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 lin ear current-voltage relationship and is not activated by protein kinas e A (PKA) plus ATP. Using the same antibody and a modified purificatio n protocol (eliminating the ion exchange chromatography steps), we iso lated and reconstituted two other anion channels from tracheal membran e vesicles. In vitro phosphorylation of these isolated proteins by PKA and ATP revealed four bands migrating at 52, 85, 120, and 174 kDa. Im munoprecipitation experiments with anti-CFTR antibodies indicate that the 174-kDa phosphoprotein was CFTR. Upon incorporation of these isola ted proteins into planar bilayers, an anion channel that exhibited a m arked outward rectification in symmetrical Cl- solutions with a slope conductance of 82 pS at depolarizing voltages was observed, PKA and AT P increased channel activity but only from one side of the bilayer. Ho wever, channel activity was unaffected by addition of ATP alone from e ither side of the membrane, DIDS (100 mu M) applied to the opposite si de 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 channe l by DIDS in the presence of PKA and ATP. This small conductance chann el was inhibited by 300 mu M diphenylamine 2-carboxylic acid. Immunode pletion of the 174-kDa phosphoprotein from the preparation prevented a ctivation of the 82-pS outwardly rectified anion channel by PKA and AT P. However, the PKA-dependent in vitro phosphorylation of the 52-, 85- , and 120-kDa phosphoproteins was unaffected by the absence of CFTR. O ur results suggest a direct regulatory relationship between an outward ly rectified anion channel and CFTR.