Introduction
Cav1.3 is a L-type voltage-gated calcium channel and an important therapeutic target for drug discovery. It has been shown that a number of drugs exhibit statedependent effects on Cav1.3 channels1 , meaning the potency of these drugs vary in response to the membrane voltage (Vm) and the consequent change of channel states (open, close, inactivated). As this likely provides highly-desired selectivity for pathologically over-activated Cav1.3 channels, there is a growing demand for the development of high-throughput assays to evaluate channel blockers under different states.
Current screening methodologies for this channel utilize either electrophysiology or fluorometric methods using potassium challenge to modulate membrane potential, yet both approaches have significant limitations. Electrophysiology directly controls Vm, but it is invasive in nature and not much amenable to highthroughput assays, whereas fluorometric assays bear poor physiological relevance and is not reversible.
To address these limitations, we report here a novel solution using optogenetic methods to control Vm via channelrhodopsin-2 (ChR2).2 ChR2 functions as a light-gated non-selective cation channel, primarily permeant to sodium (Na+ ) ions. Upon activation by blue light (470 nm), ChR2 mediates Na+ influx which elicits prolonged membrane depolarization driving the Cav1.3 channel first to the open state and then over time to the inactivated state. The membrane potential repolarizes following the termination of light, which drives the Cav1.3 channel back to its resting state.
In this study, we demonstrate the novel utility of optogenetic tools to control Vm in a reversible and precise fashion for screening state-dependent calcium channel blockers using the FLIPR Tetra® System.
Materials
- HEK-293 cell line co-expressing the ChR2D156A (derived from Chlamydomonas reinhardtii), human Cav1.3 α1 + α2δ, β subunits, and human Kir2.3 was developed by Axxam S.p.A., Milan, Italy. Expression of Kir2.3 sets the Vm initially to a hyperpolarized value, which depolarizes in response to either extracellularly-applied high potassium (acting upon Kir2.3), or blue light stimulation of ChR2 in the presence of all-trans retinal cofactor. In either case, depolarized Vm causes Cav1.3 to open, allowing calcium entry into the cell.
- FLIPR Tetra System (0310-5147), FLIPR® Calcium 6 Assay Kit (R8190), and FLIPR® Membrane Potential Assay Kit (R8042), are all from Molecular Devices, LLC.
Methods
A powerful application of optogenetics is controlling Vm using light-sensitive actuators. In this study, the light-sensitive ChR2 protein from C. reinhardtii is used to control Vm as the basis of a highthroughput assay for state-dependent Cav1.3 blockers. Specifically, the ChR2D156A mutant is activated by blue light (470 nm) from the LEDs on the FLIPR Tetra System, which induces a robust and prolonged membrane depolarization due to its permeability to Na+ , and extended time constant for the open state of about 6.9 minutes3 . As a result of depolarized Vm, the Cav1.3 channel first opens allowing Ca2+ ions to enter into the cell, (Figure 1) and over time transits to the non-conductive inactivated state (Figure 2). The kinetic changes in intracellular calcium are monitored using the FLIPR Calcium 6 Assay Kit on the FLIPR Tetra Instrument.
Figure 1. HEK-293 cells transfected with Kir2.3, Cav1.3, and ChR2. The influx of calcium ions through Cav1.3 is obtained upon depolarization of Vm which can be achieved either with a high K+ application, or with light stimulation of ChR2.
Figure 2. The Cav1.3 channel cycles among different states, depending on Vm. When the membrane potential is hyperpolarized, the channel is in its “closed” resting state. Upon Vm depolarization, the channel opens allowing Ca2+ ions to enter the cell, then immediately progresses to the “inactivated” refractory state. Upon Vm repolarization, the Cav1.3 channel recovers from the inactivation state and returns to the closed state, being then ready to respond again to Vm depolarization stimulus.
