Discovery of a potent, Kv7.3-selective potassium channel opener from a Polynesian traditional botanical anticonvulsant

The voltage-gated potassium (Kv) channel formed by Kv7.2/3 (KCNQ2/3) heteromers is a major target for anticonvulsant drug development. Here, we screened 1,444 extracts primarily from plants collected in California and the US Virgin Islands, for their ability to activate Kv7.2/3 but not inhibit Kv1.3, to select against tannic acid being the active component. We validated the 7 strongest hits, identified Thespesia populnea (miro, milo, portia tree) as the most promising, then discovered its primary active metabolite to be gentisic acid (GA). GA highly potently activated Kv7.2/3 (EC50, 2.8 nM). GA is, uniquely, 100% selective for Kv7.3 versus other Kv7 homomers; it requires S5 residue Kv7.3-W265 for Kv7.2/3 activation, and it ameliorates pentylenetetrazole-induced seizures in mice. Also an active aspirin metabolite, GA provides a molecular rationale for the use of T. populnea as an anticonvulsant in Polynesian indigenous medicine and presents novel pharmacological prospects for potent, isoform-selective, therapeutic Kv7 channel activation.

Methods
Collection of plant samples
We collected, between 2019 and 2022, aerial parts of plants under permit from Mojave National Preserve (study # MOJA-00321), Yosemite National Park (study # YOSE-00839), Santa Monica Mountains National Recreation Area (study # SAMO-00192), Muir Woods National Monument (study # MUWO-00035), Santa Cruz Island and Santa Rosa Island (study # CHIS-0023), and Boyd Deep Canyon (Indian Wells, CA) (permit through Philip L. Boyd Deep Canyon. Desert Research 376 Center, University of California, Riverside, CA) in California, US. In USVI, we collected from Virgin Islands National Park, St. John (study # VIIS-20001), Salt River Bay National Historical Park and Ecological Preserve, St. Croix (study # SARI-00056) and Buck Island Reef National Monument, St. Croix (study # BUIS-00103). Plant samples were collected in a manner designed to not kill the remaining plant, sealed in Ziploc bags (SC Johnson, Racine, WI, US), kept cold, and frozen as soon as possible. Other plant extracts were made from plants purchased from Crimson Sage Nursery (Orleans, CA) and grown in the senior author’s garden in Irvine CA, Mountain Rose Herbs (Eugene, OR) or Mother’s Market (Irvine, CA). Plant samples were stored in -20 oC freezers until extraction.

Preparation of plant extracts
Leaves and flowers were pulverized using a bead mill with porcelain beads in batches in 50 ml tubes (Omni International, Kennesaw, GA, United States), then the homogenates resuspended in 80% methanol/20% water (100 ml per 5 g solid) and incubated for 48 hours at room temperature, with occasional inversion to resuspend the particulate matter. We then filtered the extracts through Whatman filter paper #1 (Whatman, Maidstone, UK), removed the methanol using evaporation in a fume hood for 24-48 hours at room temperature, centrifuged extracts for 10 minutes at 15 oC, 4000 RCF to remove the remaining particulate matter, followed by storage (-20 oC). On the day of electrophysiological recording, we thawed the extracts and diluted them 1:50 in bath solution (see below), equivalent to 5 mg fresh plant matter starting material/ml, immediately before use.

