| Budget Amount *help |
¥14,300,000 (Direct Cost: ¥14,300,000)
Fiscal Year 2005: ¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 2004: ¥7,400,000 (Direct Cost: ¥7,400,000)
|
|---|
| Research Abstract |
We investigated intracellular sorting mechanisms of renal CLC chloride channels by their beta-subunit barttin. To solve this issue, we focused on the sorting mechanisms of barttin itself and how CLC-K and barttin were interacted.
1. Identification of CLC-K and barttin inter-acting domains.
We investigated how CLC-K and barttin form hetero-oligomer by generating several truncated mutants of CLC-K and barttin and performing co-immunoprecipitation assay. We found that barttin binds to CLC-K on its domain B and C, and domain J and K. Two membrane spanning regions of barttin are necessary for its binding to CLC-K.
2. Trial to rescue a disease-causing mutant barttin by drugs.
Barttin recruits CLC-K to the plasma membranes. R8L barttin, a disease-causing mutant, is retained in endoplasmic reticulum (ER). However, it can bind to CLC-K. Accordingly, CLC-K is also retained in ER. This is a major mechanism of Bartter syndrome caused by barttin mutaions. In order to release R8L from ER and make it translocate to plasma membranes, several drugs including chemical chaperones and curcumin were tested using R8L stably expressing MDCK cells. Curcumin (10uM), glycerol (1M), and DMSO (2%) were found to be effective in releasing R8L from ER. To test this potential therapeutic strategy in vivo, we prepared a targeting construct for making R8L knock-in mouse.
3. Identification of basolateral sorting signal of barttin.
Basolateral sorting signal of barttin was determined by expressing several truncated mutants of barttin in MDCK cells. A 15-20 amino acid stretch in the carboxy cytoplasmic region of barttin was necessary for its basolateral sorting, which may be a new type of basolateral sorting signal.
|
