Abstract
It has been conceived that NaCl absorption in the gastrointestinal tract is mediated by coupled Na+/H+ and Cl−/HCO3− exchangers. NHE3 is a major Na+/H+ exchanger contributing to NaCl absorption; however, the molecular identity of the Cl−/HCO3− exchanger involved in NaCl absorption is uncertain. Recent studies have suggested that Down Çúregulated in adenoma (DRA, also called Slc26a3) is a major Cl−/HCO3− exchanger in the colon. Since multiple isoforms of the Cl−/HCO3− exchanger are co-expressed in an intact colonic cell, complicating the functional analysis of an individual isoform, we used heterologous transfection of DRA into cell lines. We generated a construct encoding the DRA tagged with the hemagglutinin (HA) epitope and transiently transfected the cells with this construct. The expression of the HA-tagged DRA by western blotting was verified. Two immunoreactive bands were detected in the transfected cells: a major protein of 120 kDa, probably glycosylated DRA, and a smaller band of 90 kDa, which is probably un-glycosylated DRA. This assumption was validated by treatment of the cells with an N-glycosylation inhibitor resulting in the disappearance of the higher molecular band. The subcellular localization of DRA was assessed by immunofluorescence experiments; DRA was found almost exclusively in the surface membrane. Since DRA expression was not stable and disappeared over weeks, we are now trying to establish the cells stably expressing DRA by using inducible gene expression systems. [Jpn J Physiol 54 Suppl:S84 (2004)]
