The premise that the CF gene product, CFTR, is a key protein in cAMP-mediated Cl secretion has been verified by both complementation and expression studies. Furthermore, in collaboration with Dr. Jack Riordan's laboratory we have demonstrated in reconstitution studies that the purified CFTR is a kinase activated Cl channel. The general goals of this proposal are to understand the mechanism(s) of activation of the normal wild-type CFTR (wtCFTR) channel protein and how the common mutations found in CF cause impaired regulation of mutant forms of CFTR. Specifically: in our first Specific Aim we will determine whether wild-type CFTR protein and the F-delta508 mutant protein are competent activatable Cl channels at various stages of biosynthesis. Membrane vesicle fractions enriched from plasma membrane, golgi and endoplasmic reticulum from cells expressing wtCFTR and F-delta508CFTR will be used in planar lipid bilayer studies to test the effects of protein kinase A (PKA) and ATP activation on single channel kinetics. In our second Specific Aim we will test the hypothesis that nucleotides activate directly wtCFTR and determine whether this mode of activation is altered in F-delta508CFTR. The direct effects of ATP will be tested on PKA/ATP activated channels. Various nucleotide derivatives will be used to probe the nucleotide binding folds (NBF) of CFTR and their contribution to channel activation. In our third Specific Aim, we will investigate several classes of pharmacologically active agents that are known to alter the function of other nucleotide binding proteins for their effects on CFTR channel activity. Single channel kinetic studies will be performed to identify those compounds that compete for ATP binding at the NBF's as well as those binding to sites outside the NBF's. The results of Specific Aims 2 and 3 will be combined using molecular modelling techniques to construct a quantitative structure activity relationship (QSAR). This QSAR will provide a rational basis for the design of pharmacologic agents that will be effective in restoring mutant CFTR to a normal functional state.
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