Control of Vm by ChR2
As a first step to validate the control of Vm by ChR2, FLIPR Membrane Potential Dye (Ex 530 nm/Em 565 nm) is used to measure change in fluorescent signal as a result of change in Vm. HEK-293 cells transfected with ChR2, Kir2.3, and Cav1.3 are incubated with FLIPR Membrane Potential dye following the standard protocol, in the presence of all-trans retinal co-factor. Using the FLIPR Tetra System, membrane depolarization is induced by stimulating the ChR2 channel with light pulses from the blue LEDs. A second protocol is simultaneously performed which excites the membrane potential dye and measures the change in fluorescent emission over time. The membrane depolarization is reflected by the increase in fluorescent signal. After the termination of the first period of blue light stimulation, ChR2 channels progress to the closed state, allowing Vm to repolarize, resulting in decreased fluorescent signal over time.
Control of Cav 1.3 by ChR2
HEK-293 cells transfected with ChR2, Kir2.3, and Cav1.3 are first loaded with FLIPR Calcium 6 dye, in the presence of all-trans retinal co-factor. Using the FLIPR Tetra System, the cells are pulsed with blue light to stimulate ChR2 and the following change in fluorescence reflects calcium entry into cells through Cav1.3. In subsequent experiments, a dual-pulse protocol was employed, where the second light pulse was applied after varying time intervals after the first pulse to study recovery of Cav1.3 from inactivation.
Control of Cav1.3 by potassium challenge
As a reference, the standard method of analyzing Cav1.3 channel activity by potassium challenge is performed on the same cell line. When high potassium is applied extracellularly, the membrane potential depolarizes allowing the Cav1.3 channel to open and calcium ions to enter the cell (Figure 1). Over time, Cav1.3 reaches an inactive, non-conductive state. FLIPR Calcium 6 dye is used to measure the increase in intracellular calcium. This method requires the application of high concentration of K+ to the cells which is non physiological and can have many “off-target” effects. In addition, such application is irreversible as the potassium ions can’t be removed from the well.
Pharmacology
To validate the utility of optogenetics in pharmacological assays, a set of experiments were performed using the state-dependent inhibitor isradipine to block the Cav1.3 channel and to analyze IC50 results at both the closed and inactivated states. Three methods of activating the Cav1.3 channel including optogenetics, high potassium addition based on Kir2.3, and patch clamp are compared to determine isradipine IC50 values at both states
Results
Control of Vm by ChR2
Control of membrane potential by lightactivated ChR2 was validated by incubating ChR2 transfected HEK-293 cells with FLIPR Membrane Potential dye. Pulses of blue light from the FLIPR Tetra System LEDs resulted in membrane depolarization and a 3-fold increase in signal from the FLIPR Membrane Potential dye (Figure 3a). Recording the membrane potential signal over time showed that fluorescent signal returned to background levels after about 30 minutes from the initial blue light stimulation as the membrane repolarized. This data suggests that when blue light stimulated ChR2 to open, Vm was depolarized first and over time Vm repolarized as the ChR2 channel closed (Figure 3b), accordingly to its time constant of 6.9 minutes
Figure 3. (A) Light-induced depolarization. Depolarization of Vm measured as change in FLIPR Membrane Potential dye fluorescence upon the application of blue light. (B) Time course experiment showing the change in maximum FLIPR Membrane Potential dye signal over time. ChR2 Cav1.3 HEK-293 cells were incubated with FLIPR Membrane Potential dye. Stimulation of ChR2 by pulses of blue light induced membrane depolarization and resulted in an initial increase in fluorescent signal from the dye (T=0). Following the fluorescent signal over time showed a drop in signal indicating membrane repolarization.
ChR2 induced Cav1.3 response
Transfected HEK-293 cells were incubated with FLIPR Calcium 6 dye. Pulses of light from the blue LEDs triggered an increase in fluorescent signal as calcium ions entered the cell (Figure 4a). Subsequent experiments (Figure 4b) in which the cells were subjected to a dual-pulse protocol were used to identify the timedependence of Cav1.3 channel recovery from inactivation. After a 10 min interval, half the calcium flux signal was recorded. After a 45 minute interval, nearly 100% of the signal was recorded. The data suggests that 10 minutes after the first light pulse, half the channels were still in the inactivated state thus resulting in 50% of maximum calcium signal.
