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Sodium Current Measurements in HEK293 Cells
HEK293细胞中的钠电流测定   

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Abstract

This protocol is used to measure sodium currents from heterologously transfected cells lines, such as HEK293 cells. Standard whole cell patch clamp technique is used to assess ion channel function. This protocol has been used to study cardiac type Nav1.5 sodium channel, and in particular to compare wild-type and mutant channels related to Brugada Syndrome (BrS). Mutations related to BrS provoke a loss of function of the cardiac sodium channel. This is evidenced by a reduction of sodium current density and/or alterations of the activation or inactivation kinetic parameters, such as a slow recovery from inactivation. These effects could explain the alteration of the cardiac action potential that leads to the characteristic ST elevation observed in the electrocardiogram of the Brugada Syndrome patients. This protocol, with some variations, is suitable for studying other cardiac arrhythmias related to alterations in the cardiac type sodium channel function as well as for studying other voltage-dependent sodium channels.

Keywords: Sodium current(钠电流), Patch clamp(膜片钳), Heterologous transfection(外源转染), Ion channels(离子通道), Whole cell current(全细胞电流)

Materials and Reagents

  1. Human embryonic kidney HEK293 cells (Health Protection Agency Culture Collections, catalog number: 96121229 )
  2. Vector/s: mammalian expression vectors harboring the cDNA of interest (i.e. pcDNA3 + SCN5A)
  3. Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich, catalog number: D6546 )
  4. Fetal Bovine Serum (Sigma-Aldrich, catalog number: F4135 )
  5. Penicillin-streptavidin (Life Technologies, Gibco®, catalog number: 15140-122 )
  6. GlutaMAXTM (Life Technologies, Gibco®, catalog number: 35050-038 )
  7. 0.05% Trypsin-EDTA (Life Technologies, Gibco®, catalog number: 25300-062 )
  8. Dulbecco’s Phosphate Buffered Saline (Sigma-Aldrich, catalog number: D8662 )
  9. GeneCellinTM Transfection Reagent (BioCellChallenge, catalog number: GC-1000 )
  10. Opti-MEM® Reduced Serum Media + GlutaMAXTM (Life Technologies, Gibco®, catalog number: 31985-062 )
  11. Sticky wax in bars (Cera de Reus, catalog number: 25005001 )
  12. Sodium chloride (NaCl) (Sigma-Aldrich)
  13. Potassium chloride (KCl) (Sigma-Aldrich)
  14. Cesium chloride (CsCl) (Sigma-Aldrich)
  15. Calcium chloride (CaCl2) (Sigma-Aldrich)
  16. Magnesium chloride (MgCl2) (Sigma-Aldrich)
  17. N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (Sigma-Aldrich)
  18. Ethylene glycol-bis (2-amino-ethylether)-N,N,N’,N’-tetra-acetic acid (EGTA) (Sigma-Aldrich)
  19. Adenosine 5′-triphosphate magnesium salt (ATP-Mg2+) (Sigma-Aldrich)
  20. Sodium hydroxide (NaOH) (Sigma-Aldrich)
  21. Cesium hydroxide (CsOH) (Sigma-Aldrich)
  22. Glucose (Sigma-Aldrich)
  23. Bath solution (see Recipes)
  24. Pipette solution (see Recipes)

Equipment

  1. Inverted microscope Nikon Eclipse Ti fitted for epifluorescence (Nikon Instruments Inc.)
  2. Glass capillary PC-10 Puller (Narishige International USA Inc.)
  3. Micromanipulator MP-285 (Sutter Instrument Co.)
  4. Vapor pressure Osmometer VAPOR 5520 (Wescor Inc.)
  5. Axopatch 200B Capacitor Feedback Patch Clamp Amplifier (Molecular Devices)
  6. Axon Digidata 1440A Data Acquisition System (Molecular Devices)
  7. CO2 incubator
  8. Nunclon® cell culture dishes 35 x 10 mm (Sigma-Aldrich, catalog number: D7804 )
  9. Glass capillaries (Brand GmbH + CO KG, catalog number: 7493-21 )

