NBDs in the CFTR molecule impaired its processing. In addition, while the NBD1 R555K mutation is known to partially correct the processing of CFTR ΔF508 and to increase activity of both wild-type and ΔF508 individual channels, it showed no positive effect when introduced into the double NBD1 chimera. Taken together, these observations suggest that the proper folding process of CFTR results from complex interactions between NBDs and their surrounding domains (MSDs and/or R domain). Copyright (C) 2000 Elsevier Science B.V.