| Project/Area Number |
22K15074
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| Research Category |
Grant-in-Aid for Early-Career Scientists
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| Allocation Type | Multi-year Fund |
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| Review Section |
Basic Section 43040:Biophysics-related
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| Research Institution | Osaka University |
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Principal Investigator |
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| Project Period (FY) |
2022-04-01 – 2023-03-31
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| Project Status |
Discontinued (Fiscal Year 2022)
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| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2023: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2022: ¥3,120,000 (Direct Cost: ¥2,400,000、Indirect Cost: ¥720,000)
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| Keywords | ion channel / Slo3 / PIP2 / unnatural amino acid / potassium channel |
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| Outline of Research at the Start |
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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| Outline of Annual Research Achievements |
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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