MOLECULAR MECHANISMS OF CFTR FUNCTION

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DESCRIPTION: This project seeks to understand the regulatory mechanisms which control the activity of the CFTR chloride channel. Interesting proposals of how the molecule may work have arisen in the past several years based on the imagined structure of the molecule and read-out of its function, i.e. electrogenic chloride permeation. However, there has been little testing of the molecular events underlying these models. CFTR is unique among ion channels in being controlled exclusively by events on the cytoplasmic side of the membrane rather than by binding of extracellular ligands or responses of voltage sensitive elements within the membrane to potential changes. Therefore, this study focuses its attention on the smaller cytoplasmic loops (CLs) as the interface between the membrane-spanning sequences which form the pore and the larger cytoplasmic domains that are the engines of the regulation. Reconstruction and expression of 30 disease-associated mutations in the four CLs revealed that they are involved in both gating and conductance of the channel. The latter effect is consistent with the possibility that some of them, e.g., CL3, may be very near the inner mouth of the pore. Because mutations in CLs primarily alter gating kinetics which are controlled by the nucleotide binding folds (NBFs), the investigators postulate that there are interactions between the NBFs and the CLs. Experimental evidence for such interactions has been obtained from yeast 2-hybrid analysis. Consistent with the notion that these interactions occur near the bilayer surface, they have found that sensitivity to NBF mutations which change gating kinetics is dependent on the cholesterol content and hence the state of the lipid bilayer. Realizing that 3-dimensional structure information will be required to further understand mechanism, the investigators have produced the protein in a high capacity yeast expression system. Hence, the goals of the current work are: 1) to elucidate the mechanism of control of CFTR by phosphorylation. The action of protein kinase A which is a prerequisite to the gating actions of the NBFs is still not understood, nor are the significant influences of protein kinase C and tyrosine phosphorylation: 2) to determine how the action of ATP at the NBFs controls CFTR channel gating; 3) to determine the role of the cytoplasmic loops in the regulation of CFTR channel gating by the major cytoplasmic domains; and 4) to express CFTR at high levels and purify it from yeast.