Antibody against a cystic fibrosis transmembrane conductance regulator-derived synthetic peptide inhibits anion currents in human colonic cell line T84.
The cystic fibrosis (CF) phenotype is characterized by a regulatory defect in Cl- permeability in epithelia. A gene (250,000 base pairs) that is associated with this autosomal genetic disorder has been identified. To determine the cellular function of the recently cloned gene product, the cystic fibrosis transmembrane conductance regulator (CFTR), we have produced antibody against a synthetic peptide deduced from the CFTR cDNA sequence corresponding to positions 505-511. This site includes phenylalanine 508, the deletion of which is the most commonly expressed mutation in CF. We sought to determine whether the anti-CFTR505-511 peptide antibody could modulate the activation of the volume-sensitive, Ca(2+)-dependent, as well as the cAMP-dependent Cl- conductances present in the Cl(-)-secreting human colonic T84 cell line. Affinity-purified anti-CFTR505-511 antibody was introduced into the cytoplasm of individual T84 cells and its function studied using the whole-cell patch-clamp technique. Although cAMP-dependent Cl- current activation was inhibited in cells perfused with the anti-CFTR505-511 peptide antibody, Ca(2+)-dependent anion current activation remained unaffected. Chloride current activation, which accompanies cellular swelling, was partially attenuated in anti-CFTR505-511 antibody-loaded cells as compared with control cells perfused with either saline or irrelevant antibody. These results further support a role for CFTR in anion transport in epithelial cells and suggest its possible involvement in a number of anion transport pathways in chloride secretory epithelia.
Chan
H C
HC
Department of Neurology, University of Chicago, Illinois 60637.
Kaetzel
M A
MA
Nelson
D J
DJ
Hazarika
P
P
Dedman
J R
JR
D000838:Anions; D000906:Antibodies / Q000276:immunology*; D001692:Biological Transport; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D003106:Colon / Q000166:cytology / Q000502:physiology*; D000242:Cyclic AMP / Q000378:metabolism; D003550:Cystic Fibrosis / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004058:Diffusion; D006801:Humans; D008565:Membrane Proteins / Q000276:immunology*; D009842:Oligopeptides / Q000276:immunology; D010446:Peptide Fragments; D010455:Peptides / Q000276:immunology*
D000838:Anions; D000906:Antibodies; C505032:CFTR protein, human; D002413:Cations, Divalent; D002712:Chlorides; D008565:Membrane Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C074197:cystic fibrosis transmembrane conductance regulator (505-511); D000242:Cyclic AMP; D002118:Calcium