2002 Fiscal Year Final Research Report Summary
Functional analysis of Cl^- pump 55kDa subunit
| Project/Area Number |
13670103
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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 |
General pharmacology
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| Research Institution | Kansai Medical University |
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Principal Investigator |
KITAGAWA Kaori Kansai Medical University Faculty of Medicine research associate, 医学部, 助手 (10165813)
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| Project Period (FY) |
2001 – 2002
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| Keywords | Cl^- pump / rat brain plasma membrane / ClP55 / antisense oligonucleotide / Cl^-ATPase |
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| Research Abstract |
The Cl^-ATPase/pump in the plasma membrane of the rat brain is a candidate for active outwardly directed Cl^- translocating systems. We recently isolated a Cl^- pump, 520 or 580 kDa protein complex, which consisted of 51, 55, 60 and 62 kDa proteins. In this study, we cloned a cDNA encoding a 55 kDa protein, designated as ClP55, which contained an open reading frame of 1512 base pairs encoding a protein of 504 amino acids including a signal peptide of 28 amino acids. Northern blotting analysis showed a band of 4.0 kilobases rather ubiquitously with strong signals in the cerebrum, cerebellum and spinal cord. Western blotting analysis using antiserum against recombinant ClP55 demonstrated expression of ClP55 mainly in the cerebrum. Peptide N-glycosidase treatment decreased the molecular size of the protein to 53 kDa. Application of antisense phosphorothioate oligonucleotides to cultured neurons resulted in a marked increase in the intracellular Cl^- concentration ([Cl^-]i). Immunohistochemical analysis indicated that ClP55 was localized to the plasma membranes of neurons such as hippocampal pyramidal neurons and cerebellar Purkinje's cells. Taken together, these results suggest that ClP55 is one of the Cl^- pump subunits responsible for Cl^- pump activity.
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