Biochemical and biophysical identification of cystic fibrosis transmembrane conductance regulator chloride channels as components of endocytic clathrin-coated vesicles.
Cystic fibrosis results from mutations in the gene encoding the CFTR Cl- channel. Although CFTR occurs as an integral component of the plasma membrane, recent studies implicate CFTR in endocytic recycling and suggest that the protein may also exist in intracellular vesicular compartments. To test this, we analyzed CFTR in clathrin-coated vesicles (CCV) purified from cells constitutively expressing CFTR at high levels. CFTR immunoreactivity was detected in CCV by immunoblot and was identified as CFTR based on labeling of immunoprecipitates with protein kinase A and by tryptic phosphopeptide mapping. Fusion of uncoated CCV with planar lipid bilayers resulted in the incorporation of kinase- and ATP-activated Cl- channel activity (7.8 pS at 20 degrees C; 11.9 pS at 37 degrees C), with a linear current-voltage relation under symmetrical conditions. Thus, functional CFTR occurs in CCV. Moreover, CFTR interacts with the plasma membrane specific adaptor complex during endocytosis through clathrin-coated pits. Therefore, the abundance of CFTR in the plasma membrane may be regulated by exocytic insertion and endocytic recycling, and these processes may provide an augmentation to protein kinase A activation as a mechanism for regulating CFTR Cl channels in the plasma membrane.
Bradbury
N A
NA
Department of Physiology & Biophysics, University of Alabama at Birmingham 35294.
Cohn
J A
JA
Venglarik
C J
CJ
Bridges
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D018118:Chloride Channels / Q000032:analysis / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D003106:Colon / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004705:Endocytosis; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D051381:Rats
C505032:CFTR protein, human; D018118:Chloride Channels; D002966:Clathrin; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017868:Cyclic AMP-Dependent Protein Kinases