Prolonged treatment of cells with genistein modulates the expression and function of the cystic fibrosis transmembrane conductance regulator.
OBJECTIVE
Cystic fibrosis (CF) is caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. In the search for new CF therapies, small molecules have been identified that rescue the defective channel gating of CF mutants (termed CFTR potentiators). Here, we investigate the long-term effects of genistein, the best-studied CFTR potentiator, on the expression and function of CFTR.


METHODS
We pre-treated baby hamster kidney (BHK) cells expressing wild-type or F508del-CFTR (the most common CF mutant) with concentrations of genistein that potentiate (30 microM) or inhibit (100 microM) CFTR function for 2 or 24 h at 37 degrees C before examining CFTR maturation, expression and single-channel activity.


RESULTS
Using the iodide efflux technique, we found that genistein pre-treatment failed to restore function to F508del-CFTR, but altered that of wild-type CFTR. Pre-treatment of cells with genistein for 2 h had little effect on CFTR processing, whereas pre-treatment for 24 h either augmented (30 microM genistein) or impaired (100 microM genistein) CFTR maturation. Using immunocytochemistry, we found that all genistein pre-treatments increased the localization of CFTR protein to the cell surface. However, following the incubation of cells with genistein (100 microM) for 2 h, individual CFTR Cl(-) channels exhibited characteristics of channel block upon channel activation.


CONCLUSIONS
Genistein pre-treatment alters the maturation, cell surface expression and single-channel function of CFTR in ways distinct from its acute effects. Thus, CFTR potentiators have the potential to influence CFTR by mechanisms distinct from their effects on channel gating.
Schmidt
A
A
Centre of Human Genetics, National Institute of Health, Lisboa, Portugal.
Hughes
L K
LK
Cai
Z
Z
Mendes
F
F
Li
H
H
Sheppard
D N
DN
Amaral
M D
MD
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D019833:Genistein / Q000008:administration & dosage / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry; D007454:Iodides / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects; D007668:Kidney; D021381:Protein Transport / Q000187:drug effects; D013997:Time Factors
D004791:Enzyme Inhibitors; D007454:Iodides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D019833:Genistein