Basolateral localization of native CIC-2 chloride channels in absorptive intestinal epithelial cells and basolateral sorting encoded by a CBS-2 domain di-leucine motif

The Cl^- channel ClC-2 is expressed in transporting epithelia and has been proposed as an alternative route for Cl^- efflux that might compensate for the malfunction of CFTR in cystic fibrosis. There is controversy concerning the cellular and membrane location of ClC-2, particularly in intestinal tissue. The aim of this paper is to resolve this controversy by immunolocalization studies using tissues from ClC-2 knockout animals as control, ascertaining the sorting of ClC-2 in model epithelial cells and exploring the possible molecular signals involved in ClC-2 targeting. ClC-2 was exclusively localized at the basolateral membranes of surface colonic cells or villus duodenal enterocytes. ClC-2 was sorted to the basolateral membranes in MDCK, Caco-2 and LLC-PK1-μ1B, but not in LLC-PK1-μ1A cells. Mutating a di-leucine motif (L812L813) to a di-alanine changed the basolateral targeting of ClC-2 to an apical location. The basolateral membrane localization of ClC-2 in absorptive cells of the duodenum and the colon is compatible with an absorptive function for this Cl^- channel. Basolateral targeting information is contained in a di-leucine motif (L812L813) within CBS-2 domain at the C-terminus of ClC-2. It is speculated that ClC-2 also contains an apical sorting signal masked by L812L813. The proposal that CBS domains in ClC channels might behave as regulatory sites sensing intracellular signals opens an opportunity for pharmacological modulation of ClC-2 targeting.