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

在感兴趣的赖氨酸残基上使用琥珀终止密码子突变(Tag)从基因上整合光笼赖氨酸。利用非洲爪哇卵母细胞表达系统进行双电极电压钳(TEVC)记录。首先，通过观察到的Kir2.1电流，成功地将光笼赖氨酸掺入到内向整流钾(KIR)通道Kir2.1 K64TAG(位于N-末端结构域的突变)中。其次，成功地将光笼赖氨酸掺入到ATP门控的P2X2受体K71TAG中，K71TAG是一个位于ATP结合位点的突变，并在紫外光照射下成功地被取消。据报道，K71的突变使ATP的效力降低了1000倍。在不照射紫外光的情况下，30μM三磷酸腺苷没有观察到电流。在同一细胞上，在紫外线照射75秒后，可见ATP激活的P2X2电流。这表明光笼赖氨酸被成功地解离为天然赖氨酸，导致野生型P2X2受体的表型。目前，我们正在努力将光笼赖氨酸整合到先前提出的对PI(4，5)P2结合至关重要的赖氨酸残基的Slo3通道中。利用非洲爪哇卵母细胞表达系统建立离子通道(Kir2.1)和受体(P2X2)的光笼赖氨酸系统是推进Slo3通道主要研究的重要成果。到目前为止的结果也是未来将该方法应用于另一类GPCRs或离子通道/受体的重大成就。

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.