High-throughput screening for Kv7.2/3 activation
Plant extracts were applied to human Kv7.2/3 channels expressed in HEK293 cells using a FLIPR potassium assay kit and a Fluorescence Imaging Plate Reader (FLIPRTETRA™) instrument. All chemicals used in this project were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted and were of ACS reagent grade purity or higher. Stock solutions of the positive controls were prepared in dimethyl sulfoxide (DMSO) or deionized water, aliquoted, and stored frozen. Plant extracts were prepared in buffer and frozen until dilution into the assay buffers on test day. The ability of each plant extract to act as a Kv7.2/3 channel opener was evaluated in a thallium (potassium ion surrogate, Molecular Devices) flux assay by Charles River Laboratories (Cleveland, OH, US). The assay was performed with the FLIPR potassium assay kit (Molecular Devices) according to the manufacturer’s instructions. For dye loading, the growth media was removed and replaced with 20 μl of dye loading buffer for 60 minutes at room temperature. For stimulation (agonist mode): 5x (5 μL) plant extract, vehicle, or control article solutions prepared in the stimulation buffer (K+-free buffer with 5 mM Tl+) was added to each well for ~5 minutes. The agonist effects of the plant extract or control articles on KCNQ2/3 channels were evaluated. The positive control was Flupirtine (9 concentrations). Data acquisition was performed via the ScreenWorks FLIPR control software that is supplied with the FLIPR System (MDS-AT). Data were analyzed using Microsoft Excel 2013 (Microsoft Corp., Redmond, WA). For each well, the raw kinetic data were reduced to the maximum or Area Under Curve fluorescence after subtracting bias and possibly applying the negative control correction. Reduced data were analyzed as follows:

For each assay plate, a Z’ factor and Signal Window (SW) were calculated:
Z’ factor = (([Agonist Control mean] – 3 x [Agonist Control STDEV]) – ([Vehicle Control mean] + 3 x [Vehicle Control STDEV])) / ([Agonist Control mean] – [Vehicle Control mean]) SW = (([Agonist Control mean] – 3 x [Agonist Control STDEV]) – ([Vehicle Control mean] + 3 x [Vehicle Control STDEV])) / [Agonist Control STDEV]
Where the stimulation buffer was dispensed to Vehicle Control wells and a high concentration of agonist positive control was dispensed to Agonist Control wells.
Concentration-response curves were fitted to the agonist positive control. Reduced data from test article wells were normalized to the vehicle and agonist control means on each plate and expressed as normalized percent activation: Normalized % Activation = ([individual we 429 ll RLU] – [Vehicle Control mean]) / ([Agonist Control mean] – [Vehicle Control mean]) where individual well RLU = the relative light units for each well to which test article is dispensed. A significance threshold of 3 standard deviations from the vehicle control mean was calculated: Significance Threshold = 3 x [Vehicle Control STDEV] / ([Agonist Control mean] – [Vehicle Control mean]) Concentration-response curves for positive agonist controls for each plate were also conducted. The positive control results confirmed the sensitivity of the test systems to agonists. The test and control samples were prepared in the stimulation buffer (a combination of low Cl439 buffer, 5 mM Tl2SO4 and water). The signal elicited in the presence of the positive agonist control (30 or 100 μM Flupirtine) was set to 100% activation and the signal from the vehicle (stimulation buffer) was set to 0% activation.

