GLIBENCLAMIDE BLOCKADE OF CFTR CHLORIDE CHANNELS

The cystic fibrosis transmembrane conductance regulator (CFTR) is a pr otein kinase A- and ATP-regulated Cl- channel located in the apical me mbranes of epithelial cells. Previously Sheppard and Welsh (J. Gen. Ph ysiol. 100: 573-591, 1992) showed that glibenclamide, a compound which binds to the sulfonylurea receptor and thus blocks nucleotide-depende nt 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 glibenclam ide caused a reversible reduction in current carried by CFTR which was paralleled by a decrease in channel open probability (P-o). The decre ase in P-o was concentration dependent, and half-maximum inhibition (K -i) occurred at 30 mu M. Fluctuation analysis indicated a flickery-typ e block of open CFTR channels. Event duration analysis supported this notion by showing that the glibenclamide-induced decrease in P-o was a ccompanied by interruptions of open bursts [i.e., an apparent reductio n in the burst duration (tau(burst))] With only a slight reduction in closed time (tau(c)). The plot of the corresponding open-to-closed (ta u(burst)(-1)) and closed-to-open (tau(c)(-1)) rates as a function of g libenclamide concentration were consistent with a pseudo-first-order o pen-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 dissocia tion 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-block ed 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.