Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent chloride channel. The deltaF508 mutation accounts for approximately 70 percent of CF mutations and at least one copy is present in 90 percent of CF patients. The deltaF508 mutation causes increased degradation of CFTR in the endoplasmic reticulum. A significant portion of the mutant protein reaches to the plasma membrane, but the open probability (Po) of the mutant channel in response to cAMP is much lower than that of wild-type (wt) CFTR. We have demonstrated that the isoflavone genistein can increase the cAMP-dependent Po of deltaF508-CFTR by approximately 20-fold. This increase in cAMP-dependent activation suggests that genistein may be of therapeutic benefit in the treatment of CF. The molecular basis for the effects of genistein and the differences in cAMP activation of wt- and deltaF508-CFTR are poorly understood. Which step(s) of the cAMP-dependent CFTR activation differs for wt- and deltaF508-CFTR? What cellular machinery is involved? What is the molecular basis for this functional defect? What are the structural changes that account for the defect? What is a rational strategy to circumvent this defect? To address these questions, we will use a combination of approaches that incorporate biochemical, electrophysiological, pharmacological and molecular biological methodologies. These will include site-directed mutagenesis, cell-attached, excised inside-out and whole-cell configurations of the patch-clamp technique, whole-cell capacitance measurement, antisense knockout technology and phospho-peptide mapping of in vivo phosphorylated CFTR. A clear picture of CFTR regulation and CF pathogenesis at a cellular and molecular level is expected to emerge from this multidisciplinary approach. By using pharmacological agents, purified kinases and purified phosphatases, regulation of wild-type and deltaF508-CFTR via phosphorylation/dephosphorylation will be investigated. Overexpression and antisense knockout of phosphatase 2C will be used to study the physiological and pathophysiological role of this phosphatase. Combining phospho-peptide mapping and site-directed mutagenesis of phosphorylated serines, we will identify roles for individual phosphorylation sites and their pathophysiological significance in CF. A clearer understanding of the molecular mechanism for the functional defect of deltaF508-CFTR will aid in the design of pharmacological agents, such as genistein for therapeutic intervention in CF.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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General Medicine B Study Section (GMB)
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Mckeon, Catherine T
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University of Missouri-Columbia
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Hwang, Tzyh-Chang; Yeh, Jiunn-Tyng; Zhang, Jingyao et al. (2018) Structural mechanisms of CFTR function and dysfunction. J Gen Physiol 150:539-570
Zhang, Jingyao; Hwang, Tzyh-Chang (2017) Electrostatic tuning of the pre- and post-hydrolytic open states in CFTR. J Gen Physiol 149:355-372
Yeh, Han-I; Sohma, Yoshiro; Conrath, Katja et al. (2017) A common mechanism for CFTR potentiators. J Gen Physiol 149:1105-1118
Jih, Kang-Yang; Lin, Wen-Ying; Sohma, Yoshiro et al. (2017) CFTR potentiators: from bench to bedside. Curr Opin Pharmacol 34:98-104
Gao, Xiaolong; Hwang, Tzyh-Chang (2016) Spatial positioning of CFTR's pore-lining residues affirms an asymmetrical contribution of transmembrane segments to the anion permeation pathway. J Gen Physiol 147:407-22
Yu, Ying-Chun; Sohma, Yoshiro; Hwang, Tzyh-Chang (2016) On the mechanism of gating defects caused by the R117H mutation in cystic fibrosis transmembrane conductance regulator. J Physiol 594:3227-44
Lin, Wen-Ying; Sohma, Yoshiro; Hwang, Tzyh-Chang (2016) Synergistic Potentiation of Cystic Fibrosis Transmembrane Conductance Regulator Gating by Two Chemically Distinct Potentiators, Ivacaftor (VX-770) and 5-Nitro-2-(3-Phenylpropylamino) Benzoate. Mol Pharmacol 90:275-85
Yeh, Han-I; Yeh, Jiunn-Tyng; Hwang, Tzyh-Chang (2015) Modulation of CFTR gating by permeant ions. J Gen Physiol 145:47-60
Zhang, Jingyao; Hwang, Tzyh-Chang (2015) The Fifth Transmembrane Segment of Cystic Fibrosis Transmembrane Conductance Regulator Contributes to Its Anion Permeation Pathway. Biochemistry 54:3839-50
Gao, Xiaolong; Hwang, Tzyh-Chang (2015) Localizing a gate in CFTR. Proc Natl Acad Sci U S A 112:2461-6

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