High-throughput screening for Kv1.3 inhibition
Chemicals used in solution preparation were purchased from Sigma-Aldrich unless otherwise noted and were of ACS reagent grade purity or higher. Stock solutions of plant extracts and the positive controls were prepared in water and stored frozen, unless otherwise specified. Reference compound concentrations were prepared fresh daily by diluting stock solutions into a HEPES-buffered physiological saline (HB-PS) (composition in mM): NaCl, 137; KCl, 4.0; CaCl2, 4.8; MgCl2, 1; HEPES, 10; Glucose, 10; pH adjusted to 7.4 with NaOH. To minimize run-down of the Kv1.3 channel currents 0.3% DMSO was added in all reference, plant extract and control solutions. The plant extracts (diluted to 2% and 0.2%, equivalent to 5 and 0.5 mg fresh plant matter starting material/ml, respectively, were loaded into 384-well polypropylene compound plates and placed in the plate well of an automated patch-clamp (APC) system, SyncroPatchTM 384PE (SP384PE; Nanion Technologies, Livingston, NJ) immediately before application to Chinese Hamster Ovary (CHO) cells (stain source, ATCC Manassas, VA; substrain source, Chan 456 Test Corporation, Cleveland, OH, US) expressing human Kv1.3. Screening was conducted by Charles River Laboratories. Extracellular buffer was loaded into the wells of the Nanion 384-well Patch Clamp (NPC-384) chips (60 μl per well). Then, cell suspension was pipetted into the wells (20 μL per well) of the NPC-384 chip. After establishment of a whole-cell patch-clamp configuration, membrane currents were recorded using the patch clamp amplifier in the SP384PE system. Plant extracts were applied to naïve cells (n = 3, where n = the number cells/concentration). Each application consisted of addition of 40 μl of 2x concentrated test article solution to the total 80 μl of final volume of the extracellular well of the NPC-384 chip. Duration of exposure to each test article concentration was five (5) minutes. The intracellular solution was (in mM): KCl, 70; KF, 70; MgCl2, 2; EGTA, 2.5; HEPES, 10; pH adjusted to 7.2 with KOH. In preparation for a recording session, the intracellular solution was loaded into the intracellular compartment of the NPC-384 chip. The extracellular solution was the HB-PS solution described above. Kv1.3 channel currents were elicited using test pulses with fixed amplitudes: depolarization pulse to +20 mV amplitude, 200 ms duration from the holding potential of –90 mV. The test pulses were repeated with frequency 0.1 Hz: 3 min before (baseline) and 5 min after test articles addition. Kv1.3 channel current amplitudes were measured at the peak and at the end of the step to +20 mV. The positive control antagonist used was 4-aminopyridine, prepared as a 1 M stock in water; test concentrations were 1, 3, 10, 30, 100, 300, 1000 and 3000 μM.

Channel subunit cRNA preparation and Xenopus laevis oocyte injection for manual two electrode voltage-clamp (TEVC) electrophysiology
We generated cRNA transcripts encoding human Kv1.1 (KCNA1), Kv1.2 (KCNA2), Kv2.1 (KCNB1), Kv7.1, Kv7.2, Kv7.3, Kv7.4, Kv7.5 (KCNQ1-5), and KCNE3 (MiRP2) by in vitro transcription using the mMessage mMachine kit (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions, af 483 ter vector linearization, from cDNA sub-cloned into expression vectors (pTLNx, pXOOM and 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, USA) with the channel cRNAs (0.3-10 ng) and incubated the oocytes at 16 oC in ND96 oocyte storage solution containing penicillin and streptomycin, with daily washing, for 1-4 days prior to two electrode voltage-clamp (TEVC) recording. Mutant channel cDNAs were generated by GenScript Biotech (Piscataway, NJ).

Two-electrode voltage clamp (TEVC)
We conducted TEVC at room temperature with an OC-725C amplifier (Warner Instruments, Hamden, CT, USA) and pClamp10 software (Molecular Devices, Sunnyvale, CA, USA) 1-4 days after cRNA injection. We visualized oocytes in a small-volume oocyte bath (Warner) using a dissection microscope for cellular electrophysiology. We studied the effects of plant extracts and constituents solubilized directly in bath solution (in mM): 96 NaCl, 4 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES (pH 7.6). We introduced extracts or compounds into the oocyte recording bath by gravity perfusion at a constant flow of 1 ml per minute for 3 minutes prior to recording. Pipettes (1-2 MΩ resistance) were filled with 3 M KCl. We recorded currents in response to voltage pulses between -120 mV or -80 mV and +40 mV at 10 mV intervals from a holding potential of -80 mV, to yield current-voltage relationships. We analyzed data using Clampfit (Molecular Devices) and Graphpad Prism software (GraphPad, San Diego, CA, USA), stating values as mean ± SEM. We calculated the voltage dependence of activation (V0.5) by measuring currents at a voltage pulse of -30 mV (Kv7) or -50 mV (Kv1) immediately following prepulse voltages between -80 mV and + 40 mV. We plotted raw or normalized tail currents versus prepulse voltage and fitted them with a single Boltzmann function.

