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. Addition of genistein (100 μM) to the intracellular solution caused a small decrease in single‐channel current amplitude (i), but a large reduction in open probability (Po). Single‐channel analysis of channel block suggested that genistein (100 μM) may inhibit CFTR by two mechanisms: first, it may slow the rate of channel opening and second, it may block open channels. Acidification of the intracellular solution relieved channel block, suggesting that the anionic form of genistein may inhibit CFTR. Genistein inhibition of CFTR Cl− currents was weakly voltage dependent and unaffected by changes in the extracellular Cl− concentration. 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. ATP (5 mM) prevented the genistein‐induced decrease in Po, but was without effect on the genistein‐induced decrease in i. The genistein‐induced decrease in i was voltage dependent, whereas the genistein‐induced decrease in Po was voltage independent. 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.

To investigate the function of the murine cystic fibrosis transmembrane conductance regulator (CFTR), a full‐length cDNA encoding wild‐type murine CFTR was assembled and stably expressed in Chinese hamster ovary (CHO) cells. Like human CFTR, murine CFTR formed Cl− channels that were regulated by cAMP‐dependent phosphorylation and intracellular ATP. However, murine CFTR Cl− channels had a reduced single‐channel conductance and decreased open probability (Po) compared with those of human CFTR. Analysis of the dwell time distributions of single channels suggested that the reduced Po of murine CFTR was caused by both decreased residence in the open state and transitions to a new closed state, described by an intermediate closed time constant. For both human and murine CFTR, ATP and ADP regulated the rate of exit from the long‐lived closed state. 5′‐Adenylylimidodiphosphate (AMP‐PNP) and pyrophosphate, two compounds that disrupt cycles of ATP hydrolysis, stabilized the open state of human CFTR. However, neither agent locked murine CFTR Cl− channels open, although AMP‐PNP increased the Po of murine CFTR. The data indicate that although human and murine CFTR have many properties in common, some important differences in function are observed. These differences could be exploited in future studies to provide new understanding about CFTR.

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