| 研究課題/領域番号 |
22K15074
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| 研究種目 |
若手研究
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| 配分区分 | 基金 |
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| 審査区分 |
小区分43040:生物物理学関連
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| 研究機関 | 大阪大学 |
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研究代表者 |
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| 研究期間 (年度) |
2022-04-01 – 2023-03-31
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| 研究課題ステータス |
中途終了 (2022年度)
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| 配分額 *注記 |
4,680千円 (直接経費: 3,600千円、間接経費: 1,080千円)
2023年度: 1,560千円 (直接経費: 1,200千円、間接経費: 360千円)
2022年度: 3,120千円 (直接経費: 2,400千円、間接経費: 720千円)
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| キーワード | ion channel / Slo3 / PIP2 / unnatural amino acid / potassium channel |
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| 研究開始時の研究の概要 |
Voltage- and pH-gated Slo3 potassium channel is known to be regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Slo3 controls Ca2+ influx through Catsper channel under the influence of PI(4,5)P2 in sperm capacitation. However, the molecular mechanism behind PI(4,5)P2 modulation in Slo3 channel remains elusive. This research proposal aims to characterize the molecular mechanism of PI(4,5)P2 binding to Slo3 channel by introducing a new method to analyze structure-function mechanisms of membrane proteins, using genetic incorporation of unnatural amino acid, named photocaged-lysine.
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| 研究実績の概要 |
Photocaged-lysine is genetically incorporated using amber stop codon mutation (TAG) at lysine residue of interest. Following pilot experiments were conducted using Xenopus oocyte expression system in two-electrode voltage-clamp (TEVC) recording. First, photocaged-lysine is successfully incorporated into inwardly rectifying potassium (Kir) channel Kir2.1 K64TAG (a mutation located in N-terminal domain) by the observed Kir2.1 current. Second, photocaged-lysine is successfully incorporated into ATP-gated P2X2 receptor K71TAG, a mutation located in ATP binding site, and is successfully uncaged by using the UV illuminator. It has been reported that the mutation at K71 reduced the ATP potency by 1000-fold. No current was observed upon the application of 30μM ATP with no exposure of UV light. On the same cell, ATP-activated P2X2 current was seen 75 seconds after UV exposure. This showed that the photocaged lysine was successfully uncaged into native lysine that resulted in the phenotype of the wild-type P2X2 receptor. Currently, we are working to incorporate the photocaged-lysine into the Slo3 channel in the previously proposed lysine residues that are thought to be critical for PI(4,5)P2 binding. The establishment of photocaged-lysine system into ion channels (Kir2.1) and receptors (P2X2) by using Xenopus oocyte expression system is an important achievement for advancing the main research in Slo3 channel. The results so far are also significant accomplishments for the future application of this method to another class of GPCRs or ion channels/receptors.
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