Homooligomeric and heterooligomeric associations between K‐Cl cotransporter isoforms and between K‐Cl and Na‐K‐Cl cotransporters

Little is known regarding the quaternary structure of cation‐Cl‐cotransporters (CCCs) except that the Na‐dependent CCCs is organized as homooligomeric units. Given that each of the CCCs exhibits unique functional properties and that these carriers probably coexist in various cell types, it would be interesting to determine whether the K‐Cl cotransporters (KCCs) can also assemble into such units and whether they can form heterooligomers by interacting with each other or with the secretory Na‐K‐Cl cotransporter (NKCC1). To address these questions, we have conducted two‐hybrid analyses and GST pull down assays using CCC protein segments as well as coimmunoprecipitation studies using CCC‐expressing X. laevis oocytes. All of the KCCs were found to possess self‐interacting domains that can support the formation of oligomers not only comprised of the same isoform but also of different CCCs including NKCC1. Although the carboxy‐termini of KCC2, KCC4 and NKCC1 appeared to play a role in oligomerization, it was not possible to identify interacting regions for all of the KCCs, indicating that the residues or factors required for certain associations to occur could differ among the isoforms. These findings could change our views on how CCCs operate or are regulated in mammalian tissues and suggest that cation‐Cl cotransport achieves higher levels of functional diversity than foreseen in certain cell types. Supported by CIHR