Seizure studies
The mouse study was performed under an approved Institutional Animal Care and Use Committee protocol at the University of California, Irvine. Male C57Bl/6 mice at 2 months of
age were injected intraperitoneally (IP) with either vehicle (saline) or gentisic acid at 2, 10 or 20 mg/kg (pH7.5). After 30 minutes, mice were next injected IP with pentylene tetrazole (80 mg/kg) and then observed by a scorer blinded to the treatment, who measured the latency to first clonic seizure.

In silico docking
We plotted and viewed chemical structures and electrostatic surface potential using Jmol, an open-source Java viewer for chemical structures in 3D: http://jmol.org/. For in silico ligand docking predictions of binding to Kv7.3, we performed unguided docking to predict potential binding sites, using SwissDock with CHARMM forcefields and the AlphaFold-predicted human Kv7.3 monomer structure. We prepared channel structures for docking using DockPrep in UCSF Chimera (https://www.rbvi.ucsf.edu/chimera), with which we also generated docking figures.

Statistics and Reproducibility
All values are expressed as mean ± SEM. One-way ANOVA (with Dunnett correction for multiple comparisons in the case of seizure studies) or t-test was applied for all tests; all p values were two-sided. Electrophysiological data were confirmed in at least two batches of oocytes. Biological replicates are defined as numbers of oocytes; sample sizes are given in the figure legends.

由Kv7.2/3（KCNQ2/3）异聚体构成的电压门控钾（Kv）离子通道是抗惊厥药物研发的主要靶点。本研究针对从美国加利福尼亚州及美属维尔京群岛（USVI）采集的1444份植物提取物，筛选其激活Kv7.2/3通道且不抑制Kv1.3通道的能力，并排除单宁酸作为活性组分的可能性。我们验证了7个活性最强的阳性命中物，鉴定出桐棉（Thespesia populnea，又名miro、milo、波多树）为最具潜力的样本，并发现其主要活性代谢产物为龙胆酸（GA）。龙胆酸（GA）可强效激活Kv7.2/3通道，半数有效浓度（EC50）为2.8 nM。尤为独特的是，相较于其他Kv7同源多聚体通道，GA对Kv7.3具有100%的亚型选择性；其激活Kv7.2/3通道的作用依赖于Kv7.3的S5位点残基W265，且可缓解戊四氮诱导的小鼠癫痫发作。作为阿司匹林的活性代谢产物，GA为波利尼西亚本土医学中使用桐棉作为抗惊厥药物提供了分子层面的理论依据，同时也为强效、亚型选择性的治疗性Kv通道激活提供了全新的药理学研究方向。

## 实验方法
### 植物样本采集
2019年至2022年间，我们依托许可在美国加利福尼亚州的莫哈韦国家保护区（研究编号MOJA-00321）、优胜美地国家公园（研究编号YOSE-00839）、圣塔莫尼卡山脉国家游乐区（研究编号SAMO-00192）、缪尔森林国家纪念碑（研究编号MUWO-00035）、圣克鲁斯岛与圣罗莎岛（研究编号CHIS-0023）以及博伊德深峡谷保护区（加州印第安韦尔斯，许可由加州大学河滨分校菲利普·L·博伊德深峡谷沙漠研究中心376号项目提供）采集植物地上部分。在美属维尔京群岛，我们分别于圣约翰岛的维尔京群岛国家公园（研究编号VIIS-20001）、圣克罗伊岛的盐河湾国家历史公园与生态保护区（研究编号SARI-00056）以及圣克罗伊岛的巴克岛礁国家纪念碑（研究编号BUIS-00103）开展采集。植物样本采集过程以不损伤植株剩余部分为原则，采集后密封于Ziploc密封保鲜袋（庄臣公司，美国威斯康星州拉辛市），低温保存并尽快冷冻。其余植物提取物的原材料购自加州奥尔良市的Crimson Sage苗圃，或采自通讯作者位于加州欧文市的私人花园，另有部分来自俄勒冈州尤金市的Mountain Rose Herbs以及加州欧文市的Mother’s Market。所有植物样本均保存于-20℃冰箱直至提取物制备。

