BASOLATERAL SORTING OF CHLORIDE CHANNEL 2 IS MEDIATED BY INTERACTIONS BETWEEN A DILEUCINE MOTIF AND THE CLATHRIN ADAPTOR AP-1

In spite of the many key cellular functions of chloride channels, the mechanisms that mediate their subcellular localization are largely unknown.  is a ubiquitous chloride channel usually localized to the basolateral domain of epithelia that regulates cell volume, ion transport and acid-base balance; mice knocked-out for ClC-2 are blind and sterile. Previous work has suggested that CLC-2 is sorted basolaterally by TIFSLL, a dileucine motif in CLC-2’s C-terminal domain. However, our in silico modelling of ClC-2 suggested that this motif was buried within the channel’s dimerization interface and identified two cytoplasmically exposed dileucine motifs, ESMILL and QVVALL, as candidate sorting signals. Alanine mutagenesis and trafficking assays support a scenario in which ESMILL acts as the authentic basolateral signal of ClC-2. Silencing experiments and yeast three hybrid assays demonstrated that both ubiquitous (AP-1A) and epithelial-specific (AP-1B) forms of the tetrameric clathrin adaptor AP-1 are capable of carrying out basolateral sorting ClC-2 through interactions of ESMI623LL with a highly conserved pocket in their γ1-σ1A hemicomplex.