5'-ADENYLYLIMIDODIPHOSPHATE DOES NOT ACTIVATE CFTR CHLORIDE CHANNELS IN CELL-FREE PATCHES OF MEMBRANE

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- cha nnel requires both phosphorylation of its R domain and the presence of nucleoside triphosphates for activation. Our previous work suggested that hydrolysis of nucleoside triphosphates may be required to support channel activity. However, recent studies have suggested that the non hydrolyzable adenosine triphosphate analogue, 5'-adenylylimidodiphosph ate (AMP-PNP), may support some Cl- channel activity in sweat gland du ct epithelia in the presence of low ATP concentration and in Cl- chann els associated with expression of the P-glycoprotein multidrug resista nce transporter. To examine the effect of AMP-PNP, we applied it to th e cytosolic surface of phosphorylated CFTR Cl- channels contained in e xcised, cell-free patches of membrane. We found that preparations of 1 0 mM AMP-PNP opened phosphorylated CFTR Cl- channels. However, this ef fect was due to contaminating ATP: high-pressure liquid chromatography analysis of AMP-PNP demonstrated that 10 mM AMP-PNP could contain up to 50 muM ATP, which could account for the observed stimulation of CFT R Cl- channel activity. When contaminating ATP was hydrolyzed with hex okinase, AMP-PNP was unable to support CFTR channel activity. AMP-PNP (10 mM) also failed to attenuate or potentiate the current induced by 0.3 mM ATP. These results suggest that AMP-PNP has no direct effect on CFTR Cl- channels.