### 植物提取物制备
采用瓷珠珠磨仪（Omni International，美国佐治亚州肯尼索市）在50ml离心管中分批次对叶片与花进行粉碎，随后将匀浆重悬于80%甲醇/20%水溶液中（每5g固体样品加100ml溶剂），室温孵育48小时，期间每隔一段时间倒置混匀以重悬沉淀颗粒。随后使用Whatman 1号滤纸（Whatman，英国梅德斯通市）过滤提取物，于通风橱内室温蒸发去除甲醇（耗时24~48小时），之后将提取物以4000相对离心力、15℃离心10分钟以去除残留颗粒，最终保存于-20℃冰箱。在电生理实验当日，将提取物解冻后以浴液（详见下文）按1:50比例稀释，终浓度相当于每毫升溶液对应5mg新鲜植物原材料，且于使用前即刻配制。

### Kv7.2/3通道激活的高通量筛选
采用FLIPR钾离子检测试剂盒与荧光成像平板阅读器（FLIPRTETRA™），将植物提取物作用于表达于人胚肾293（HEK293）细胞的人源Kv7.2/3通道。本研究所用化学品除特别注明外均购自西格玛奥德里奇公司（Sigma-Aldrich，美国密苏里州圣路易斯市），纯度均达到ACS试剂级及以上。阳性对照的储备液分别以二甲基亚砜（DMSO）或去离子水配制，分装后冷冻保存。植物提取物以缓冲液配制后冷冻保存，直至实验当日稀释至检测缓冲液中。本研究委托查尔斯河实验室（Charles River Laboratories，美国俄亥俄州克利夫兰市）采用铊离子（钾离子替代物，Molecular Devices公司）通量检测法，评估每份植物提取物作为Kv7.2/3通道开放剂的能力。检测操作严格按照FLIPR钾离子检测试剂盒（Molecular Devices公司）的说明书进行。
染色步骤：移除细胞培养液，每孔加入20μl染色缓冲液，室温孵育60分钟。刺激（激动剂模式）步骤：将配制于刺激缓冲液（无钾离子缓冲液，含5mM Tl+）的5倍浓缩植物提取物、溶媒对照或阳性对照溶液各5μL加入每孔，孵育约5分钟。本步骤用于评估植物提取物或对照品对KCNQ2/3通道的激动作用。阳性对照为氟吡汀（设置9个浓度梯度）。数据采集通过FLIPR系统配套的ScreenWorks FLIPR控制软件（MDS-AT）完成。数据分析采用Microsoft Excel 2013软件（微软公司，美国华盛顿州雷德蒙德市）。对于每孔数据，在扣除本底偏差并经阴性对照校正后，将原始动力学数据简化为荧光峰值或曲线下面积（AUC）。简化后的数据按以下方式分析：
对于每一块检测板，均计算Z’因子与信号窗口（SW）：
Z’因子 = （[激动剂对照均值] – 3 × [激动剂对照标准差]） – （[溶媒对照均值] + 3 × [溶媒对照标准差]） / （[激动剂对照均值] – [溶媒对照均值]）
信号窗口SW = （[激动剂对照均值] – 3 × [激动剂对照标准差]） – （[溶媒对照均值] + 3 × [溶媒对照标准差]） / [激动剂对照标准差]
其中，溶媒对照孔加入刺激缓冲液，激动剂对照孔加入高浓度激动型阳性对照。
将阳性对照的浓度-反应曲线进行拟合。将待测样本孔的简化数据以每块板的溶媒对照与激动剂对照均值进行归一化，以归一化激活百分率表示：归一化激活百分率 = （单孔相对光单位（RLU） – [溶媒对照均值]） / （[激动剂对照均值] – [溶媒对照均值]），其中单孔RLU为加入待测样本的每孔的相对光单位数值。设定与溶媒对照均值相差3个标准差作为显著性阈值，计算公式为：显著性阈值 = 3 × [溶媒对照标准差] / （[激动剂对照均值] – [溶媒对照均值]）。每块检测板均对阳性激动剂对照进行浓度-反应曲线检测，阳性对照结果可验证检测系统对激动剂的响应灵敏度。待测样本与对照品均配制于刺激缓冲液中（由低氯缓冲液、5mM硫酸铊与纯水混合而成）。以阳性激动剂对照（30μM或100μM氟吡汀）诱导的信号设为100%激活，以溶媒（刺激缓冲液）诱导的信号设为0%激活。

