Conifer metabolite pisiferic acid restores activity in human Kv1.2 potassium channels carrying pathogenic sequence variants

Sequence variants in KCNA2, the gene encoding voltage-gated potassium channel Kv1.2, cause epilepsy, intellectual disability, and movement disorders. Drugs that directly correct mutant Kv1.2 function are lacking.  Kv1.2 downregulation is also implicated in pain and amyotrophic lateral sclerosis (ALS). We recently found that the abietane diterpenoid pisiferic acid (PA) from conifer Chamaecyparis pisifera beneficially restores activity in pathogenic loss-of-function (LOF)-variant Kv1.1 channels.  Here, using cellular electrophysiology, we classified 19 human Kv1.2 gene variants (pathogenic or of unknown significance) into LOF, gain of function (GOF), or mixed LOF/GOF. By hyperpolarizing their voltage dependence of activation, PA improved function in 13/13 LOF and 1/1 LOF/GOF pathogenic Kv1.2 variants tested, using cRNA ratios representative of autosomal dominant KCNA2 disorders. In silico docking, mutagenesis, and electrophysiology identified a PA binding site in the Kv1.2 volta..., Channel subunit cRNA preparation and Xenopus laevis oocyte injection
Wild-type Kv1.1 and wild-type and mutant Kv1.2 cDNAs were generated (Genscript, Piscataway, NJ) in the pMAX and pTLNx expression vectors.  As previously described, we generated cRNA transcripts encoding human Kv1.2 (wild-type and mutant) by in vitro transcription using the mMessage mMachine kit (Thermo Fisher Scientific), after vector linearization, from cDNA subcloned into plasmids (pMAX) incorporating Xenopus laevis β-globin 5’ and 3’ UTRs flanking the coding region to enhance translation and cRNA stability.  We injected defolliculated stage V and VI Xenopus laevis oocytes (Xenoocyte, Dexter, MI, US) with Kv1 cRNAs (0.1-10 ng). We incubated the oocytes at 16 oC in ND96 oocyte storage solution containing penicillin and streptomycin, with daily washing, for 1-2 days prior to two-electrode voltage-clamp (TEVC) recording.
Two-electrode voltage clamp (TEVC)
We performed TEVC at room temperature using an OC-725C..., ## Conifer metabolite pisiferic acid restores activity in human Kv1.2 potassium channels carrying pathogenic sequence variants

The datasets included are the original Excel files used to generate each panel for figures 2-9 in this manuscript. The title of each Excel file is labeled to directly correspond to the panel of each figure in the manuscript:

**Figure Number & panel > Channel Investigated (including mutant and wild type (WT)) > Condition > Parameter Measured.**

Example:

**Figure 2A, B - Kv1.2 12 uM Pisiferic acid - IV.xlsx**

The data contained within this repository are those obtained from cellular electrophysiology recordings. We used two-electrode voltage clamp (TEVC) electrophysiology and the *Xenopus laevis* oocyte expression system to record the electrical activity of wild-type Kv1.2 and Kv1.1/Kv1.2 channels in response to pisiferic acid. We also studied nineteen Kv1.2 disease-linked mutations. Using TEVC, we biophysically characterized homomeric homozygous patient Kv1....,

KCNA2基因（编码电压门控钾通道Kv1.2）的序列变异可引发癫痫、智力障碍与运动障碍。目前尚无能够直接矫正突变型Kv1.2功能的治疗药物。此外，Kv1.2表达下调还与疼痛及肌萎缩侧索硬化症（ALS）密切相关。我们此前的研究发现，来自日本花柏（Chamaecyparis pisifera）的松香烷二萜类化合物松脂酸（pisiferic acid, PA）可有效恢复致病性功能丧失型（loss-of-function, LOF）变异Kv1.1通道的活性。本研究通过细胞电生理学技术，将19个人类KCNA2基因变异（致病性或意义未明）划分为功能丧失型、功能获得型（gain-of-function, GOF）以及混合型LOF/GOF三类。实验采用常染色体显性遗传性KCNA2疾病特征性的互补RNA（cRNA）比例体系，结果显示，通过使通道激活电压依赖性发生超极化，PA可改善全部13个功能丧失型致病性Kv1.2变异以及1个混合型LOF/GOF致病性Kv1.2变异的通道功能。通过计算机分子对接、诱变实验与电生理学实验，我们鉴定出Kv1.2上的PA结合位点……

### 通道亚基cRNA制备与非洲爪蟾（Xenopus laevis）卵母细胞注射
野生型Kv1.1、野生型与突变型Kv1.2的互补DNA（cDNA）由金斯瑞（Genscript, 美国新泽西州皮斯卡塔韦）合成，并克隆至pMAX与pTLNx表达载体中。如前所述，我们将编码人类Kv1.2（野生型与突变型）的cDNA亚克隆至带有非洲爪蟾β-球蛋白5'与3'非翻译区（UTR）的pMAX质粒中，通过体外转录使用mMessage mMachine试剂盒（赛默飞世尔科技, Thermo Fisher Scientific）制备cRNA转录本，以提升翻译效率与cRNA稳定性，转录前需先对载体进行线性化处理。我们向脱滤泡的V、VI期非洲爪蟾卵母细胞（Xenoocyte, 美国密歇根州德克斯特）注射Kv1 cRNA（0.1~10 ng）。将卵母细胞置于添加了青霉素与链霉素的ND96卵母细胞保存液中，于16℃孵育，每日换液，1~2天后进行双电极电压钳（two-electrode voltage clamp, TEVC）记录。

### 双电极电压钳技术（TEVC）
本研究在室温下使用OC-725C型号设备开展双电极电压钳实验……

## 针叶树代谢物松脂酸恢复携带致病性序列变异的人类Kv1.2钾通道活性
本研究附带的数据集为生成本文手稿中图2至图9各面板的原始Excel文件。每个Excel文件的命名规则直接对应手稿中各图表的面板：**"图表编号及面板>研究通道类型（包含突变型与野生型（WT））>实验条件>测量参数"**。
示例：**"图2A、B - Kv1.2 12 μM 松脂酸 - 电流-电压曲线.xlsx"**
本仓库包含的所有数据均来自细胞电生理学记录实验。我们采用双电极电压钳电生理学技术与非洲爪蟾卵母细胞表达系统，记录野生型Kv1.2及Kv1.1/Kv1.2通道在松脂酸处理下的电活动。我们同时研究了19个与疾病相关的Kv1.2突变体。通过双电极电压钳技术，我们对纯合子患者来源的Kv1.2通道进行了生物物理特性表征……