Figure 4. (A) ChR2 induced Cav1.3 response after blue light stimulation. The signal trace from the FLIPR Tetra System records the increase in fluorescence of FLIPR Calcium 6 dye as calcium enters the cell though Cav1.3. (B) Cav1.3 recovery from the inactivated state. A dual-pulse protocol was applied here, where the first pulse of blue light was applied at t = 0, then cells received a second pulse after incremental intervals as indicated and calcium flux was measured. Data showing half the initial calcium signal suggests that the half-inactivated state of the channel occurs with the second light pulse delivered 10 minutes after the first stimulation.
Potassium Challenge
To control the Cav1.3 channel state with K+ , the transfected HEK-293 cell line was incubated in FLIPR Calcium 6 dye loading buffer with increasing potassium concentrations from 4 mM to 75 mM for one hour at 37°C resulting in different membrane potentials (Vm). Then a high K+ concentration of 75 mM was added during detection of calcium signal on the FLIPR Tetra System. Representative calcium signal responses are shown in Figure 5a. A K+ dependent inactivation curve plotting the K+ loading concentration vs. the signal from the FLIPR Calcium 6 dye revealed the concentration of K+ to induce 50% of maximum Ca2+ signal is at 22 mM K+ (Figure 5b), suggesting that half the channels were in the inactivated state in this K+ loading condition.
Figure 5. (A) Cells loaded with FLIPR Calcium 6 dye in different [K+ ] to induce different starting voltages. 75 mM K+ added during detection of calcium signal on FLIPR Tetra System. (B) K+ inactivation curve. By incubating the cells in dye with 22 mM K+ , 50% of the channels are in the inactivated state.
Pharmacology
Isradipine, an anti-hypertension drug that may also be useful in slowing the progression of Parkinson’s disease, was chosen for this study due to its known state-dependent effect on Cav1.3. Using the optogenetic protocol with ChR2, HEK-293 cells expressing Cav1.3 were incubated with FLIPR Calcium 6 dye for one hour at 37°C, in the presence of different concentrations of isradipine. Blue light pulses were given on the FLIPR Tetra System and increase in fluorescent signal with calcium influx at the closed state was measured. After 10 minutes, a second pulse of blue light was given recording the change in fluorescence with calcium influx at the inactivated state. Only half of the original calcium signal was seen. Results are shown as % Cav1.3 response on the graph in Figure 6a. Isradipine IC50 curves from experiments following the high K+ protocol are shown in Figure 6b. Based on earlier potassium experiments, 4 mM K+ or 22 mM K+ was added to the FLIPR Calcium 6 dye loading buffer to induce closed and inactivated states, respectively. 75 mM K+ was added during detection of calcium signal on the FLIPR Tetra System. In Figure 6c, IC50 curves of isradipine block of Cav1.3 at both the closed and inactivated states using a patch clamp protocol are shown.
Figure 6. Isradipine state dependency impact upon pharmacology. (A) Optogenetic protocol. At the closed state (first blue light stimulation), isradipine IC50 = 149 nM. At the inactivated state (cells re-pulsed with blue light after 10 min), isradipine IC50 = 15 nM. (B) High potassium protocol. 75 mM K+ was added during detection. At the Cav1.3 closed state (4 mM K+ load), isradipine IC50 = 26 nM. At the inactivated state (22 mM K+ load), isradipine IC50 = 4 nM. (C) Patch clamp protocol. At the Cav1.3 closed state (-90mV holding potential) the isradipine IC50 = 362 nM, and at the inactivated state (-60mV holding potential) the isradipine IC50 = 32 nM.
