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Introduction

Carole Crittenden | Applications Scientist | Molecular Devices

Voltage-gated ion channels are present in the excitable cell membranes of heart, skeletal muscle, brain and nerve cells. Blocking or modulating such channels can have a therapeutic effect or may interfere with normal cell function. As a result, compounds that affect voltage-gated ion channels are important targets in drug discovery. Cardiac Na_V1.5 channels are classified as “Tetrodotoxin (TTX)-resistant.”¹ The pharmacological significance of the Na_V1.5 channel is that it is a target for the action of antiarrhythmic drugs and is also blocked by local anesthetics such as lidocaine².

In terms of traditional electrophysiology, Na_V1.5 channels exhibit state- and use-dependent inhibition by some compounds such as lidocaine. State-dependent inhibition means that inhibitors bind more readily to a channel when it is in a particular voltage-dependent conformational state (e.g., closed, open, inactivated). Use-dependent inhibition refers to an accumulation of inhibition by a particular compound with repetitive stimuli, such as a train of voltage pulses. The pulse train causes the channel to cycle through the voltage-dependent conformational states thus giving the use-dependent compound more cumulative access to its binding site.³ State- and use-dependence phenomena result in a shift of IC₅₀ concentrations of some compounds between membrane potential assays and patch clamp data.

FLIPR^® Membrane Potential (FMP) Assay Kits provide a rapid and reliable fluorescence-based method to detect changes in membrane potential brought about by compounds that modulate or block voltage-gated ion channels. It has been previously shown that interaction between test compounds and membrane potential dyes may cause changes in fluorescence response leading to altered estimation of compound activity.⁴

Molecular Devices has developed two different no-wash formulations of FLIPR Membrane Potential Assay Kits: Blue and Red. Each kit uses the same proprietary indicator dye, combined with different quencher to maximize cell line/channel/compound applicability while eliminating causes of variability in the data. Depending on the chemical properties of compounds in a library, differences in compound auto-fluorescence, or effect of dye on cell type or receptor, one kit may provide better response than the other. During assay development, optimal compound response can be empirically determined by testing both kits with a representative sample of the library to be screened. In this application note, CHL cells transfected with the cardiac Na_V1.5 channel are used to demonstrate differences in membrane potential assay functionality with tetrodotoxin and lidocaine and show optimization results with both the FLIPR Membrane Potential Red and Blue Assay Kits on the FLIPR Tetra^® System.

Materials

• Cell line: Voltage-gated sodium channel Na _V 1.5 stably expressed in CHL (Chinese hamster lung cells)
• Culture Media: Dulbecco’s Modified Eagle’s Media (Thermo Fisher cat. #11995), 5% Fetal Bovine Serum (Hyclone cat. #SH30071.03), 1% Pen/Strep/Glutamine (Thermo Fisher cat. #103786), 450 μg/mL Geneticin (Thermo Fisher cat. #10131-035)
• Assay Buffer: HBSS with no phenol red (Thermo Fisher cat. #14025092) and 20 mM HEPES (Thermo Fisher cat. # 15630080)
• FLIPR Membrane Potential Assay Kits (Molecular Devices, Red cat. #R8123, Blue cat. #R8034)
• 384-well black wall, sterile, TC treated, clear bottom plates (Corning cat. #3072)
• Sodium channel modulators:
  • Veratridine (Sigma Cat. #V-5754)
  • Lidocaine (Sigma Cat. #L-7757)
  • Tetrodotoxin (Sigma Cat. #T-8024)
• FLIPR Tetra System (Molecular Devices)

Methods

Assay plate preparation

To create 384-well assay plates, NaV1.5 cells were trypsinized, re-suspended in culture media, and plated at 12,500 cells/well in 25 μL per well in black-wall, clear-bottom, 384-well plates. Microplates were incubated overnight at 37°C in 95% humidity, and 5% CO₂.

