| Project/Area Number |
12671030
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Kidney internal medicine
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| Research Institution | Kitasato University |
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Principal Investigator |
SAKAMOTO Hisato Kitasato University, School of Medicine, Assistant Professor, 医学部, 講師 (80187046)
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| Co-Investigator(Kenkyū-buntansha) |
NAITO Ichiro Shigei Medical Institute, director, 超微病理部門, 部長
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2000: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | CIC-5 channel / CIC-3 channel / sorting / endocytosis / Osteoclast / gastric cells / H^+-ATPase / intercalated cells / ClCー5クロライドチャネル / 細胞内小胞 / デント病 / 遺伝子形質導入 / ClC-3クロライドチャネル / ClC-5クロライドチャネル / H^--ポンプ |
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| Research Abstract |
A number of mutations in the human CIC-5 chloride (CI-) channel genes might be responsible for Dent's disease. In the present study, we determined whether mutant CIC-5 is involved in the trafficking disorder using stable transfection system in the mammalian cultured cells, CHO-K1. When expressed in CHO-K1 cells, two kinds of mutants CIC-5 (S270R and R280P) with naturally occurring missense mutations in Dent's disease were correctly processed and trafficked to specific destination, early endosome, even if associated with aberrant structure. In contrast, the trafficking of the channel to plasma membrane was only detected in wild type. Because the sites of both mutations belong to the specific loop between the predicted transmembrane domain D5 and D6 of CIC-5, these findings suggest that the structural modification in this region affect on the assembling and subsequent trafficking of the channel in the endosome rather than intracellular sorting of channel. Furthermore, we identified a new
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mutant with flame-shift in C-terminal region might result in retention and rapid degradation in an intracellular compartment. Taken together, these findings suggest that to presume the function of defective gene product at the level of intracellular sorting is of fundamental importance to assess the possibility of therapeutic intervention.
We also identified and characterized that CIC-5 channel expressed in osteoclast and gastric parietal cells, in both of witch the channel co-localized with H^+-ATPase. A novel finding is that calcitonin might inhibit the function of osteoclast through the internalization of CIC-5 related channel in an actin-associated manner.
The specific anti-ClC-3 monoclonal antibody was successfully established to evaluate the interrelationship between two homologous channels, CIC-3 and CIC-5, in the intercalated cells (IC). CIC-3 was differentially localized in type C IC compared with CIC-5 localized in type A IC. Further experiments are necessary to confirm the role ofCIC-5 and CIC-3 in IC. Less
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