| Budget Amount *help |
¥3,950,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2007: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2006: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
We have determined Kv1.5 and Kvβ1.2 are two of major oxygen-sensitive Kvs expressed in the neonatal ductus arteriosus (DA) and pulmonary artery (PA). In order to elucidate mechanism that regulates open/closure of Kv induced by change of PO2, Kv1.5-Kvβ1.2 interaction and their post-translational modification were investigated.
We have established HEK 293 cells stably expressed Kv1.5. Kv complexes from the HEK 293-Kv1.5 cells transfected with or without Kvβ1.2 were isolated, concentrated, and separated using blue native polyacrylamide gel-electrophoresis (BN-PAGE). Apparent molecular sizes of the Kv1.5, Kvβ1.2, and Kv1.5/KvB1.2 complexes on the BN-PAGE are 630, 530, and 800 kDa, respectively. Functional Kv is composed of a tetramer of Kv alpha subunits, which co-assemble with Kv beta subunits. Since deduced molecular sizes of tetramer of Kv1.5, Kvβ1.2, and Kv1.5/KvB1.2 are 263, 182, and 445 kDa, respectively, apparent molecular sizes of Kv complexes were much bigger than these deduced mol
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ecular sizes, which suggested Kv complexes might contain interacting protein(s) and/or post-translational modification(s). Analysis of the Kv1.5 complex by Western blotting showed the bands at 90-95 kDa and 300-400 kDa were relevant to Kv1.5 monomers and polymers. PNGase F treatment of the Kv1.5 complex increased mobility of these bands, suggesting Kv1.5 is glycosylated in the HEK 293 cells. The KvB1.2 monomer appeared as band at 45 kDa. Inter-protein disulphide might play an important role to form Kv1.5/KvB1.2 complex. Immunocytochemistry was used to verify the co-localization of Kv1.5 and KvB1.2 on plasma membrane in the HEK 293 cells.
Glutathionylation and nitrotyrosination in the fetal DA and PA under hypoxic condition were examined by immunohistochemistry. No difference was observed in the glutathionylation experiments. Characteristic immunostaining with anti-nitrotyrosine was observed on inner media of the DA but not on the PA, and a slightly extent staining is present in the DA treated with hypoxic condition. Less
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