Characterizing the molecular mechanism of PI(4,5)P2 modulation in Slo3 voltage- and pH-gated potassium channel using photoactivable amino acid

使用光敏氨基酸表征 Slo3 电压门控钾通道和 pH 门控钾通道中 PI(4,5)P2 调节的分子机制

• 批准号: 22K15074
• 负责人: ANDRIANI RIZKITSARI
• 金额: $ 3万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Early-Career Scientists
• 财政年份: 2022
• 资助国家: 日本
• 起止时间: 2022-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-22K15074/
• 关键词: ion channel; Slo3; PIP2; unnatural amino acid; potassium channel

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.

在感兴趣的赖氨酸残基处使用琥珀终止密码子突变（TAG）遗传地掺入光笼化赖氨酸。本研究采用非洲爪蟾卵母细胞表达系统进行双电极电压钳记录。首先，通过观察到的Kir2.1电流，光笼化赖氨酸被成功地掺入到内向整流钾（Kir）通道Kir2.1 K64 TAG（位于N端结构域的突变）中。第二，光笼化的赖氨酸成功地掺入到ATP门控的P2 X2受体K71 TAG中，K71 TAG是位于ATP结合位点的突变，并且通过使用UV照明器成功地解开。据报道，K71的突变使ATP效力降低了1000倍。在施加30μM ATP且未暴露于UV光时未观察到电流。在同一细胞上，在UV暴露后75秒观察到ATP激活的P2 X2电流。这表明光笼化的赖氨酸成功地被释放到天然赖氨酸中，导致野生型P2 X2受体的表型。目前，我们正致力于将光笼化赖氨酸掺入到先前提出的赖氨酸残基中的Slo 3通道中，这些赖氨酸残基被认为对PI（4，5）P2结合至关重要。利用非洲爪蟾卵母细胞表达系统建立了光笼-赖氨酸离子通道（Kir2.1）和受体（P2 X2）表达系统，是推进Slo 3通道研究的重要成果。到目前为止的结果也是该方法未来应用于另一类GPCR或离子通道/受体的重要成就。