Dibasic phosphorylation sites in the R domain of CFTR have stimulatory and inhibitory effects on channel activation.
To better understand the mechanisms by which PKA-dependent phosphorylation regulates CFTR channel activity, we have assayed open probabilities (P(o)), mean open time, and mean closed time for a series of CFTR constructs with mutations at PKA phosphorylation sites in the regulatory (R) domain. Forskolin-stimulated channel activity was recorded in cell-attached and inside-out excised patches from transiently transfected Chinese hamster ovary cells. Wild-type CFTR and constructs with a single Ser-to-Ala mutation as well as octa (Ser-to-Ala mutations at 8 sites) and constructs with one or two Ala-to-Ser mutations were studied. In cell-attached patches, Ser-to-Ala mutations at amino acids 700, 795, and 813 decreased P(o), whereas Ser-to-Ala mutations at 737 and 768 increased P(o). In general, differences in P(o) were due to differences in mean closed time. For selected constructs with either high or low values of P(o), channel activity was measured in excised patches. With 1 mM ATP, P(o) was similar to that observed in cell-attached patches, but with 10 mM ATP, all constructs tested showed elevated P(o) values. ATP-dependent increases in P(o) were due to reductions in mean closed time. These results indicate that R-domain phosphorylation affects ATP binding and not the subsequent steps of hydrolysis and channel opening. A model was developed whereby R-domain phosphorylation, in a site-dependent manner, alters equilibrium between forms of CFTR with low and high affinities for ATP.
Vais
Horia
H
Division of Medical Genetics, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Zhang
Rugang
R
Reenstra
William W
WW
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000502:physiology*; D004594:Electrophysiology; D004789:Enzyme Activation / Q000502:physiology*; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*
D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases