PHOSPHATE STIMULATES CFIR CL- CHANNELS

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel s appear to be regulated by hydrolysis of ATP and are inhibited by a p roduct of hydrolysis, ADP. We assessed the effect of the other product of hydrolysis, inorganic phosphate (P-i), on CFTR Cl- channel activit y using the excised inside-out configuration of the patch-clamp techni que. Millimolar concentrations of P-i caused a dose-dependent stimulat ion of CFTR Cl- channel activity. Single-channel analysis demonstrated that the increase in macroscopic current was due to an increase in si ngle-channel open-state probability (p(o)) and not single-channel cond uctance. Kinetic modeling of the effect of P-i using a linear three-st ate model indicated that the effect on p(o) was predominantly the resu lt of an increase in the rate at which the channel passed from the lon g closed state to the bursting state. P-i also potentiated activity of channels studied in the presence of 10 mM ATP and stimulated Cl- curr ents in CFTR mutants lacking much of the R domain. Binding studies wit h a photoactivatable ATP analog indicated that P-i decreased the amoun t of bound nucleotide. These results suggest that P-i increased CFTR C l- channel activity by stimulating a rate-limiting step in channel ope ning that may occur by an interaction of P-i at one or both nucleotide -binding domains.