Glibenclamide is well known to interact with the sulphonylurea receptor (SUR) and has been shown more recently to inhibit the cystic fibrosis transmembrane conductance regulator protein (CFTR), both proteins that are members of the ABC [adenosine 5'-triphosphate (ATP)-binding cassette] transporters. The effect of glibenclamide and two synthetic sulphonylcyanoguanidine derivatives (dubbed BM-208 and BM-223) was examined on P-glycoprotein, the major ABC transporter responsible for multidrug resistance (MDR) in cancer cells. To this end, we employed different cell lines that do or do not express P-glycoprotein, as confirmed by Western blotting: first, a tumour cell line (VBL600) selected from a human T-cell line (CEM) derived from an acute leukaemia; second, an epithelial cell line derived from a rat colonic adenocarcinoma (CC531(mdr+)) and finally, a non tumour epithelial cell line derived from the proximal tubule of the opossum kidney (OK). Glibenclamide and the two related derivatives inhibited P-glycoprotein because firstly, they acutely increased [3H]colchicine accumulation in P-glycoprotein-expressing cell lines only; secondly BM-223 reversed the MDR phenomenon, quite similarly to verapamil, by enhancing the cytotoxicity of colchicine, taxol and vinblastine and thirdly, BM-208 and BM-223 blocked the photoaffinity-labelling of P-glycoprotein by [3H]azidopine. Furthermore, glibenclamide is itself a substrate for P-glycoprotein, since the cellular accumulation of [3H]glibenclamide was low and substantially increased by addition of P-glycoprotein substrates (e. g., vinblastine and cyclosporine) only in the P-glycoprotein-expressing cell lines. We conclude that glibenclamide and two sulphonylcyanoguanidine derivatives inhibit P-glycoprotein and that sulphonylurea drugs would appear to be general inhibitors of ABC transporters, suggesting an interaction with some conserved motif.
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 DeltaF508 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 DeltaF508 and to increase activity of both wild-type and DeltaF508 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).
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