(Taken directly from the application) The long-term goal of this research is to understand the function and regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) as a chloride channel. Phosphorylation by protein kinase A of serine residues in the intracellular regulatory (R) domain is required for opening of the CFTR channel. It is believed that the R domain acts as a putative gating particle, serving to close the channel when not phosphorylated, but undergoing a conformational change with phosphorylation, thus removing the block to facilitate chloride ion flow. To search for the minimum gating particle for the CFTR channel (Aim 1), the study will use a reconstitution system of native CFTR or it-domain deleted CFTR captured in the lipid bilayer membrane, to test various peptides from the R domain sequence for their ability to promote channel closure. Using the phosphorylation-dependent interaction between the exogenous R domain protein and the CFTR channel, the experiments will test whether the exogenous R domain protein bind to the CFTR channel in the open or the closed state. The intracellular loops (ICLs) joining the transmembrane segments of CFTR are highly hydrophilic (with positive and negative charged amino acids), which might display intramolecular interactions with other charged portions of the CFTR molecule (R and NBDs), or intermolecular interactions with adjacent CFTR molecules, thus forming part of the binding site(s) for the R domain to control opening of the chloride channel. These ICLs are important for the processing of CFTR, as removal of part of the first (delta exon5) and second (delta 19) ICLs leads to expression of CFTR proteins which are mostly core-glycosylated and retained in the endoplasmic reticulum (ER) membranes. To determine the contribution of intracellular loops to CFTR function (Aim 2), plasma and ER membrane vesicles will be isolated from HEK 293 cells transfected with delta exon5 and delta 19 CFTR cDNAs, and point-mutations in the ICLs, to study the CFTR channel in both processed and unprocessed form using the bilayer reconstitution system. Through elucidating the mechanisms of how R domain regulates the CFTR channel, and how the intracellular loops interact with other hydrophilic domains (R and NBDs) of CFTR, the proposed research will arrive at a better understanding of the molecular mechanism of the opening and closing of the CFTR channel, information which can be, applied to studies of activation of the mutant CFTR channels for therapeutic purposes. In addition, the studies may lead to engineering of CFTR channels with higher activity, which will be useful for high efficiency gene therapy for CF.