Summary
A comparison of isradipine IC50 values at both closed state and inactivated state including historical patch clamp results is shown in Table 1. An approximately 10-fold left shift in IC50 values at the inactivated state of Cav1.3 compared to the values at the closed state is preserved with all methods. This is important because compound effects upon ion channels can have different clinical implications at closed and inactivated states. With the optogenetic protocol, the IC50 values are closer to the IC50 values obtained with “gold standard” electrophysiology methods. The “ratio block” value obtained with optogenetics is very similar to that obtained with electrophysiology methods suggesting high biorelevance. Unlike electrophysiology assays, the cells in the optogenetics assay are intact at the end of the experiment. In addition, the light-driven optogenetic protocol is reversible, a highly desirable feature for flexible assay protocols.
| Isradipine IC50 | Optogenetics | High K+ | Patch Clamp | Literature4 |
|---|---|---|---|---|
| Closed State | 149 nM | 26 nM | 362 nM | 300 nM (-90 mV) |
| Inactivated State | 15 nM | 4 nM | 32 nM | 30 nM (-50 mV) |
| RATIO block Closed/Inactivated | 9.9 | 6.5 | 11.3 | 10 |
Table 1. Summary of isradipine IC50 results at both closed and inactivated states. The optogenetic protocol results are the closest to electrophysiological and literature results.
Acknowledgement
Experimental work and data figures were provided by Axxam S.p.A., Milan, Italy
Reference
- Koschak, A., et al, J. Biol. Chem. 2001, 276:22100-22106
- Prigge, M., Rossler, A., Heggeman, P., Channels. 2010, May/June, 4:3, 241-247
- Berndt, A., Yizhar, O., Gunaydin, L., Hegemann, P., Deisseroth, K., Nat Neurosci. 2009, Feb;12(2):229-234.
- Catterall WA, Perez-Reyes E, Snutch TP, Striessnig J. Pharmacol Rev. 2005 Dec;57(4):411-25.
介绍
