| Co-Investigator(Kenkyū-buntansha) |
UEDA Kazumitsu Kyoto University, Graduate School of Agriculture, Professor (10151789)
KATO Hiroaki Kyoto University, Faculty Graduate School of Pharmaceutical Sciences, Professor (90204487)
YAMADA Katsuya Hirosaki University, School of Medicine, Associate Professor (40241666)
NAGASHIMA Kazuaki Kyoto University, Graduate School of Medicine, Assistant Professor (40324628)
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| Research Abstract |
Our aim in this study is to clarify the molecular basis of novel membrane transport mechanisms by investigating not only functions of ABC proteins in membrane transport but also on their structures and pathophysiological roles. In this study
1) By investigating the function and pathophysiology of ABCA3 in patients and ABCA3-deficient mice, we determined that ABCA3 is critical in pulmonary surfactant production and lamellar body biogenesis, probably by transporting these lipids as substrates.
2) We cloned a full-length cDNA of ABCA2, and showed that ABCA2 is expressed at high levels in brain white matter regions, mainly in oligodendrocytes. In addition, we showed using ABCA2-defiocient mice, that ABCA2 is involved in the intracellular metabolism of sphingolipids, particularly sphingomyelin and gangliosides GM1, in the brain.
3) We measured the ATP hydrolysis activity of purified ABCA1 and demonstrated that it is stimulated preferentially by phosphatidylcholine. In addition, ABCG1-mediated
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efflux of cholesterol is dependent on the cellular sphingomyelin level and correlates with the efflux of sphingomyelin, suggesting the cooperative roles of ABCA1 and ABCG1 in HDL formation.
4) The mechanism how ABC proteins handle enormous numbers of hydrophobic compounds with various structures and molecular weights, or phospholipids and cholesterol, is not known. We demonstrated that cholesterol plays an important role in substrate recognition, especially by MDR1, where cholesterol fills the empty space in the substrate binding site when small drugs bind to it, and proposed the cholesterol fill-in model.
5) The function and pathophysiological role of regulator-type ABC protein, SUR, are determined. Using Kir 6.2-deficient mice, we showed that Kir6.2-containing KATP channels are critically involved in the maintenance of hypoxic gasping and depression of respiratory frequency. Furthermore, we have shown that Kir6.1-containing KATP channels are involved in the neural activity-regulated blood flow in small arterioles in the brain.
6) The system to purify human MDR1 protein which will be used in obtaining crystal structure has been established. In addition, the system to predict the ability of ABC proteins to crystallize based on the localization of the GFP-fused proteins has been developed. By screening 19 ABC proteins, we obtained the crystal of 3 proteins, 2 of which will be suitable to analyze the X ray crystal structure. Furthermore, we have established large-scale preparation and purification of KATP channel (SUR1-Kir 6.2 complex), and obtained the electron microscopy of purified and negatively stained SUR1-Kir 6.2 complex. Less
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