Molecular mechanisms of Bartter syndrome caused by mutations in the BSND gene

Barttin, a gene product of BSND, was identified as a fourth gene responsible for Bartter syndrome. The co-expression of barttin with CLC-K chloride channels has been demonstrated to dramatically induce the expression of CLC-K current. However, it remains unknown how barttin interacts with CLC-K channels in mammalian cells and how the mutations of barttin lead to Bartter syndrome. In an attempt to clarify the effect of barttin expression on CLC-K2 cellular localization, we examined the expression of the CLC-K2 chloride channel and barttin, solely and in combination, in transient and stable expression systems in mammalian cells. In addition, we generated several stable cell lines expressing mutant barttins to clarify the consequence of the previously reported barttin mutations in Bartter syndrome. In immunocytochemistry, CLC-K2 was retained in the Golgi in the absence of barttin expression, but delivered to the plasma membrane when barttin was present. Barttin was coprecipitated with CLC-K2, suggesting a protein-protein interaction. Disease-causing mutant barttins, especially R8L, were retained intracellularly, but their binding ability to CLC-K2 was preserved. This led to a retention of CLC-K2 in intracellular organelles with barttin, and a loss of plasma membrane localization. The stability of the CLC-K2 protein was also markedly increased by coexpression with barttin. These results clarified that barttin determined cellular localization of CLC-K2 by protein-protein interaction. Thus, the mislocalization of CLC-K2 was identified as the molecular pathogenesis of Bartter syndrome by mutant barttins.