Cav 1.3 是一种 L 型电压门控钙通道，是药物 发现的重要治疗靶点。有研究表明，一些药 物对 Cav 1.3 通道产生了状态依赖性的效应1 ， 这意味着这些药物的效应随着膜电位的变化 (Vm) 以及随之而来的通道状态的改变 ( 开放、 关闭、失活 ) 而发生变化。由于这可能为病理 上过度激活的 Cav 1.3 通道提供了高度期望的 选择性，因此，对高通量分析方法的开发需 求日益增长，以评估通道阻断剂的不同状态 下的效应。
目前的筛选方法采用电生理学或荧光法，利 用钾离子来调节膜电位，但两种方法都有很 大的局限性。电生理学直接控制 Vm，但它是 侵入性的且并不是很适合高通量的检测分析 模式，而荧光测定法则具有较差的生理相关 性，而且是不可逆的。
为了解决这些局限性，我们在这里报告了一种 新的解决方案，它利用光遗传学方法通过光 敏感通道 -2 (channelrhodopsin-2，ChR2)2 来控制 Vm。ChR2 是一种光门控机制的非选 择 性 阳 离 子 通 道 ， 主 要 用 于 通 透 钠 离 子 (Na+ )。当通过蓝光 (470 nm) 激活时，ChR2 介导 Na+ 的流入，从而延长了膜的去极化，使 Cav 1.3 通道首先进入开放状态，然后逐渐进 入失活状态。在光激活终止后，膜电位再极 化，这将使 Cav 1.3 通道回到它的静息状态。 在这项研究中,我们将使用 FLIPR 系统展示一 种光遗传学方法的新应用来控制 Vm，进行可 逆且精确的方式来筛选状态依赖性钙通道阻 断剂。
材料
- HEK-293 细胞系联合表达 ChR2D156A ( 来源于莱茵衣藻 )，人源的 Cav 1.3 α1 + α 2δ,Β 亚基，以及人源的 Kir2.3 是由意大 利米兰的 Axxam S.p. A 提供。Kir2.3 的 表达首先将 Vm 设置为超极化值，在全 反式视网膜辅因子存在的情况下，它可 以对胞外高钾离子 ( 作用于 Kir2.3 )，或 蓝色光刺激的 ChR2 进行去极化。在这两 种情况下，去极化的 Vm 都会导致 Cav 1.3 打开，允许钙进入细胞。
- FLIPRTETRA 系统 (0310 - 5147), FLIPR® 钙 6 检测试剂盒 (R8190) 和 FLIPR® 膜电位 检 测试剂盒 (R8042)，均来自于都 从 Molecular Devices 公司。
方法
光遗传学的一个强大应用是利用光敏驱动 器控制 Vm。在本研究中，C. reinhardtii 的光敏 ChR2 蛋白被用于控制 Vm，作为 对状态依赖性 Cav 1.3 阻断剂的高通量分析 检测的基础。具体地说，ChR2D156A 突变体 在 FLIPR Tetra 系统上被其 LED 所发出的 蓝光 (470 nm) 激活，这使得由于其对 Na+ 的渗透性和开放状态下约为 6.9 分钟的延 长的时间常数3，导致了一种超强的且被 延长的膜去极化。由于Vm的去极化， Cav 1.3通道首先打开允许 Ca2+ 离子进入细 胞，( 图 1 )，并随着时间的推移进入失活 状态 ( 图 2 )。使用 FLIPR 钙 6 检测试剂盒 观察细胞内钙离子的动态变化。
图 1 Kir2.3、Cav1.3 和 ChR2 转染至 HEK-293 细胞上。 高K+ 的应用，或者是对 ChR2 的光刺激对 Vm 进行去极化，从而获得 了钙离子通过 Cav 1.3 流入胞内。
图 2 依据 Vm，不同状态下 Cav1.3 的通道周期。当膜电位超极化时，通道处于其“闭合”静息状 态。在 Vm 去极化后，通道打开，允许 Ca2+ 离子进入细胞，然后立即进入“未激活”的不应状态。 在 Vm 重新极化后，Cav 1.3 通道从失活状态恢复，返回到封闭状态，然后准备再次响应 Vm 去极化 刺激。
用 ChR2 控制 Vm
作为验证 Vm 控制的第一步，用 FLIPR 膜 电位染料 (Ex 530 nm/Em 565 nm) 测量 荧光信号的变化，这是Vm变化的结果。 HEK-293 细胞经 ChR2、Kir2.3、Cav 1.3 转 染后，在全反式视网膜辅因子存在的情况 下，按照标准流程 FLIPR 膜电位染料孵 育。利用 FLIPRTETRA 系统，利用蓝色发光 二极管的光脉冲刺激 ChR2 通道来诱导膜 去极化。第二种方案是同时进行的，它能 激发膜电位染料，并随着时间的推移测量 荧光发射的变化。荧光信号的增加反映了 膜的去极化。在第一次蓝光刺激结束 后，ChR2 通道进入关闭状态，允许 Vm 重新极化，从而导致荧光信号随时间减 少。
ChR2 对 Cav 1.3的控制