### Kv1.3通道抑制的高通量筛选
溶液配制所用化学品除特别注明外均购自西格玛奥德里奇公司，纯度达ACS试剂级及以上。除特别说明外，植物提取物与阳性对照的储备液均以纯水配制，冷冻保存。对照化合物的工作浓度于当日新鲜配制：将储备液稀释于HEPES缓冲生理盐溶液（HB-PS，组分浓度单位为mM：NaCl 137、KCl 4.0、CaCl2 4.8、MgCl2 1、HEPES 10、葡萄糖10，以NaOH调节pH至7.4）中。为减少Kv1.3通道电流的衰减，所有对照溶液、植物提取物溶液及对照品溶液中均加入0.3%的DMSO。将植物提取物分别稀释至2%与0.2%（对应每毫升溶液相当于5mg与0.5mg新鲜植物原材料），点样至384孔聚丙烯复合板中，并即刻置于全自动膜片钳（APC）系统SyncroPatchTM 384PE（SP384PE；Nanion Technologies，美国新泽西州利文斯顿市）的板架上，随后作用于表达人源Kv1.3通道的中国仓鼠卵巢（CHO）细胞（细胞来源：美国典型培养物保藏中心（ATCC），弗吉尼亚州马纳萨斯；亚株来源：Chan Test公司，美国俄亥俄州克利夫兰市）。本筛选由查尔斯河实验室完成。
将细胞外缓冲液加入Nanion 384孔膜片钳芯片（NPC-384）的各孔中（每孔60μl），随后移取20μl细胞悬液至NPC-384芯片的每孔中。待形成全细胞膜片钳构型后，通过SP384PE系统的膜片钳放大器记录膜电流。将植物提取物作用于未处理的细胞（n=3，n为每个浓度对应的细胞数）。每次加样时，向NPC-384芯片细胞外孔的80μl总体系中加入40μl 2倍浓缩的待测样本溶液，终浓度对应稀释比例。每个浓度的待测样本暴露时长为5分钟。细胞内液组分（浓度单位为mM）：KCl 70、KF 70、MgCl2 2、EGTA 2.5、HEPES 10，以KOH调节pH至7.2。实验前将细胞内液加载至NPC-384芯片的细胞内腔室。细胞外液为前述HB-PS缓冲液。
采用固定幅度的测试脉冲诱发Kv1.3通道电流：从-90mV的钳制电位去极化至+20mV，脉冲时长200ms。测试脉冲以0.1Hz的频率重复：在加入待测样本前记录3分钟作为基线，加入后记录5分钟。分别于去极化至+20mV的电流峰值与脉冲末期测量Kv1.3通道电流幅值。本实验所用阳性对照拮抗剂为4-氨基吡啶，以纯水配制1M储备液，工作浓度设置为1、3、10、30、100、300、1000与3000μM。

