Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain.
Phosphorylation by cAMP-dependent protein kinase (PKA) regulates the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. We previously showed that in vivo PKA phosphorylated 4 serines (Ser-660, Ser-737, Ser-795, and Ser-813) within the R domain. Here we show that a mutant CFTR lacking all 4 serines can still be phosphorylated by PKA to yield an activated Cl- channel, but channel open-state probability was substantially reduced. We also observed phosphorylation and Cl- channel activity in another mutant lacking all 8 consensus PKA serines in the R domain. We were unable to identify the residual phosphorylation sites by tryptic phosphopeptide mapping. These data suggest two possible interpretations: (a) additional, as yet unidentified, phosphorylation sites within CFTR may also open the channel, or (b) the 4 serines, previously identified as in vivo PKA phosphorylation sites, are the primary regulatory sites within CFTR, but in their absence, other sites can be phosphorylated to open the channel. The additional sites are likely located within the R domain: CFTR delta R-S660A, which lacks much of the R domain (residues 708-835) and replaces Ser-660 with an alanine, was no longer regulated by PKA. Substitution of aspartate for consensus PKA phosphorylation sites in the R domain mimicked the effect of phosphorylation. Mutants containing six or more serine-to-aspartate substitutions generated Cl- channels that opened without PKA phosphorylation. These results suggest that the R domain keeps the channel closed and that phosphorylation of the R domain or insertion of the negatively charged aspartate opens the channel, perhaps by electrostatic interactions.
Rich
D P
DP
Howard Hughes Medical Institute, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242.
Berger
H A
HA
Cheng
S H
SH
Travis
S M
SM
Saxena
M
M
Smith
A E
AE
Welsh
M J
MJ
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D002460:Cell Line; D018118:Chloride Channels; D003550:Cystic Fibrosis / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006367:HeLa Cells; D006801:Humans; D015640:Ion Channel Gating; D007473:Ion Channels / Q000502:physiology*; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D008715:Methionine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012694:Serine; D013462:Sulfur Radioisotopes; D014162:Transfection
C505032:CFTR protein, human; D018118:Chloride Channels; D007473:Ion Channels; D008565:Membrane Proteins; D010748:Phosphopeptides; D013462:Sulfur Radioisotopes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D012694:Serine; D008715:Methionine; D011494:Protein Kinases; D000409:Alanine