FLIPR Membrane Potential assays

To prepare the membrane potential assay, dye-loading buffer for two plates was prepared by completely dissolving the contents of one FMP Red Assay explorer-kit reagent vial and one FMP Blue Assay explorer-kit reagent vial, each with 10 mL assay buffer. Cell plates were removed from the incubator, and 25 μL of red dye-loading buffer was added directly to each well in the plate without removing culture media or washing. Dye-loaded plates were incubated for 30 minutes at 37°C prior to assay on the FLIPR Tetra System. The FLIPR Tetra System was prepared using the setup parameters, as listed in Table 1. In polypropylene 384-well plates, a concentration-response series containing 5X concentrations of veratridine, a voltage dependent sodium channel opener, and 5X concentrations of sodium channel blockers lidocaine, and TTX, were prepared. On the FLIPR Tetra System, veratridine was added to a Na_V1.5 cell plate to open the sodium channel and record the change in fluorescence as the cell membranes depolarized. The effective concentration for an 80% maximal response (EC₈₀) for veratridine was determined. Offline, 12.5 μL of 10X channel blocking compounds were added to additional dye-loaded cell plates and incubated at room temperature for 15 minutes. On the FLIPR Tetra System, 5X veratridine was added at EC₈₀ concentration, and the change in fluorescence was recorded during membrane depolarization. After the assay was complete, the IC₅₀ concentrations of lidocaine and tetrodotoxin were determined. Results were graphed using GraphPad Prism 6 graphing software.

Table 1. FLIPR Tetra System Settings for Membrane Potential Assays (384-well plate).

Results

Veratridine: channel opener

In an electrophysiology assay, voltage is applied to the cell membrane to drive the sodium channel into its open state followed by a rapid movement into the inactivated state. By comparison, in membrane potential fluorescence assays, veratridine is used to hold the sodium channel in its open state, preventing inactivation through binding to site two of the six topologically separated toxin binding sites that have been described.⁵ Rapid influx of Na⁺ into the cell subsequently depolarizes the membrane leading to an increase in fluorescence. As shown in Figure 1, comparison of the cellular response to veratridine using the FMP Red and Blue Assay Kits is similar. EC₅₀ concentration using the FMP Red Assay Kit is slightly right-shifted at 26.6 μM compared to 20.6 μM with the FMP Blue Assay Kit.

Figure 1. Na_v1.5 channel response to veratridine. The EC80 concentration was determined to be 33 µM and veratridine was used as the voltage channel opener in the blocking assay.

Tetrodotoxin: channel blocker

Tetrodotoxin (TTX) is a potent sodium channel blocker isolated from Japanese puffer fish. A 5X dose-response series is added to the Na_V1.5 cells to block channel opening 15 minutes prior to addition of the EC₈₀ dose of veratridine. Results showed that FMP Red IC₅₀ was 6.2 μM and FMP Blue IC₅₀ was 6.3 μM. The change in fluorescence was equivalent between FMP Assays (Figure 2). In addition, Z factors⁶ for FMP Red Assay Kit were 0.68 and 0.78 for the blue Assay Kit. A dose concentration response curve for TTX collected using an electrophysiological assay, generated using the IonWorks^® Quattro System from Molecular Devices (Figure 3), shows an IC₅₀ value of 2.4 μM. The membrane potential assay results are similar to published conventional patch-clamp data.¹

Figure 2. Modulation of Na_v1.5 channel in CHL cells by tetrodotoxin. Comparison of tetrodotoxin IC₅₀ curves using FLIPR Membrane Potential Red and Blue Kits.

Figure 3. Tetrodotoxin concentration-response curve by electrophysiological assay. Dose-response curve for TTX using an electrophysiological assay. An IC₅₀ value of 2.4 μM was obtained using an IonWorks HT instrument. The single hole per well PatchPlate^™ substrate was used and 12 wells were pooled for each data point. Mean ± SD of the fraction of current remaining after compound addition is plotted.

Lidocaine: use-dependent channel blocker

Lidocaine is a local anesthetic that works by blocking sodium channels in a use dependent manner. Use dependent inhibition is seen in IonWorks electrophysiological data in Figure 4, Panel A comparing currents from Pulse 1 to Pulse 10 and showing pulse train and raw Na⁺ currents. Dose-response curves for lidocaine are shown in Figure 4, Panel B. IC₅₀ values measured at Pulse 1 vs. Pulse 10 are 886 μM and 262 μM, respectively. Evidence of use dependence is present because, as the pulse train progresses from Pulse 1 to Pulse 10, the closed, open, and inactivated states are cycled and the cumulative amount of time that lidocaine is exposed to the open and inactivated state increases. Increased exposure to lidocaine manifests itself as a leftward shift in the measured dose-response curve for Pulse 10.

