The current proposal is aimed at providing the PI with extensive training in advanced molecular biology along with formal courses at the graduate level to enhance his research and laboratory skills. The experienced sponsor- and co-sponsor, along with a highly interactive basic-research environment at the section of Gastroenterology at the Univ. of IL at Chicago, offer a great training opportunity for the PI to achieve his career goals in becoming an independent basic-research investigator in the area of the physiology of intestinal transport. The proposed studies are aimed at examining the exact role of the DRA (Down Regulated in Adenoma) gene in the intestinal Cl- absorption. The DRA gene was shown to be mutated in patients with Congenital Chloride Diarrhea (CLD) disease, where the basic defect is an impaired intestinal apical membrane Cl-/HCO3 exchange process. However, DRA cDNA has a high sequence homology with sulfate transporters. DRA was initially shown to be a sulfate/oxalate transporter. Data from few recent studies also indicated that DRA might function as a Cl- transporter. However, in contrast, our recent studies along with studies from other laboratories provide strong evidence that DRA itself may not encode for the conventional intestinal luminal Cl-/HCO3' exchanger. Therefore, it is critical to investigate the precise functional role of DRA in Cl- transport.
In Specific Aim 1, we will examine the role of DRA utilizing the anti-sense technique to attenuate its endogenous expression in Caco2 cells and study its effect on SO4--and Cl- transport processes, along with Structure/Function studies to reveal the mechanism(s) by which DRA mutations affects the intestinal Cl- transport.
Specific Aim 2 will focus on the mechanism(s) involved in DRA targeting to the plasma membrane.
In Specific Aim 3, we will elucidate the transcriptional regulation of DRA, whereas in Specific Aim 4, we will identify proteins interacting with DRA, and possibly a novel intestinal apical membrane Cl- /HCO3 exchanger by screening human colonic cDNA library utilizing the Yeast Two-Hybrid System. The proposed studies will significantly enhance our understanding of the pathophysiological basis of Congenital Chloride Diarrhea and diarrhea in other colonic diseases.
|Gill, Ravinder K; Saksena, Seema; Tyagi, Sangeeta et al. (2005) Serotonin inhibits Na+/H+ exchange activity via 5-HT4 receptors and activation of PKC alpha in human intestinal epithelial cells. Gastroenterology 128:962-74|
|Gill, Ravinder K; Saksena, Seema; Alrefai, Waddah A et al. (2005) Expression and membrane localization of MCT isoforms along the length of the human intestine. Am J Physiol Cell Physiol 289:C846-52|
|Saksena, Seema; Gill, Ravinder K; Tyagi, Sangeeta et al. (2005) Involvement of c-Src and protein kinase C delta in the inhibition of Cl(-)/OH- exchange activity in Caco-2 cells by serotonin. J Biol Chem 280:11859-68|
|Alrefai, Waddah A; Sarwar, Zaheer; Tyagi, Sangeeta et al. (2005) Cholesterol modulates human intestinal sodium-dependent bile acid transporter. Am J Physiol Gastrointest Liver Physiol 288:G978-85|
|Alrefai, W A; Tyagi, S; Gill, R et al. (2004) Regulation of butyrate uptake in Caco-2 cells by phorbol 12-myristate 13-acetate. Am J Physiol Gastrointest Liver Physiol 286:G197-203|