INTERACTION BETWEEN CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR AND OUTWARDLY RECTIFIED CHLORIDE CHANNELS

We have previously described a protocol for the simultaneous isolation and reconstitution of a protein kinase A (PKA)-sensitive outwardly re ctified chloride channel (ORCC) and the cystic fibrosis transmembrane conductance regulator (CFTR) from bovine tracheal epithelium, immunopr ecipitation of CFTR from this preparation prevented PKA activation of the ORCC, suggesting that CFTR regulated the ORCC and that this regula tory relationship was preserved throughout the purification procedure. We now report the purification of CFTR from bovine tracheal epithelia and the purification of a CFTR conduction mutant (G551D CFTR) from re trovirally transduced mouse L cells using a combination of alkali stri pping, Triton-X extraction, and immunoaffinity chromatography. Immunop urified CFTR proteins were reconstituted in the absence and presence o f ORCC. To test the hypothesis that only functional CFTR can support a ctivation of ORCC by PKA and ATP, we used an inhibitory anti-CFTR(505- 511) peptide antibody or G551D CFTR. When anti-CFTR(505-511) peptide a ntibodies were present prior to the addition of PKA and ATP, activatio n of both the ORCC and CFTR was prevented. If the antibody was added a fter activation of the ORCC and CFTR Cl- channels by PRA and ATP, only the CFTR Cl- channel was inhibited. When ORCC and G551D CFTR were co- incorporated into planar bilayers, only the ORCC was recorded and this channel could not be further activated by the addition of PRA and ATP . Thus, functional CFTR is required for activation of the ORCC by PKA and ATP. me also tested the hypothesis that PKA activation of ORCC was dependent-on the extracellular presence of ATP. We added ATP on the p resumed extracellular side of the lipid bilayer under conditions where it was not possible to activate the ORCC, i.e. in the presence of inh ibitory anti-CFTR(505-511) antibody or G551D CFTR. In both cases the O RCC regained PRA sensitivity, Moreover, the addition of hexokinase + g lucose to the extracellular side prevented activation of the ORCCs by PKA and ATP in the presence of CFTR. These experiments confirm that bo th the presence of CFTR as well as the presence of ATP on the extracel lular side is required for activation of the ORCC by PKA and ATP.