Figure 4. Measuring use dependence. (A) Voltage pulse protocol used to measure use-dependence (left). A ten-pulse protocol is used to elicit currents from a holding potential of -100 mV to the test potential of -20 mV. Raw Na⁺ current traces for Pulse 1 (top right) and Pulse 10 of a pulse train voltage protocol. Data was collected using an IonWorks Quattro^™ instrument running in Population Patch Clamp^™ (PPC) mode. PPC measures the average current from the cells sealed to 64 holes present in each well. Each data point represents the current in a single PPC well showing the fraction of current remaining after compound addition is plotted

The FLIPR Membrane Potential Assay Kit results in Figure 5 show that the FMP Red IC₅₀ is approximately 52.4 μM and FMP Blue IC₅₀ is approximately 135.6 μM. Z’ factors were equivalent between the FMP Red Assay Kit and FMP Blue Assay Kit. Higher potency measurements in this assay is expected compared to the electrophysiology assay because veratridine locks the channel in the open conformational state, maximizing the exposure of the channel to lidocaine and thus increasing its apparent potency. Follow-up screening by automated patch clamp methods on compounds of interest identified in the FLIPR assay is recommended.

Figure 5. Modulation of Na_v1.5 channel in CHL cells by lidocaine. Comparison of lidocaine IC₅₀ curves. Note that, due to use dependence in the Na_V1.5 channel, IC₅₀ values from the FLIPR Membrane Potential Assay Kits are left-shifted when compared to patch-clamp results shown in Figure 4.

Conclusion

Veratridine can be used instead of voltage to open sodium channels as part of a high throughput membrane potential screening strategy to identify modulating compounds. Compared to the IC₅₀ value at Pulse 1 in the electrophysiology assay, IC₅₀ values of lidocaine in FMP assays are much smaller, which is consistent with previous publications and suggests most Na_V1.5 channels in CHL are not at closed state. With two kit options available, it is possible to optimize a membrane potential assay to provide the best results for specific cell types or compound classes. In this study, both FLIPR Membrane Potential Red and Blue Assay Kits provided good results with veratridine, as well as with tetrodotoxin and lidocaine.

References

1. J. Satin, J.W. Kyle, M. Chen, P. Bell, L.L. Cribbs, H.A. Fozzard, et al. A mutant of TTX-resistant cardiac sodium channels with TTX-sensitive properties. Science 1992; 256:1202-1205.
2. W.A. Catterall, A.L. Goldin, S.G. Waxman. International Union of Pharmacology. XLVII. Nomenclature and structure-function relationships of voltage-gated sodium channels. Pharmacol Rev 2005; 57:397-409.
3. B. Hille. Ion Channels of Excitable Membranes, Edn. Third. (Sinauer Associates, Sunderland; 2001).
4. C. Wolff, B. Fuks, P. Chatelain. (2003) Comparative Study of Membrane Potential Sensitive Fluorescent Probes and Their Use in Ion Channel Screening Assays. J Biomol Screen, 8, 533 – 543.
5. S. Cestele, R.B. Ben Khalifa, M. Pelhate, H. Rochat, D. Gordon. Alpha-scorpion toxins binding on rat brain and insect sodium channels reveal divergent allosteric modulations by brevetoxin and veratridine. J Biol Chem 1995; 270: 15153-15161.
6. J. Zhang, et. al. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays, J Biomol Screen 1999, Vol. 4, (2) 60-73.

简介

Carole Crittenden | Applications Scientist | Molecular Devices

电压门控离子通道表达于心脏、骨骼肌、脑神经细胞的可兴奋性细胞膜上。阻断或调控这类通道可能会有治疗效应，或也许干扰到正常细胞功能。从而，可以影响电压门控离子通道的化合物成为药物研发中重要的靶标。心肌Nav1.5通道被归类为

“河豚毒素（TTX）耐受型”。1 Nav1.5 通道药理学意义在于其是抗心律失常药激 活的靶标且被局麻药如利多卡因阻断。2

状态依赖性与使用依赖性

Nav1.5通道展现对一些化合物例如利多 卡因的抑制效应具有出状态依赖性和使用 依赖性。状态依赖性抑制意味着阻断剂更 易结合处于某种特殊电压依赖性构象状态 （例如关闭、开放、失活）的离子通道。 使用依赖性抑制指的是特殊化合物在重复 性刺激条件下抑制效应的蓄积，比如一串 电压脉冲。 串刺激脉冲可引起通道循环变 化，使得电压依赖性构象状态给予了使用 依赖性化合物更多蓄积效应结合到相应的 结合位点。3 状态和使用依赖性现象导致 了一些化合物在膜电位实验和膜片钳数据 之间IC50浓度的改变。