Software

  1. pCLAMP 10.2 Electrophysiology Data Acquisition and Analysis Software (Molecular Devices)
  2. OriginPro 8 software (OriginLab Corporation)

Procedure

  1. Cell culture and transfection
    1. The day before transfection, plate HEK293 cells in 35 mm dishes at a density that will result in 70% confluence 24 h later (this is 2.1 x 105 cells but it may vary depending on how fast your cell line grows), and maintain in a CO2 incubator at 37 °C in DMEM supplemented with 10% Fetal Bovine Serum, 1% Penicillin-streptomycin and 1% GlutaMAXTM.
    2. Perform the transfection for each 35 mm dish of HEK293 cells with a mix of 200 μl Opti-MEM® Medium, 3 μg (in total) of the vectors of interest and 4 μl of GeneCellinTM Transfection Reagent. The inclusion of a vector harboring the cDNA encoding a fluorescent protein will allow the identification of transfected cells.
      Note: Other transfection reagents and methods have been successfully used for transfecting sodium channels.
    3. Twenty-four hours after transfection, wash the each dish with 1.5 ml DPBS, then add 0.2 ml 0.05% Trypsin-EDTA and incubate at 37 °C for 2 min, cells should look separated from each other, add 1.5 ml of DMEM to the dish, disperse with a fine tip pipette and plate 200-300 μl in a new dish with 1.5 ml of media.
      Note: You should get individual cells for recording.

  2. Patch-clamp procedure (48 h after transfection)
    1. Pull pipettes from glass capillaries and finely coat the tip with melted dental wax (be careful not to occlude the tip hole). Their resistance should range from 2 to 4 MΩ when filled with the Pipette solution.
    2. Remove DMEM from the transfected cells dish that you will use and, after rinsing the cells twice with the Bath solution (approximately 3 ml in total), fill it to about one third of the height with the same solution.
    3. Choose the cells that you will record from individual fluorescent cells.
    4. Fill the pipette with the pipette solution and place it in the electrode holder. Lower the pipette to place it in the Bath solution. After compensating offsets, approach the pipette to the chosen cell with the help of the remote micromanipulator to form a high resistance cell-attached seal.
    5. Once the seal is formed and the whole cell configuration is established, compensate series resistance at 80-90%.
    6. Wait five minutes before starting to record. This allows the cell content to equilibrate with the pipette solution.
    7. For acquisition, set your filter at 5 kHz and your sampling rate at 20-25 kHz.

  3. Sodium measurements protocols
    1. Macroscopic sodium current: Currents are elicited by 50 ms depolarizing steps (from -80 to +80 mV in 5 mV increments) from a holding potential of -120 mV.

    2. Steady-state inactivation (h∞): Current is measured with -20 mV pulses (20 ms), following 50 ms pre-pulses to different potentials (-140 to +5 mV in 5 mV increments).

    3. Recovery from inactivation: current is elicited by a -20 mV, 20 ms, pulse (P2), preceded by a 50 ms depolarizing pre-pulse to -20 mV (P1) from a holding potential of -120 mV, followed by a hyperpolarizing pulse to -120 mV of increasing duration (1-100 ms).

    4. Slow inactivation: Double pulse protocol 1: Current is measured with a test pulse to -20 mV during 20 ms (P2), preceded by a depolarizing pulse from -120 mV to -20 mV (P1) of increasing duration (10-100 ms in 10 ms increments), followed by a hyperpolarizing pulse to -120 mV during 20 ms, to remove fast inactivation. Double pulse protocol 2: This protocol is the same as the previous one except for that the duration of the first depolarizing pulse to -20 (P1) runs from 100 to 2,000 ms in 200 ms increments.


