ClC-K chloride channels belong to the CLC chloride channel family and play an important role in transepithelial chloride transport in the kidney. To be functional, ClC-K channels need to be translocated to the plasma membranes after synthesis; the translocation requires the binding to its beta-subunit, barttin. The binding interaction between barttin and ClC-K channels has not been characterized, although the crystal structure of CLC was resolved. In the present study, we sought to clarify the binding sites of barttin in ClC-K2 by co-immunoprecipitation and immunofluorescence microscopy using various ClC-K2 mutants. The deletion of the carboxy-terminal portion of ClC-K2 up to leucine 91, a construct which contains the B domain alone, showed the binding ability to barttin. Since the CLC channel forms an internal antiparallel structure, domain J corresponds to domain B in the carboxy-terminal half of ClC-K. Accordingly, we made the carboxy-terminal half of ClC-K2 containing domain J and thereafter and its deletion mutants, and performed a similar co-immunoprecipitation study. As a result, only domain J was enough for binding to barttin. Immunofluorescence microscopy confirmed that the domains B and J as well as the full length ClC-K2 could be localized to the plasma membranes only when co-expressed with barttin. These results showed that barttin was able to bind to the domains that constitute the outer lateral surfaces of ClC-K2. This information regarding the binding sites will be useful for designing a new class of diuretics or anti-hypertensive agents that inhibit the interaction of CIC-K and barttin. (c) 2007 Elsevier Inc. All rights reserved.