The hypothesis of this proposal is that the functional activities of the cAMP transporter (MRP4) and CFTR Cl- channel are physically and functionally coupled within the gut epithelial cells. The long-term objectives of this laboratory are (I), To define the mechanism of how protein-protein interactions regulate CFTR Cl- channel function and (II), To dedicate our efforts toward making a long-term contribution in understanding gastrointestinal disorders related to diarrheal diseases.
The specific aims of this proposal are:
Specific Aim 1. To test the hypothesis that MRP4 is an apical cAMP transporter in the gut and inhibition of this transporter potentiates cholera toxin (CTX) induced diarrhea. Four subaims will be tested, they are (1a). To test whether MRP4 is an apical cAMP transporter in gut epithelial cells and to characterize the cyclic nucleotide transport using HPLC. (1b). To test whether MRP4 inhibition and MRP4 silencing (using Si-RNA) augments CFTR-dependent short circuit currents in the apical membrane of polarized gut epithelial cells and in excised mouse intestine. (1c). To test whether MRP4 inhibition can potentiate cholera toxin (CTX)-induced and CFTR-dependent secretory diarrhea in mice and to test if CFTR knock out mice fail to respond to CTX and MRP4 inhibition. (1d). To test whether MRP4 knock out mice are more susceptible to CTX-induced secretory diarrhea and to test if MRP5 inhibitors fail to induce secretion.
Specific Aim 2. To test the hypothesis that there is a physical and functional coupling of cAMP transporter (MRP4) and CFTR Cl- channel at or near the apical plasma membrane of gut epithelial cells. Three subaims will be tested, they are (2a). To test whether the cAMP transporter (MRP4) is in a macromolecular complex with PDZK1 and CFTR and to define the stoichiometries of CFTR:PDZK1 and MRP4:PDZK1 complex in the plasma membrane of gut epithelial cells. (2b). To test if the disruption of the cAMP transporter containing macromolecular complex inhibits CFTR function and to test if the lateral mobility of CFTR and MRP4 increases (high diffusion rates) at the plasma membrane. (2c). To test whether cAMP accumulates at or near the plasma membrane (using a membrane associated cAMP sensor) upon inhibition of the cAMP transporter. These studies will demonstrate that the two ABC transporters (CFTR and MRP4) can be functionally and physically coupled and that MRP4 inhibition can augment CFTR transporter function. The results of these studies will provide us with possible alternative methods and targets for treating certain diseases of the gastrointestinal tract such as secretory diarrhea and IBD. These studies will, therefore, have clinical relevance in individuals suffering from certain forms of diarrhea.

Public Health Relevance

The proposed research will test the hypothesis that the two ABC transporters MRP4 (functions as cAMP transporter) and CFTR Cl- channel are physically and functionally coupled within the gut epithelial cell. This proposal will also test if inhibition of the MRP4 transporter function augments CFTR Cl- channel function thus potentiating cholera toxin (CTX) induced diarrhea in mice.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
7R01DK080834-06
Application #
8755665
Study Section
Clinical, Integrative and Molecular Gastroenterology Study Section (CIMG)
Program Officer
Grey, Michael J
Project Start
2008-04-01
Project End
2018-03-31
Budget Start
2013-09-18
Budget End
2014-03-31
Support Year
6
Fiscal Year
2013
Total Cost
$223,848
Indirect Cost
$80,356
Name
Cincinnati Children's Hospital Medical Center
Department
Type
DUNS #
071284913
City
Cincinnati
State
OH
Country
United States
Zip Code
45229
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Arora, Kavisha; Moon, Changsuk; Zhang, Weiqiang et al. (2014) Stabilizing rescued surface-localized ?f508 CFTR by potentiation of its interaction with Na(+)/H(+) exchanger regulatory factor 1. Biochemistry 53:4169-79
Holcomb, Joshua; Jiang, Yuanyuan; Lu, Guorong et al. (2014) Structural insights into PDZ-mediated interaction of NHERF2 and LPA(2), a cellular event implicated in CFTR channel regulation. Biochem Biophys Res Commun 446:399-403
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Ren, Aixia; Zhang, Weiqiang; Yarlagadda, Sunitha et al. (2013) MAST205 competes with cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand for binding to CFTR to regulate CFTR-mediated fluid transport. J Biol Chem 288:12325-34
Sinha, Chandrima; Ren, Aixia; Arora, Kavisha et al. (2013) Multi-drug resistance protein 4 (MRP4)-mediated regulation of fibroblast cell migration reflects a dichotomous role of intracellular cyclic nucleotides. J Biol Chem 288:3786-94
Arora, Kavisha; Sinha, Chandrima; Zhang, Weiqiang et al. (2013) Compartmentalization of cyclic nucleotide signaling: a question of when, where, and why? Pflugers Arch 465:1397-407
Zhang, Weiqiang; Fujii, Naoaki; Naren, Anjaparavanda P (2012) Recent advances and new perspectives in targeting CFTR for therapy of cystic fibrosis and enterotoxin-induced secretory diarrheas. Future Med Chem 4:329-45
Ray, Ramesh M; Li, Chunying; Bhattacharya, Sujoy et al. (2012) Spermine, a molecular switch regulating EGFR, integrin ýý3, Src, and FAK scaffolding. Cell Signal 24:931-42
Sellers, Zachary M; Naren, Anjaparavanda P; Xiang, Yang et al. (2012) MRP4 and CFTR in the regulation of cAMP and ?-adrenergic contraction in cardiac myocytes. Eur J Pharmacol 681:80-7

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