Glibenclamide blockade of CFTR chloride channels

The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A- and ATP-regulated Cl- channel located in the apical membranes of epithelial cells. Previously Sheppard and Welsh (J. Gen. Physiol. 100: 573-591, 1992) showed that glibenclamide, a compound which binds to the sulfonylurea receptor and thus blocks nucleotide-dependent K+ channels, reduced CFTR whole cell current. The aim of this study was to identify the mechanism underlying this inhibition in cell-free membrane patches containing CFTR Cl- channels. Exposure to glibenclamide caused a reversible reduction in current carried by CFTR which was paralleled by a decrease in channel open probability (P-o). The decrease in P-o was concentration dependent, and half-maximum inhibition (K-i) occurred at 30 mu M. Fluctuation analysis indicated a flickery-type block of open CFTR channels. Event duration analysis supported this notion by showing that the glibenclamide-induced decrease in P-o was accompanied by interruptions of open bursts [i.e., an apparent reduction in the burst duration (tau(burst))] With only a slight reduction in closed time (tau(c)). The plot of the corresponding open-to-closed (tau(burst)(-1)) and closed-to-open (tau(c)(-1)) rates as a function of glibenclamide concentration were consistent with a pseudo-first-order open-blocked mechanism and provided estimates of the on rate (k(on) = 1.17 mu M(-1)s(-1)), the off rate (k(off) = 16 s(-1)), and the dissociation constant (K-d = 14 mu M). The difference between the K-i (30 mu M) and the K-d (14 mu M) is the result expected for a closed-open-blocked model with an initial P-o of 0.47. Since the initial P-o was 0.50 +/- 0.02 (n = 12), we can conclude that glibenclamide blocks CFTR by a closed-open-blocked mechanism.