HEK-293 细胞经 ChR2、Kir2.3、Cav 1.3 转 染后，首先在全反式视网膜辅因子存在的 情况下，加入了 FLIPR 钙 6 染料进行孵 育。使用 FLIPR Tetra 系统，细胞用蓝光 脉冲刺激 ChR2，而荧光的变化则通过 Cav 1.3 反映钙进入细胞。在随后的实验中， 使用了双脉冲刺激方案，在第一次脉冲 后，第二次光脉冲在第一次脉冲后应用于 失活的 Cav 1.3 恢复。
高钾条件下对 Cav 1.3 的调控
作为参考，在同一细胞系上进行了高钾条 件下分析 Cav 1.3 通道活性的标准方法。 当高钾离子被添加到细胞外液中时，膜电 位去极化使得 Cav 1.3 通道打开，钙离子进 入细胞 ( 图 1 )。随着时间的推移，Cav 1.3 进入失活的非导电状态。用 FLIPR 钙 6 染 料测量细胞内钙的增加。该方法要求高浓 度的 K+ 对非生理的细胞具有许多“非靶 向”效应。此外，这种应用是不可逆转 的，因为钾离子不能从孔板中去除。
药理学
为了验证光遗传学在药理学实验中的效 用，一组实验采用了状态依赖性抑制剂依 拉地平来阻断 Cav 1.3 通道，并在关闭和失 活状态下分析 IC50 的结果。三种方法激活 Cav 1.3 通道，包括光遗传学、基于 Kir2.3 的高钾添加、和膜片钳检测方法，比较两 种状态下的依拉地平的 IC50 值。
结果
通过 ChR2 调控 Vm
通过用 FLIPR 膜电位染料孵育 ChR2 转染 的 HEK-293 细胞，验证了光激活 ChR2 对膜电位的调控。由 FLIPRTETRA 系统LED 发 出的蓝光脉冲导致膜去极化，而 FLIPR 膜 电位染料的信号增加了 3 倍 ( 图 3a )。随 着时间的推移，膜电位信号的记录显示， 当细胞膜重新极化时，从最初的蓝光刺激 到大约 30 分钟后，荧光信号返回到背景水 平。这一数据表明，当蓝色光刺激 ChR2 打开时，Vm首先被去极化，随着时间的 推移，随着 ChR2 通道关闭 Vm 重新极化 ( 图 3b )，它的时间常数为 6.9 分钟。
图 3 (A) 光刺激诱导去极化。利用蓝色光将 Vm 的去极化作为 FLIPR 膜电位染料荧光强度改变的定 量检测。(B) 时程实验表明随着时间的推移，最大的 FLIPR 膜电位染料信号的变化。ChR2 Cav 1.3 HEK -293 细胞用 FLIPR 膜电位染料孵育。通过脉冲蓝光诱导膜去极化，使荧光信号从染料 (T = 0) 开始逐步增加。随着时间的推移，荧光结果显示了信号的下降，表明膜的再极化。
ChR2 对 Cav 1.3的控制
HEK-293 细胞经 ChR2、Kir2.3、Cav 1.3 转 染后，首先在全反式视网膜辅因子存在的 情况下，加入了 FLIPR 钙 6 染料进行孵 育。使用 FLIPR Tetra 系统，细胞用蓝光 脉冲刺激 ChR2，而荧光的变化则通过 Cav 1.3 反映钙进入细胞。在随后的实验中， 使用了双脉冲刺激方案，在第一次脉冲 后，第二次光脉冲在第一次脉冲后应用于 失活的 Cav 1.3 恢复。
图 4 (A) 在蓝光刺激后，ChR2 诱导的 Cav 1.3 反应。从 FLIPRTETRA 系统记录的信号中发现，随着钙 进入细胞，钙 6 染料的荧光信号增强。(B) 从失活态恢复的 Cav 1.3。在这里应用了双脉冲检测方案， 在 t = 0 处应用了第一个蓝色光脉冲，然后在增加间隔后，细胞获得了第二个脉冲，并测量了钙流信 号。数据表明基于半数初始钙流信号的标准，通道的半数激活状态出现于在第一次刺激 10 分钟后第 二次光脉冲诱导的信号。
ChR2 诱导的 Cav 1.3 反应
转染的 HEK-293 细胞与 FLIPR 钙 6 染料 孵育。随着钙离子进入细胞 ( 图 4A )，蓝 色发光二极管发出的光脉冲触发了荧光信 号的增加。随后的实验 ( 图 4B ) 中，使用 双脉冲检测方案的细胞来识别 Cav 1.3 通道 从失活状态恢复的时间依赖性。10 分钟间 隔后，记录到了原始钙流一半的信号。 45 分钟间隔后，几乎 100% 的信号被记录 下来。数据显示，在第一次光脉冲 10 分 钟后，一半的通道仍处于失活状态，从而 产生 50% 的最大钙信号。
钾离子的调控
