| Co-Investigator(Kenkyū-buntansha) |
ISOMOTO Shojiro Faculty of Medicine, Osaka University, 医学部, 助手 (80273671)
YAMADA Mitsuhiko Faculty of Medicine, Osaka University, 医学部, 助手 (10263237)
TAKUMI Toru School of Medicine, Kobe University, 医学部, 講師 (00222092)
HORIO Yoshiyuki Faculty of Medicine, Osaka University, 医学部, 講師 (30181530)
DAVID Chella S Mayo Clinic, U.S.A.Immunology, 免疫学, 教授
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| Research Abstract |
To analyze inwardly rectifying potassium channels (IRKs) in the brain, we have cloned cDNAs of GIRK1B and GIRK2B.These channels may participate in the reguration of GTP-binding protein-regulated potassium channels (K_G). We also cloned SUR2B (sulfonyl urea receptor 2B) cDNA which is a subunit of the ATP-regulated potassium channel expressed in vascular smooth muscle. Expression of SUR2B was ubiquitous and was also found in the brain. The genes for IRK3, K_<AB>-2, and GIRK1 were isolated. These genes will be used for knockout studies.
To investigate the distribution of IRLs in the central nervous system, we performed in situ hybridization. These IRK channels were specifically expressed in special sets of neurons. Large amount of IRK2 mRNA was detected in the granular cell later of cerebellum, on the other hand, IRK3 mRNA was localized in the forebrain. Immunohistochemical studies showed that GIRK1 was expressed in presynapses in the paraventricular nubleus. We have previously demonstrate
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d that K_<AB>-2 was predominantly expressed in glial cells. Using anti-K_<AB>-2 antibody, we further analized the distribution of K_<AB>-2, and found that K_<AB>-2 was expressed in Muller cells, retinal glial cells, and marginal cells, which are thought to contribute elevation of endolymph potential of inner ear. In these cells, K_<AB>-2 may have a key role inthe transport of K^+ from cells to cells. We also found that K_<AB>-2 was clustered in the membrane of Muller cells. K_<AB>-2 was associated with PSD-95/SAP90 and clustered when K_<AB>-2 was co-expressed with PSD-95/SAP90 in HEK293T cells. We also found that teh expression of PSD-95/SAP90 family proteins enhanced tha K_<AB>-2 current in HEK293T cells. Electrophysiological properties of inwardly rectifying potassium channels were studied to investigate the function and regulation of these channels in the brain. We found that inward rectification of K_G channels and cloned IRK2 channels was caused by intracellular polyamines. We also found that the binding of more than two molecules (could be four) of GTP-binding protein betagamma heterodimer to a single K_G channel was needed to open the channel. As a K_G channel was composed of four subunits of GIRK channels, our data suggested that each subunit binds one molecule of betagamma heterodimer. Less
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