Arbeitsgruppe Intestinale Mukosafunktion

Funktion und Regulation des Anionentransporters SLC26A3 - Professor Dr. med. Georg Lamprecht

We study the role and the regulation of the intestinal anion exchanger down regulated in adenoma (DRA). DRA belongs to the SLC26 family of anion transporters and is isoform A3 (SLC26A3).

DRA is predominantely expressed in the distal ileum and proximal colon, where it mediates electroneutral NaCl absorption together with the Na/H echanger NHE3 (SLC9A3).

About 9 l of fluid arrive in the proximal small intestine as secretions from the salivary glands, the stomach, the pancreas, the liver (bile) and as orally consumed fluids. Most of this fluid is reabsorbed as part of nutrient absorption. The remaining salt and water (~ 2-3 l) is reabsorbed in the ileum and proximal colon by electroneutral NaCl absorption and by electrogenic Na absorption through the epithelial sodium chanel ENaC in the distal colon. Fluid absorption varies anlong the postprandial and interprandial phase and therefore must be tighly regulated. Disturbances – for instance by pathogens – result ín diarrhea which may be live threatening.

The quantitative importance of DRA and NHE3 is evident from the rare genetic disease chloride losing diarrhea (CLD), which results from loss of function mutations in the DRA gene, and from mouse knock out models of DRA or NHE3.

Both NHE3 and DRA interact with so called PDZ adapter proteins of the NHERF family, which comprises NHERF1 – 4 (NHE3 regulatory factors, NHERF) and SNX27 (sorting nexing 27). Ample data show that this PDZ interaction is important for the localization and regulation of both transporters, which in the ileum and colon probably occurs by common or at least similar mechanisms. But the exact role of the individual PDZ adapter proteins is not fully understood though.

The PDZ adapter proteins are located in different intracellular compartments: different domains of the plasma membrane, early endosomes, recycling endosomes. Both DRA and NHE3 are also found in these compartments and endocytic recycling has been shown for both transporters. Both arms of endocytic recycling – endocytic retrival from and exocytic insertion into the plasma membrane – may be regulated resulting in various numbers of transporters in the apical plasmam membrane facilitating various transport activity.

SNX27 is predominantely located in early endosomes where it is part of a large multiproetin complex, the retromer. Given the complex movement of DRA through the cell we speculate that is present in other multiprotein complexes as well and that these may contain some of the PDZ adapter proteins. The composition of those complexes may even be determined by the PDZ adapter proetins (or vice versa). In our current project we want to address the role of the PDZ interaction of DRA in such complexes using molecular cell biology techniques such as endocytosis and exocytosis assays, subcellular fractionation and untargetted proteomics of multiprotein complexes.

For our studies we mostly use transfected HEK and Caco-2 cells, because these model systems allow us to compare wilde type DRA with a mutant that lacks the PDZ interaction motiv, which are the C-terminal 4 amino acids glutamate-threonine-lysine-phenylalanine (ETKF).

DRA mediates the exchange of chloride and bicarbonate. We measure DRA activity as intracellular alkalinization induced by removal of extracellular chloride using the highly pH sensitive fluorescent dye BCECF.