2001 Fiscal Year Final Research Report Summary
Cloning and functional expression of a novel C1 channel that interacts with EBP50
| Project/Area Number |
12670083
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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 | CHIBA UNIVERSITY |
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Principal Investigator |
OGURA Takehiko CHIBA UNIVERSITY, GRADUATE SCHOOL OF MEDICINE, Assistant, 大学院・医学研究院, 助手 (00292673)
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| Co-Investigator(Kenkyū-buntansha) |
NAKAYA Haruaki CHIBA UNIVERSITY, GRADUATE SCHOOL OF MEDICINE, PROFESSOR, 大学院・医学研究院, 教授 (60113594)
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| Project Period (FY) |
2000 – 2001
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| Keywords | C1C-3 / EBP50 / PDZ DOMAIN / CFTR / ORCC |
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| Research Abstract |
We have cloned C1C-3B, a novel alternative splicing variant of C1C-3 (C1C-3A) that is expressed predominantly in epithelial cells. CIC-3B has a different, slightly longer C-terminal end than C1C-3A, and contains a consensus motif for binding to ,the second PDZ domain of the epithelium-specific scaffolding protein EBP50. Both in vitro and in vivo binding assays demonstrate interaction between C1C-3B and EBP50. C127 mouse mammary epithelial cells transfected with C1C-3B alone showed diffuse immunoreactivity for C1C-3B in the cytoplasmic region. In contrast, when EBP50 was co-transfected with C1C-3B, strong immunoreactivity for C1C-3B appeared at the leading edges of membrane ruffles. Patch-clamp experiments revealed that co-transfectipn of C1C-3B and EBP50 resulted in a remarkable increase in outwardly rectifying C1-channel (ORCC) activities at the leading edges of membrane ruffles in C127 cells. The electrophysiological properties of the C1C-3B-induced ORCCs are similar to those of ORCCs described in native epithelial cells. When cystic fibrosis transmembrane conductance regulator (CFTR) was co-transfected with C1C-3B and EBP50, C1C-3B-dependent ORCCs were activated via the protein kinase A-dependent pathway. These findings indicate that C1C-3B is itself a CFTR-regulated ORCC molecule or its activator. Because PKA-dependent activation of the ORCC by mutated CFTR is defective in CF epithelia, ORCCs and their regulation by CFTR have been well studied. Normal ORCC activities can be induced by means independent of CFTR and PKA, such as depolarization, extracellular ATP, and an src-like kinase, all of which represent potential therapeutic targets for the treatment of CF. In the present study, we demonstrate that co-transfection of C1C-3B and EBP50 stimulates ORCC activity which is regulated by CFTR. These findings are important to better understand both epithelial C1- channels and the pathophysiology of CF.
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Research Products
(23 results)
