Cystic fibrosis transmembrane conductance regulator is required for protein kinase A activation of an outwardly rectified anion channel purified from bovine tracheal epithelia.
Our laboratory has developed a protocol for the isolation of a 140-kDa protein that forms an anion-selective channel when reconstituted into planar lipid bilayers. Polyclonal antibodies have been raised against the 38-kDa component of this purified protein. This channel has a linear current-voltage relationship and is not activated by protein kinase A (PKA) plus ATP. Using the same antibody and a modified purification protocol (eliminating the ion exchange chromatography steps), we isolated and reconstituted two other anion channels from tracheal membrane vesicles. In vitro phosphorylation of these isolated proteins by PKA and ATP revealed four bands migrating at 52, 85, 120, and 174 kDa. Immunoprecipitation experiments with anti-CFTR antibodies indicate that the 174-kDa phosphoprotein was CFTR. Upon incorporation of these isolated proteins into planar bilayers, an anion channel that exhibited a marked outward rectification in symmetrical Cl- solutions with a slope conductance of 82 pS at depolarizing voltages was observed. PKA and ATP increased channel activity but only from one side of the bilayer. However, channel activity was unaffected by addition of ATP alone from either side of the membrane. DIDS (100 microM) applied to the opposite side of the bilayer to which PKA and ATP act, blocked channel activity. A linear anion-selective channel with a conductance of 16 pS could be also resolved after inhibition of the outwardly rectified anion channel by DIDS in the presence of PKA and ATP. This small conductance channel was inhibited by 300 microM diphenylamine-2-carboxylic acid. Immunodepletion of the 174-kDa phosphoprotein from the preparation prevented activation of the 82-pS outwardly rectified anion channel by PKA and ATP. However, the PKA-dependent in vitro phosphorylation of the 52-, 85-, and 120-kDa phosphoproteins was unaffected by the absence of CFTR. Our results suggest a direct regulatory relationship between an outwardly rectified anion channel and CFTR.
Jovov
B
B
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294.
Ismailov
I I
II
Benos
D J
DJ
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D000906:Antibodies; D001345:Autoradiography; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000187:drug effects / Q000302:isolation & purification / Q000502:physiology*; D002852:Chromatography, Ion Exchange; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004848:Epithelium / Q000378:metabolism; D007700:Kinetics; D008051:Lipid Bilayers; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000502:physiology*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D014132:Trachea / Q000378:metabolism*
D000906:Antibodies; D018118:Chloride Channels; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010761:Phosphorus Radioisotopes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid