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 #
5R01DK080834-03
Application #
8018145
Study Section
Gastrointestinal Cell and Molecular Biology Study Section (GCMB)
Program Officer
Carrington, Jill L
Project Start
2009-01-01
Project End
2012-12-31
Budget Start
2011-01-01
Budget End
2011-12-31
Support Year
3
Fiscal Year
2011
Total Cost
$319,144
Indirect Cost
Name
University of Tennessee Health Science Center
Department
Physiology
Type
Schools of Medicine
DUNS #
941884009
City
Memphis
State
TN
Country
United States
Zip Code
38163
Zhang, Weiqiang; Zhang, Zhihong; Zhang, Yanhui et al. (2017) CFTR-NHERF2-LPA? Complex in the Airway and Gut Epithelia. Int J Mol Sci 18:
Sinha, Chandrima; Arora, Kavisha; Naren, Anjaparavanda P (2016) Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration. J Vis Exp :53973
Zhang, W; Zhang, X; Zhang, Y H et al. (2016) Lumacaftor/ivacaftor combination for cystic fibrosis patients homozygous for Phe508del-CFTR. Drugs Today (Barc) 52:229-37
Giridhar, Premkumar Vummidi; Bell, Sheila M; Sridharan, Anusha et al. (2016) Airway Epithelial KIF3A Regulates Th2 Responses to Aeroallergens. J Immunol 197:4228-4239
Arora, Kavisha; Yarlagadda, Sunitha; Zhang, Weiqiang et al. (2016) Personalized medicine in cystic fibrosis: genistein supplementation as a treatment option for patients with a rare S1045Y-CFTR mutation. Am J Physiol Lung Cell Mol Physiol 311:L364-74
Sinha, Chandrima; Zhang, Weiqiang; Moon, Chang Suk et al. (2015) Capturing the Direct Binding of CFTR Correctors to CFTR by Using Click Chemistry. Chembiochem 16:2017-22
Balogh, Andrea; Shimizu, Yoshibumi; Lee, Sue Chin et al. (2015) The autotaxin-LPA2 GPCR axis is modulated by ?-irradiation and facilitates DNA damage repair. Cell Signal 27:1751-62
Moon, Changsuk; Zhang, Weiqiang; Ren, Aixia et al. (2015) Compartmentalized accumulation of cAMP near complexes of multidrug resistance protein 4 (MRP4) and cystic fibrosis transmembrane conductance regulator (CFTR) contributes to drug-induced diarrhea. J Biol Chem 290:11246-57
Riazanski, Vladimir; Gabdoulkhakova, Aida G; Boynton, Lin S et al. (2015) TRPC6 channel translocation into phagosomal membrane augments phagosomal function. Proc Natl Acad Sci U S A 112:E6486-95
Moon, Changsuk; Zhang, Weiqiang; Sundaram, Nambirajan et al. (2015) Drug-induced secretory diarrhea: A role for CFTR. Pharmacol Res 102:107-112

Showing the most recent 10 out of 36 publications