### 通道亚基cRNA制备与非洲爪蟾卵母细胞注射用于手动双电极电压钳（TEVC）电生理实验
我们采用mMessage mMachine体外转录试剂盒（赛默飞世尔科技，美国马萨诸塞州沃尔瑟姆市），按照说明书操作，从亚克隆至表达载体（pTLNx、pXOOM与pMAX）的cDNA合成编码人源Kv1.1（KCNA1）、Kv1.2（KCNA2）、Kv2.1（KCNB1）、Kv7.1~Kv7.5（KCNQ1~KCNQ5）以及KCNE3（MiRP2）的cRNA转录本。上述表达载体在编码区两侧整合了非洲爪蟾β-珠蛋白的5’与3’非翻译区（UTR），以提升翻译效率与cRNA稳定性。在转录前需先将载体线性化。我们将通道cRNA（0.3~10ng）注射至去滤泡膜的V、VI期非洲爪蟾卵母细胞（Xenoocyte，美国密歇根州德克斯特市），随后将卵母细胞置于含青霉素与链霉素的ND96卵母细胞保存液中，于16℃培养，每日更换培养液，培养1~4天后开展双电极电压钳（TEVC）记录。突变型通道cDNA由金斯瑞生物科技（GenScript Biotech，美国新泽西州皮斯卡塔韦市）合成。

### 双电极电压钳（TEVC）实验
我们于cRNA注射后1~4天，在室温下采用OC-725C放大器（Warner Instruments，美国康涅狄格州哈姆登市）与pClamp10软件（Molecular Devices，美国加利福尼亚州桑尼维尔市）开展TEVC实验。采用解剖显微镜在小体积卵母细胞浴槽（Warner）中定位卵母细胞以进行细胞电生理实验。我们将植物提取物与活性成分直接溶解于浴液中（组分浓度单位为mM：NaCl 96、KCl 4、MgCl2 1、CaCl2 1、HEPES 10，pH 7.6），以研究其对通道的作用。在记录前，以1ml/min的恒定流速通过重力灌流将提取物或化合物加入卵母细胞记录浴槽，灌流时长3分钟。玻璃微电极（电阻1~2MΩ）内填充3M KCl溶液。从-80mV的钳制电位出发，施加-120mV/-80mV至+40mV、间隔10mV的电压脉冲，记录对应的电流以绘制电流-电压关系曲线。数据采用Clampfit（Molecular Devices）与Graphpad Prism软件（GraphPad，美国加利福尼亚州圣迭戈市）分析，结果以均值±标准误（SEM）表示。我们通过测量-80mV至+40mV的预脉冲后即刻在-30mV（Kv7通道）或-50mV（Kv1通道）电压脉冲下的电流，计算通道激活的电压依赖性（V0.5）。将原始或归一化尾电流随预脉冲电压的变化作图，并采用单Boltzmann函数进行拟合。

### 癫痫发作实验
本小鼠实验按照加州大学欧文分校动物实验伦理委员会批准的方案开展。选取2月龄雄性C57Bl/6小鼠，分别腹腔注射（IP）溶媒（生理盐水）或2、10、20mg/kg的龙胆酸（pH7.5）。30分钟后，再次腹腔注射戊四氮（80mg/kg），随后由对处理分组不知情的评分者观察并记录首次阵挛性癫痫发作的潜伏期。

### 计算机虚拟对接
我们采用开源Java三维化学结构查看工具Jmol（http://jmol.org/）绘制与查看化学结构及静电表面电势。针对Kv7.3通道的配体虚拟对接预测，我们采用搭载CHARMM力场的SwissDock工具，以AlphaFold预测的人源Kv7.3单体结构为模板，开展无引导对接以预测潜在结合位点。我们采用UCSF Chimera软件（https://www.rbvi.ucsf.edu/chimera）中的DockPrep工具预处理用于对接的通道结构，并使用该软件生成对接可视化图。

### 统计学与实验可重复性
所有数据均以均值±标准误（SEM）表示。所有统计学检验均采用单因素方差分析（癫痫实验中采用Dunnett法校正多重比较）或t检验，所有P值均为双侧检验。电生理实验数据至少在2批卵母细胞中重复验证。生物学重复定义为卵母细胞的数量，样本量详见图注。