Activity and Surface Expression of the Intestinal Anion Exchanger Down-Regulated in Adenoma (DRA)

Abstract

Down-regulated in adenoma (DRA; SLC26A3) is an intestinal chloride/bicarbonate exchanger that is functionally coupled to NHE3 (Na/H exchanger-isoform 3) in the ileum and proximal colon to mediate electroneutral NaCl absorption. This NaCl absorption is a major regulator of total body volume and electrolyte homeostasis. Knock out of either transporter (DRA or NHE3) causes chronic diarrhea [10,11]. Furthermore, a nonfunctional DRA leads to life-threatening diarrhea (human genetic disorder congenital chloride diarrhea) [2]. NHE3 and several other transport proteins are associated with lipid rafts. These microdomains contain a special subset of lipids, mainly sphingolipids and cholesterol [8]. They are involved in cellular processes that include membrane sorting and recycling, protein trafficking [3] and cell signaling [12]. In case of NHE3 the raft association is necessary for its normal activity as well as for its basal and stimulated trafficking [5]. Lipid rafts are involved in phosphatidylinositol-3-(PI3)-kinase-dependent exocytosis of NHE3 as well as in its basal endocytosis [7]. Since the function of NHE3 and DRA appears to be regulated in parallel [4] DRA may be associated with lipid rafts as well. Both transporters posses PDZ binding motifs that facilitate binding to PDZ adaptor proteins of the NHERF family [4]. The four members of the NHERF family (NHERF, E3KARP, PDZK1 and IKEPP) appear to play important roles for the regulation of intestinal ion transport [4]. However, the potential interplay of raft association and PDZ interaction of both transporters has not been studied. To this end, we have generated HEK293 cells stably expressing DRA (EGFP-DRA) or a DRA mutant that lacks the PDZ binding motif (EGFP-DRA-ETKFminus). We have studied whether DRA associates with lipid rafts – independent or dependent of its PDZ interaction and whether the activity of DRA depends on the raft association. Furthermore, we examined the possibility that DRA is inserted into the plasma membrane in a lipid raft and PI3-kinase dependent manner.