The overall goal of this project is to understand at the molecular level the role of protein-protein interactions in the function of the CFTR Cl- channel. CFTR contains a large intracellular regulatory (R) domain with multiple consensus phosphorylation sites for PKA that are the basis for physiological regulation of the Cl- channel. A short negatively charged segment with in the R domain (NEG2, a.a. 817-838) has been implicated in the control of the Cl- channel function. This NEG2 region constitutes an amphipathic alpha-helical structure with a negatively charged face. Deletion of NEg2 from CFTR eliminates PKA dependence of the Cl- channel, and the exogenous NEG2 peptide exhibits both stimulatory and inhibitory effects on CFTR function. To elucidate the function of NEG2 in CFTR channel (Aim 1), the effects of mutating the NEG2 sequence on CFTR function will be assayed in a bilayer reconstitution system, and the interaction of NEG2 peptide with the nucleotide-binding folds (NBFs) of CFTR will be tested using an enzymatic assay. Combining the structural analysis with NMR, the experiments will attempt to identify the features in the NEG2 sequence that are responsible for activation and inhibition of the CFTR channel. Comprehensive genotype-phenotype studies have indicated possible contribution of protein-protein interactions to the severity of the cystic fibrosis disease, but little is known on the stoichiometry of CFTR as a Cl- channel. To test whether a monomer of CFTR is sufficient to produce a functional Cl- channel, or whether two CFTR molecules are required to form the full conductance state of the Cl- channel, tandem-linked CFTR heterodimers with distinct gating properties (e.g. G551D-wt) or distinct conduction properties (e.g. R334C-wt) will be expressed in HEK 293 cells, with a unique enzymatic cleavage site engineered in the linker sequence. Through analysis of the functional properties of the CFTR dimers before and after the enzymatic cleavage, the experiments will determine the contribution of individual CFTR molecules to the overall conduction and gating processes of the Cl- channel (Aim 2). If CFTR is capable of forming stable oligomers in the membrane, one expects to see suppression of Cl- transport activity by an inactive mutant co-expressed with the wild type CFTR (negative dominance) or restoration of Cl- transport activity by co-expression of two inactive mutants (functional complementation). This will be used to test the functional interaction between deltaF508 and the various mutants of CFTR (Aim #3). The experimental methods employed in this project include molecular cloning and expression of eukaryotic genes, structural and biochemical assays of recombinant proteins, confocal microscopic imaging of fluorescent-tagged molecules, and electrophysiological characterization of ion channel activities. Fulfillment of the experiments proposed in this project should provide further insights into the function and regulation of the CFTR Cl- channel, as well as the potential therapeutic interventiosn in cystic fibrosis.
