To learn the mechanisms of function and regulation of the Cystic Fibrosis (CF) Transmembrane conductance Regulator (CFTR) channel, we will use electrophysiology and protein biochemistry (and mass spectrometry) to examine its function at the molecular level, and molecular biology and crystallography to manipulate and analyze its structure, for correlation with these functional measurements. The goal is to examine the precise mechanisms by which specific kinases and phosphatases act on the regulatory (R) domain of wild-type an mutant CFTR to regulate the function of its nucleotide binding folds (NBFs) which, in turn, effect the conformational changes that control ion flow through the channel pore. We will build on our recent findings that different phosphorylation sites on CFTR, susceptible to attack by distinct phosphatases, independently regulate the ability of the two NBFs to bind and hydrolyze ATP. A particularly labile phosphorylation site appears to control the length of time the channel stays open, for example, so that identifying, and pharmacologically targeting., the specific phosphatase that regulates that site ought to permit the """"""""rescue"""""""" of diseased cells with inadequate ion flow due to expression of mutant CFTR channels; this includes those mutants that fail to reach the cell surface in sufficient numbers, those that have a diminished single-channel conductance, and those that are not open for a large enough fraction of time. There are two specific aims. The first addresses the questions: How do the NBFs function, and what are the mechanisms of interactions between the two NBFs? The working hypothesis is that the two NBFs are similar, in that they both hydrolyze ATP, but t hey differ in function and mechanism one becoming """"""""active"""""""" upon nucleotide hydrolysis, the other requiring only nucleotide binding likely reflecting documented differences in primary sequence and, hence, three-dimensional structure. The second addresses the questions; How does phosphorylation of the R domain (and at which site or sites) control the function of NBF1 to permit channel opening? How does phosphorylaiton of an additional labile site (or sites)? If ATP hydrolysis at NBF1 causes channel opening, but ATP hydrolysis at NBF2 prompts channel closing, then the mechanism underlying any alteration of the open probability of a channel bearing a CF-associated mutation will be ambiguous unless the opening and closing rates of individual channels are monitored. That is what we will measure, for wild-type and mutant channels.
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