用 K+ 调控 Cav 1.3 通道的状态，转染 HEK293 细胞系中加载 FLIPR 钙 6 染在 37 °C 下孵育一小时，添加缓冲液增加钾离子浓 度从 4 mM 到 75 mM 以形成不同的膜电 位 (Vm)。然后在 FLIPRTETRA 系统中检测 钙信号时添加 75 mM 的高浓度 K+ 。代表 性的钙信号响应如图5A 所示。K+ 依 赖的 失活曲线展示了与 K+ 浓度对应的 FLIPR 钙 6 染料信号，数据显示诱发的 50% 最 大 Ca2+ 信号的钾离子浓度是 22 mM ( 图 5B )，这表明在添加了此浓度的钾离子条 件下有一半的 Cav 1.3 通道处于失活状 态。
图 5 (A)+ 加载有 FLIPR 钙 6 染料的细胞在不同的 [K+ ] 条件诱导产生不同的起始电压。在 FLIPRTETRA 系统检测钙信号时添加 75 mM K+ 。(B) K+ 失活曲线。通过在染料中孵育 22 mM K+ 的细胞，50% 的 通道处于失活状态。
药理学
依拉地平是一种抗高血压药物，它也可能 有助于减缓帕金森疾病症状的发展，因其 对 Cav 1.3 的状态依赖性影响而被选为这 项研究的对象。采用 ChR2 的光遗传学检 测方案，表达了 Cav 1.3 的 HEK-293 细胞 在添加不同浓度依拉地平的情况下，用 FLIPR 钙 6 染料在 37 °C 下孵育一小时。 在 FLIPRTETRA 系统上采用蓝色光脉冲进行 刺激，检测到通道关闭状态下钙流荧光信 号的强度的增加。10 分钟后，给予了第二 次蓝色光脉冲刺激，记录到了通道失活状 态下钙流荧光信号的变化。仅观察到原始 钙流信号一半强度的荧光信号。在图 6A 中，结果显示为 Cav 1.3 通道效应的 %。高 K+ 调控方案记录的依拉地平的 IC50 曲线如 图 6B 所示。在较早的钾离子调控实验基 础上，将 4 mM K+ 或 22 mM K+ 添加到 FLIPR 钙 6 染料的加载缓冲液中，分别诱 导关闭和失活状态。在 FLIPRTETRA 系统中 检测钙信号时添加了 75 mM K+ 。膜片钳方 法下检测的 Cav1.3 通道关闭和失活状态 下依拉地平的 IC50 曲线如图 6C 所示。
图 6 依拉地平 (Isradipine) 状态依赖性对药理学的影响。 (A) 光遗传学检测方案。在通道关闭状态 下 ( 第一次蓝光刺激 )，依拉地平的 IC50 = 149 nM。在失活状态下 ( 10分钟后，细胞以蓝光重新刺激 )， 依拉地平的 IC50 = 15nm。(B) 高钾检测方案。在检测过程中添加了 75 mM K+ 。在 Cav 1.3 关闭状态 下 (4 mM K+ load)， 依拉地平的 IC50 = 26 nM。在失活状态下 (22 mM K+ load)，依拉地平的 IC50 = 4 nM。(C) 膜片钳检测方案。在 Cav 1.3 关闭状态下 ( -90 mV 钳制电位 )中，依拉地平的 IC50 = 362 nM，在失活状态 ( -60 mV 钳制电位 ) 中，依拉地平的 IC50 = 32nm。
总结
包括膜片钳结果在内的所有 Cav 1.3 通道关 闭和失活状态下依拉地平的 IC50 值得比较 如表 1 所示。与关闭状态下的值相比，所 有检测方法下得到的在失活状态下的 IC50 值的左移大约为 10 倍。这很重要，因为 对离子通道的复合效应在通道关闭和失活 状态下可能具有不同的临床意义。通过光 遗传学检测方案，IC50 值更接近于以电生 理方法获得的 IC50 值。光遗传学所获得的 “阻断比例”值与电生理学方法所获得的 值高度相似，从而提示其高生物相关性。 与电生理学不同的是，光遗传学检测中的 细胞在实验结束时完好无损。此外，光驱 动的光遗传检测是可逆的，这是灵活的分 析方法的一个非常理想的特性。
| Isradipine IC50 | Optogenetics | High K+ | Patch Clamp | Literature4 |
|---|---|---|---|---|
| Closed State | 149 nM | 26 nM | 362 nM | 300 nM (-90 mV) |
| Inactivated State | 15 nM | 4 nM | 32 nM | 30 nM (-50 mV) |
| RATIO block Closed/Inactivated | 9.9 | 6.5 | 11.3 | 10 |
表 1 在通道关闭和失活状态下依拉地平的 IC50 结果总结。光遗传检测方案的结果是最接近电生理 和文献的结果。
致谢
实 验 工 作 和 数 据 数 据 由 意 大 利 米 兰 的 Axxam S.p.A 提供。
参考文献
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