RESUMO
Treatment of cystic fibrosis relies so far on expensive and sophisticated drugs. A logical approach to rescuing the defective ΔF508-CFTR protein has not yet been published. Therefore, virtual docking of ATP and CFTR activators to the open conformation of the CFTR protein was performed. A new ATP binding site outside of the two known locations was identified. It was located in the cleft between the nucleotide binding domains NBD1 and NBD2 and comprised six basic amino acids in close proximity. Citrate and isocitrate were also bound to this site. Citrate was evaluated for its action on epithelial cells with intact CFTR and defective ΔF508-CFTR. It activated hyaluronan export from human breast carcinoma cells and iodide efflux, and recovered ΔF508-CFTR from premature intracellular degradation. In conclusion, citrate is an activator for ΔF508-CFTR and increases export by defective ΔF508-CFTR into the extracellular matrix of epithelial cells.
Assuntos
Ácido Cítrico , Regulador de Condutância Transmembrana em Fibrose Cística , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Isocitratos , Iodetos , Ácido Hialurônico , Nucleotídeos , Trifosfato de Adenosina , Aminoácidos BásicosRESUMO
Hyaluronan is synthesized within the cytoplasm and exported into the extracellular matrix through the cell membrane of fibroblasts by the MRP5 transporter. In order to meet the law of electroneutrality, a cation is required to neutralize the emerging negative hyaluronan charges. As we previously observed an inhibiting of hyaluronan export by inhibitors of K(+) channels, hyaluronan export was now analysed by simultaneously measuring membrane potential in the presence of drugs. This was done by both hyaluronan import into inside-out vesicles and by inhibition with antisense siRNA. Hyaluronan export from fibroblast was particularly inhibited by glibenclamide, ropivacain and BaCl(2) which all belong to ATP-sensitive inwardly-rectifying K(ir) channel inhibitors. Import of hyaluronan into vesicles was activated by 150 mM KCl and this activation was abolished by ATP. siRNA for the K(+) channels K(ir)3.4 and K(ir)6.2 inhibited hyaluronan export. Collectively, these results indicated that hyaluronan export depends on concurrent K(+) efflux.
