Shrinkage activates a nonselective conductance: involvement of a Walker-motif protein and PKC.
The ability of all cells to maintain their volume during an osmotic challenge is dependent on the regulated movement of salt and water across the plasma membrane. We demonstrate the phosphorylation-dependent gating of a nonselective conductance in Caco-2 cells during cellular shrinkage. Intracellular application of exogenous purified rat brain protein kinase C (PKC) resulted in the activation of a current similar to that activated during shrinkage with a Na(+)-to-Cl- permeability ratio of approximately 1.7:1. To prevent possible PKC- and/or shrinkage-dependent activation of cystic fibrosis transmembrane regulator (CFTR), which is expressed at high levels in Caco-2 cells, a functional anti-peptide antibody, anti-CFTR505-511, was introduced into the cells via the patch pipette. Anti-CFTR505-511, which is directed against the Walker motif in the first nucleotide binding fold of CFTR, prevented the PKC/shrink-age current activation. The peptide CFTR505-511 also induced current inhibition, suggesting the possible involvement of a regulatory element in close proximity to the channel that shares sequence homology with the first nucleotide binding fold of CFTR and whose binding to the channel is required for channel gating.
Nelson
D J
DJ
Department of Medicine, University of Chicago, Illinois 60637, USA.
Tien
X Y
XY
Xie
W
W
Brasitus
T A
TA
Kaetzel
M A
MA
Dedman
J R
JR
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002468:Cell Physiological Phenomena; D003550:Cystic Fibrosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000276:immunology / Q000502:physiology*; D004553:Electric Conductivity; D005796:Genes; D006801:Humans; D010446:Peptide Fragments / Q000276:immunology / Q000502:physiology*; D017510:Protein Folding; D011493:Protein Kinase C / Q000494:pharmacology / Q000502:physiology*; D051381:Rats; D014162:Transfection; D014407:Tumor Cells, Cultured; D014882:Water-Electrolyte Balance
D000906:Antibodies; C505032:CFTR protein, human; D010446:Peptide Fragments; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011493:Protein Kinase C