DIBASIC PROTEIN-KINASE-A SITES REGULATE BURSTING RATE AND NUCLEOTIDE SENSITIVITY OF THE CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATORCHLORIDE CHANNEL

1. The relationship between phosphorylation of the cystic fibrosis tra nsmembrane conductance regulator (CFTR) chloride channel and its gatin g by nucleotides was examined using the patch clamp technique by compa ring strongly phosphorylated wild-type (WT) channels with weakly phosp horylated mutant channels lacking four (4SA) or all ten (10SA) dibasic consensus sequences for phosphorylation by protein kinase A (PKA). 2. The open probability (P-o) of strongly phosphorylated WT channels in excised patches was about twice that of 4SA and 10SA channels, after c orrecting for the number of functional channels per patch by addition of adenylylimidodiphosphate (AMP-PNP). The mean burst durations of WT and mutant channels were similar, and therefore the elevated P-o of WT was due to its higher bursting rate. 3. The ATP dependence of the 10S A mutant was shifted to higher nucleotide concentrations compared with WT channels. The relationship between P-o and [ATP] was noticeably si gmoid for 10SA channels (Hill coefficient, 1.8), consistent with posit ive co-operativity between two sites. Increasing ATP concentration to 10 mM caused the P-o of both WT and 10SA channels to decline. 4. Wild- type and mutant CFTR channels became locked in open bursts when expose d to mixtures of ATP and the non-hydrolysable analogue AMP-PNP. The ra te at which the low phosphorylation mutants became locked open was abo ut half that of WT channels, consistent with P-o being the principal d eterminant of locking rate in WT and mutant channels. 5. We conclude t hat phosphorylation at 'weak' PKA sites is sufficient to sustain the i nteractions between the ATP binding domains that mediate locking by AM P-PNP. Phosphorylation of the strong dibasic PKA sites controls the bu rsting rate and P-o of WT channels by increasing the apparent affinity of CFTR for ATP.