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Approximately 61,000 statements.
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1. The isoflavone genistein may either stimulate or inhibit cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. To investigate how genistein inhibits CFTR, we studied CFTR Cl- channels in excised inside-out membrane patches from cells expressing wild-type human CFTR. 2. Addition of genistein (100 microM) to the intracellular solution caused a small decrease in single-channel current amplitude (i), but a large reduction in open probability (Po). 3. Single-channel analysis of channel block suggested that genistein (100 microM) may inhibit CFTR by two mechanisms: first, it may slow the rate of channel opening and second, it may block open channels. 4. Acidification of the intracellular solution relieved channel block, suggesting that the anionic form of genistein may inhibit CFTR. 5. Genistein inhibition of CFTR Cl- currents was weakly voltage dependent and unaffected by changes in the extracellular Cl- concentration. 6. Channel block was relieved by pyrophosphate (5 mM) and ATP (5 mM), two agents that interact with the nucleotide-binding domains (NBDs) of CFTR to greatly stimulate channel activity. 7. ATP (5 mM) prevented the genistein-induced decrease in Po, but was without effect on the genistein-induced decrease in i. 8. The genistein-induced decrease in i was voltage dependent, whereas the genistein-induced decrease in Po was voltage independent. 9. The data suggest that genistein may inhibit CFTR by two mechanisms. First, it may interact with NBD1 to potently inhibit channel opening. Second, it may bind within the CFTR pore to weakly block Cl- permeation.
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2014-07-03T14:29:50.737+02:00
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