Down Regulated Adenoma (DRA) gene has been shown to be a candidate for a rare autosomal recessive disease, Congenital Chloride Diarrhea. The basic defect in these patients has been demonstrated to be in ileal and colonic luminal membrane C1-HCO3 exchange. However, there is no direct functional evidence that DRA is Cl-HCO3 exchanger. Functional studies, to date have shown that DRA encodes for a sulfate/oxalate transporter. In preliminary studies, our laboratory has demonstrated the presence of an oxalate/anion exchanger inhibited by cis sulfate and distinct from the Cl-HCO3 exchanger in the human distal colonic apical membrane vesicles. Therefore, we hypothesize that DRA encodes for a sulfate/oxalate anion exchanger but not for a luminal CL-HCO3 exchanger. We will approach this issue as follow: I) study the mechanism(s) of sulfate and oxalate in the human ileal and colonic luminal membranes; and ii) Elucidate the function of the DRA by: a) analyze its mRNA and protein expression in the human intestine and compare it with the Cl-HCO3 exchange as well as sulfate/oxalate transport activity.; b) examine the functional roles of DRA in anion transport by transfection of DRA cDNA in 293 cells (human embryonic kidney line) which lacks the endogenous Cl-HCO3 exchange activity; and c) To confirm the transport role of DRA in apical sulfate/oxalate or CL-HCO3 transport via utilizing antisenses oligonucleotides designed against the DRA cDNA to attenuate DRA expression in human colonic cancer cell line Caco2. The proposed studies will be of great importance in identifying the functional roles of DRA, its relationship to chloride/bicarbonate exchange activity in the luminal membrane of the human intestine epithelial cells and to better understand the pathophysiological basis of congenital chloride diarrhea.
