PHOSPHORYLATION BY CAMP-DEPENDENT PROTEIN-KINASE CAUSES A CONFORMATIONAL CHANGE IN THE R-DOMAIN OF THE CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR

Individuals with cystic fibrosis have a defect in the CFTR protein, a chloride channel regulated by CAMP-dependent protein kinase (PKA). The majority of the phosphorylation sites of PKA are located in the R dom ain of CFTR. It has been postulated that this domain may act as a gate for the chloride channel. Of the many possible mechanisms whereby the R domain could gate the channel, including interdomain interactions, charge distribution, or conformational change, we investigated the pos sibility that phosphorylation leads to conformational changes in the R domain. To test this hypothesis, a protocol for purification of human R domain peptide synthesized in a bacterial expression system was dev eloped. Purified R domain was phosphorylated by PKA, and CD spectra we re obtained. As a result of phosphorylation by PKA, a significant spec tral change, indicative of a reduction in the alpha-helical content, w as found. CD spectra of the R domain of a shark homologue of CFTR indi cated similar changes in conformation as a result of phosphorylation b y PKA. In contrast, phosphorylation of the human R domain by PKC, whic h has only a small influence on CFTR channel activity, failed to elici t CD spectral changes, indicating no conformational change comparable to those induced by PKA phosphorylation. These observations provide th e first structural characterization of the R domain and suggest that t he gating of the CFTR chloride channel by PKA may involve a conformati onal change in the R domain.