The broad goal of this renewal proposal is to define the basic principles of CFTR channel gating and how the most common CF mutations disrupt this process. Like other ABC transporters, CFTR has two nucleotide binding domains (NBDs) that bind ATP in pockets at the interface of an NBD dimer. CFTR also has a unique regulatory domain (R domain) that inhibits channel opening unless phosphorylated by protein kinase A (PKA). Outstanding issues include the degree of coupling between ATP binding and channel opening and how R domain phosphorylation stimulates channel activity. During the current funding period we made several significant discoveries that shed light on the CFTR gating mechanism;notably;(i) certain point mutations in the cytosolic loops that connect the NBDs to the pore promote constitutive (ATP-independent) channel opening;(ii) these mutations rescue the defective gating of a common CF mutant channel (G551D);(iii) CFTR gating is well described by an allosteric mechanism in which ATP binding shifts the equilibrium between pre-existing closed and open states and (iv) the R domain regulates channel activity independent of either ATP binding or NBD dimerization. These findings set the stage for the next phase of our project in which we will pursue three specific aims.
Aim 1 : Test a model in which the cytosolic loops function as a compression spring that strongly resists unliganded CFTR channel opening, and determine whether our CFTR findings can be generalized to a related ABC transporter (yeast YOR1 exporter).
Aim 2 : Test predictions of an allosteric gating model that address the link between channel opening and nucleotide occupancy.
Aim 3 : Define the link between R domain phosphorylation and CFTR channel gating. This project should significantly improve our understanding of the basic principles of CFTR channel gating, and may lead to new approaches for treating CFTR-related diseases.
Mutations in the CFTR chloride channel cause cystic fibrosis (CF), one of the most common genetic diseases in the U.S. The main goals of this project are to define how this channel operates normally and how CF-causing mutations disrupt its operation. The resulting information may provide better clues as to how to correct defective CFTR channel function in CF patients.
|Wang, Wei; Hong, Jeong S; Rab, Andras et al. (2016) Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators. PLoS One 11:e0152232|
|Wei, Shipeng; Roessler, Bryan C; Icyuz, Mert et al. (2016) Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels. FASEB J 30:1247-62|
|Wei, Shipeng; Roessler, Bryan C; Chauvet, Sylvain et al. (2014) Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps. J Biol Chem 289:19942-57|
|Wang, Wei; Roessler, Bryan C; Kirk, Kevin L (2014) An electrostatic interaction at the tetrahelix bundle promotes phosphorylation-dependent cystic fibrosis transmembrane conductance regulator (CFTR) channel opening. J Biol Chem 289:30364-78|
|Hwang, Tzyh-Chang; Kirk, Kevin L (2013) The CFTR ion channel: gating, regulation, and anion permeation. Cold Spring Harb Perspect Med 3:a009498|
|Kirk, Kevin L (2013) Being positive: revisiting the elevated sodium permeability hypothesis in cystic fibrosis. J Physiol 591:3675-6|
|Okeyo, George; Wang, Wei; Wei, Shipeng et al. (2013) Converting nonhydrolyzable nucleotides to strong cystic fibrosis transmembrane conductance regulator (CFTR) agonists by gain of function (GOF) mutations. J Biol Chem 288:17122-33|
|Wang, Wei; Okeyo, George O; Tao, Binli et al. (2011) Thermally unstable gating of the most common cystic fibrosis mutant channel (?F508): ""rescue"" by suppressor mutations in nucleotide binding domain 1 and by constitutive mutations in the cytosolic loops. J Biol Chem 286:41937-48|
|Bernard, Karen; Kirk, Kevin L (2010) Cross-linking of ?F508-CFTR promotes its trafficking to the plasma membrane. Channels (Austin) 4:251-4|
|Bernard, Karen; Wang, Wei; Narlawar, Rajeshwar et al. (2009) Curcumin cross-links cystic fibrosis transmembrane conductance regulator (CFTR) polypeptides and potentiates CFTR channel activity by distinct mechanisms. J Biol Chem 284:30754-65|
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