FLIPR® 膜电位 (FMP) 检测试剂盒提供快 速、可靠的基于荧光方法的检测化合物调 节或阻断电压门控离子通道引起的膜电位 变化。早前实验已表明检测化合物与膜电 位染料之间的相互作用可能引起荧光效应 的变化，从而导致对化合物活性评估的改 变。4 Molecular Devices已开发出两种 不同类型的免洗FLIPR膜电位检测试剂 盒：蓝色和红色。每种试剂盒采用专有的 指示剂染结合不同的淬灭剂，以最大化细 胞系/通道/化合物适用性以消除数据中存 在的差异性。基于化合物库中不同的化学 特性、化合物自发荧光的差异、或染料在 细胞类型或受体上的效应，一种类型染料 的效应可能明显强于另外一种。

N在方法开发阶段，可以通过使用化合物 库中一种代表性的样品来检测两种试剂盒 的表现，从而评估最优的化合物效应。心 肌 Nav1.5通道被用来在FLIPR TETRA® 系统上演示膜电位功能性检测试验，以展 示FLIPR膜电位红色和蓝色检测试剂盒的 最佳结果。

材料

• 细胞系: 电压门控钠离子通道Nav1.5 稳 定表达的CHL（中国仓鼠肺细胞）
• 培养基：Dulbecco’s Modified Eagle’s Media (Invitrogen Cat. #11995)， 5%胎牛血清(FBS，Hyclone Cat #SH30071.03)，1% 青/链霉素/谷氨酰 胺 (Invitrogen Cat. #103786), 450 µg/mL Geneticin (Invitrogen Cat. #10131-035)
• 缓冲溶液：10X HBSS (Invitrogen Cat. #1406-5056)溶于灭菌水用于加样 (Irvine Scientific Cat. 9309)，含20 mM HEPES (Invitrogen Cat. #15630- 080)。 调节pH至7.4。
• FLIPR膜电位检测试剂盒(Molecular Devices, 红色 Cat. #R8123，蓝色 Cat. #R8034)
• 黑壁、底透、无菌、表面经过处理的 384-孔板 (Corning Cat. #3072)
• 钠通道调节剂：
  • 藜芦定(Sigma Cat. #V-5754)
  • 利多卡因Sigma Cat. #L-7757)
  • 河豚毒素(TTX，Sigma Cat. #T-8024)
• FLIPR Tetra系统 (Molecular Devices)

方法

实验板准备

准备 384-孔实验记录板，Nav1.5细胞用 胰蛋白酶消化、在培养基中重悬并按照每 孔25 µL、12500个细胞铺在黑壁底透的 384孔板中。37°C、95% 湿度和5% CO2 条件下在培养箱中过夜培养。

FLIPR膜电位实验

步骤1：准备10块板加载染料的缓冲液，100mL缓冲液可以完全溶解一瓶FMP红色大包装试剂盒。

表1. FLIPR Tetra系统在膜电位检测试验中的设置（384孔）

结果

藜芦定：通道开放剂

在电生理实验中，在细胞膜上施加电压刺 激以开放钠离子通道，然后快速进入失活 状态。作为对比，在膜电位荧光实验中， 藜芦定通过结合相应位点被用来维持钠通 道开放状态以阻止其失活，这两个位点在 文献中已有报道5，是六个跨膜螺旋中已 分离出的毒性结合位点。Na+快速内流进 入细胞引起细胞膜去极化，从而导致荧光 信号增强。如图1所示，FMP红色与蓝色 试剂盒检测的藜芦定细胞效应的结果是相 似的。相比于FMP红色试剂盒20.4 µM 的 EC50值，FMP蓝色试剂盒的EC50值是 37.2 µM ，有轻微的右移。

图1. 藜芦定在Nav1.5 通道上的效应。藜芦定EC50曲线的比较 (A) FLIPR 膜电位红色试剂盒结果。 (B) FLIPR 膜电位蓝色试剂盒结果显示藜芦定效应存在轻微的差异。

河豚毒素：通道阻断剂

河豚毒素是一种源自日本河豚鱼的强效的 钠通道阻断剂。加入藜芦定15分钟前， 10X剂量效应的梯度浓度被加入细胞中以 阻断通道开放。结果显示FMP红色试剂 盒IC50值为0.79 µM，FMP蓝色试剂盒 IC50值为2 µM 。FMP红色试剂盒检测过 程中的荧光信号变化更强烈(图2)。另 外，FMP红色试剂盒检测结果中的Z因子 值6也比FMP蓝色试剂盒更高。TTX量效 曲线结果来自于Molecular Devices公司 的IonWorks®系统的电生理实验数据(图 3)，IC50值为2.4 µM。膜电位实验结果与 已发表的传统膜片钳数据1是一致的。

