Intracellular trafficking mechanisms of renal C1C and AQP channels

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We investigated intracellular sorting mechanisms of renal CLC chloride-channels and AQP water-channels, especially focusing on the disease-causing mutants of these channels.1. Molecular pathogenesis of autosomal-dominant type nephrogenic diabetes insipidus (AD-NDI) in AQP2. We found five families of AD-NDI. Sequencing of AQP2-gene revealed the heterozygous frame-shift mutations in all patients, resulting in addition of 61 amino-acids to carboxy-terminus of AQP2. In contrast to the apical localization of wild-type AQP2 expressed in MDCK cells, the disease-causing mutants basolaterally distributed. Furthermore, the mutants recruited the wild-type AQP2 to the basolateral membrane when co-expressed, showing dominant-negative effect. We also found that a di-luecine motif in the mutant was responsible for the basolateral sorting. To confirm whether this mechanism worked in vivo, we generated by gene-targeting a knock-in mouse expressing the mutant AQP2. As expected, the mice showed AD-NDI. A … More pical localization of wild-type AQP2 was significantly impaired by the mutant AQP2-expression. Further analysis of this mouse model will help to clarify the molecular mechanisms of AD-NDI and to develop strategy for treatments of AD-NDI.2. Molecular mechanisms of Bartter syndrome caused by mutations in the BSND-gene. The BSND-gene encodes barttin, which functions as beta-subunit of Cl-channel C1C-K. We determined intracellular localization of C1C-K2 with or without co-expressing barttin in MDCK cells. Barttin clearly recruited C1C-K2 to the plasma membranes. R8L barttin, a disease-causing mutant, was retained in endoplasmic reticulum (ER). However, it could bind C1C-K2. Accordingly, C1C-K2 was also retained in ER. This might be a major mechanism of Bartter syndrome caused by barttin mutaions.3. Chloride-shunt theory as a cause of pseudohypoaldosteronism type II (PHAII). Mutaions of WNK-kinases were identified to be responsible for PHAII. However, its molecular pathogenesis was not determined. We demonstrated that a disease-causing WNK4-mutant increased paracellular chloride-permeability and claudin-phosphorylation. Less