PYROPHOSPHATE STIMULATES WILD-TYPE AND MUTANT CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR CL- CHANNELS

A unique feature of the cystic fibrosis transmembrane conductance regu lator (CFTR) Cl- channel is regulation by ATP through the two cytoplas mic nucleotide-binding domains (NBDs). To better understand this proce ss, we asked how channel activity is affected by inorganic pyrophospha te (PPi), a compound that binds to NBDs in other proteins. PPi and thr ee nonhydrolyzable PPi analogs reversibly stimulated the activity of p hosphorylated channels. Kinetic modeling of single channel data demons trated that PPi affected two distinct steps in channel regulation. Fir st, PPi increased the rate at which channels opened. Second, once chan nels were open, PPi delayed their closure. PPi could only stimulate ch annels when it was applied in the presence of ATP. PPi also increased the photolabeling of CFTR by an ATP analog. These two findings suggest that PPi modifies the activity of ATP dependent CFTR channel gating. Based on these and previous data, we speculate that the effects of PPi are mediated by binding of PPi, to NBD2 where it regulates channel op ening by NBD1, and then, because it is not hydrolyzed, it slows the ra te of NBD2-mediated channel closing. Because PPi stimulated wild-type channels, we tested its effect on CFTR containing the cystic fibrosis mutations: Delta F508, R117H, and G551S. PPi stimulated all three. PPi also stimulated endogenous CFTR in the apical membrane of permeabiliz ed T-84 epithelia. These results suggest that PP, or an analog might b e of value in the development of new approaches to the treatment of cy stic fibrosis.