图2. TTX对CHL细胞上表达的Nav1.5 的调控。河豚毒素IC50曲线的比较。 (A) FLIPR 膜电位红色试 剂盒。 (B) FLIPR 膜电位蓝色试剂盒。在FMP红色试剂盒检测结果中可得到更高的Z因子值

图3. 电生理实验结果的TTX量效曲线。量效曲线结果来自于Molecular Devices公司的IonWorks®系 统的电生理实验数据，IC50值为2.4µM。

利多卡因：使用依赖性的通道阻断剂

利多卡因是一种局麻药，其以使用依赖性 的方式阻断钠离子通道。图4中展示了 IonWorks电生理数据的使用依赖性特 点。其中A图中比较了脉冲1和脉冲10并 展示了一串电压刺激脉冲以及Na+原始电 流。图4的B图中显示了利多卡因的量效 曲线。脉冲1和脉冲10测得的IC50值分别 为886 µM和262 µM。这可以作为一个明 显的使用依赖性的证据。因为在从脉冲1 到脉冲10的一串电压刺激过程中，关闭、 开放、和失活状态循环出现，从而利多卡 因暴露在开放和失活状态下的时间长度逐 渐增加。显示在脉冲10的量效曲线中，利 多卡因的效价显著左移。

图4. 使用依赖性检测。 (A) 用于检测使用依赖性的电压刺激脉冲方案（左），共10个脉冲。脉冲1的 原始Na+电流图（右上）和10个脉冲串刺激的电压方案。(B) 显示了利多卡因的量效曲线。脉冲1和脉 冲10测得的IC50值分别为886 µM和262 µM。

FLIPR膜电位检测试剂盒在图5中的结果 显示FMP红色试剂盒的IC50是955 µM以 及FMP蓝色试剂盒的IC50是51.3 µM。 FMP红色试剂盒结果中的Z因子值也高于 其它FMP蓝色试剂盒结果。本试验中得 到的高效价检测结果可能是由于藜芦定锁 定了通道保持在开放构象状态下，最大限 度增加了通道对利多卡因和TTX的暴露， 从而显著增加了两者的效价。推荐采用全 自动膜片钳方法在FLIPR实验完成验证后 对感兴趣的化合物进行二次筛选。

图5. 利多卡因对CHL细胞上表达的Nav1.5的调控。利多卡因IC50曲线的比较。FLIPR 膜电位红色试剂 盒(A) 产生更强的荧光信号变化以及更敏感的利多卡因效应，相比于FLIPR膜电位蓝色试剂盒(B)。

结论

作为高通量膜电位筛选策略的一部分，藜 芦定用来代替电压去开放钠通道，去确证 调节性化合物。通过通道阻断剂TTX和使 用依赖性阻断剂利多卡因的证实，IC50浓 度可能产生变化，因而在FLIPR Tetra系 统上被确证的感兴趣化合物需要通过全自 动或传统膜片钳方法进一步验证。FLIPR 膜电位检测试剂盒有两种模式可供选择， 其可以针对特殊的细胞类型或化合物种类 提供最优的膜电位检测结果。在本研究 中，FMP红色和蓝色试剂盒在藜芦定基 础上均产生了好的结果。

当加入TTX和利多卡因之类的化合物之后，FMP红色试剂盒具有更好的结果。

参考文献

1. J. Satin, J.W. Kyle, M. Chen, P. Bell, L.L. Cribbs, H.A. Fozzard, et al. A mutant of TTX-resistant cardiac sodium channels with TTX-sensitive properties. Science 1992; 256:1202-1205.
2. W.A. Catterall, A.L. Goldin, S.G. Waxman. International Union of Pharmacology. XLVII. Nomenclature and structure-function relationships of voltage-gated sodium channels. Pharmacol Rev 2005; 57:397-409.
3. B. Hille. Ion Channels of Excitable Membranes, Edn. Third. (Sinauer Associates, Sunderland; 2001).
4. C. Wolff, B. Fuks, P. Chatelain. (2003) Comparative Study of Membrane Potential Sensitive Fluorescent Probes and Their Use in Ion Channel Screening Assays. J Biomol Screen, 8, 533 – 543.
5. S. Cestele, R.B. Ben Khalifa, M. Pelhate, H. Rochat, D. Gordon. Alpha-scorpion toxins binding on rat brain and insect sodium channels reveal divergent allosteric modulations by brevetoxin and veratridine. J Biol Chem 1995; 270: 15153-15161.
6. J. Zhang, et. al. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays, J Biomol Screen 1999, Vol. 4, (2) 60-73.

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