  4. Sodium measurements analysis (pClamp 10.2 and OriginPro 8 software)
    1. Current density-voltage (pA/pF): The measured peak current at the different voltages applied is normalized by the cell capacitance.
    2. Activation curve: Whole cell conductance (G) is obtained from the current- density relationship (I-V) by dividing the peak current obtained at each potential by the driving force (V-Eion) at each potential. These values are normalized to the maximum conductance (Gmax) and plotted against each voltage. Data is fitted to a Boltzmann equation of the form G=Gmax/ (1 + exp[(V1/2-V)/k]) where V is the applied potential, V1/2 is the voltage at which half of the channels are activated, and k is the slope factor.
    3. Inactivation time constants: time constants (t), tslow and tfast, are obtained from fitting the currents elicited with the macroscopic sodium current protocol to a second order exponential function. At some voltages, when the current is either too fast or too small, it is not possible to fit a second order exponential. In this case a first order exponential is used. The region analyzed to obtain the time constants is comprised from the peak of the current to a point where the current has reached a plateau near zero. tslow and tfast are plotted against voltage.
    4. Steady-state inactivation curve: Peak current amplitude (I) is normalized to the maximum peak current amplitude (Imax). The I/Imax values from the test pulse are plotted against the voltage during the pre-pulse. Experimental data is fitted to a Boltzmann equation of the form I = Imax/ (1 + exp[(V-V1/2)/k]), where V is the applied voltage, V1/2 is the voltage at which half of the channels are inactivated and k is the Boltzmman constant.
    5. Recovery from inactivation: Recovery current values are obtained by dividing the peak current from P2 by the peak current at P1. P2/P1 ratios are plotted against the recovery interval times. The recovery from inactivation curve is fitted to a mono-exponential function to obtain the time constants (t).
    6. Slow inactivation: Peak current is measured at P2 and P1. The P2/P1 ratio is plotted against the depolarizing pulse interval times. The slow inactivation curve is fitted to a mono-exponential function to obtain the slow inactivation constant (t).

Recipes

  1. Bath solution
    140 mM NaCl
    3 mM KCl
    10 mM HEPES
    1.8 mM CaCl2
    1.2 mM MgCl2
    The pH is adjusted to 7.4 with NaOH
    Osmolality is adjusted by the addition of glucose to approximately 325 mOsm.
  2. Pipette solution
    130 mM CsCl
    1 mM EGTA
    10 mM HEPES
    10 mM NaCl
    2 mM ATP-Mg2+
    The pH is adjusted to 7.2 with CsOH
    Osmolality is adjusted by the addition of glucose to approximately 308 mOsm.
    Note: The difference in osmolality between Pipette and Bath solutions should be near 5%.

Acknowledgments

This protocol is adapted from Riuro et al. (2013) and Tarradas et al. (2013).

References

  1. Riuró, H., Beltran‐Alvarez, P., Tarradas, A., Selga, E., Campuzano, O., Vergés, M., Pagans, S.,Iglesias, A., Brugada, J. and Brugada, P. (2013). A Missense Mutation in the Sodium Channel β2 Subunit Reveals SCN2B as a New Candidate Gene for Brugada Syndrome. Hum Mutat 34(7):961-966.
  2. Tarradas, A., Selga, E., Beltran-Alvarez, P., Perez-Serra, A., Riuro, H., Pico, F., Iglesias, A., Campuzano, O., Castro-Urda, V., Fernandez-Lozano, I., Perez, G. J., Scornik, F. S. and Brugada, R. (2013). A novel missense mutation, I890T, in the pore region of cardiac sodium channel causes Brugada syndrome. PLoS One 8(1): e53220. 

Additional suggested bibliography

  1. Sakmann, Bert and Neher, Erwin. (1995). Single-Channel Recording. Springer-Verlag, 2nd Edition. New York.
  2. Molleman, Areles (reprinted 2008). Patch Clamping. An Introductory guide to patch clamp electrophysiology. John Wiley and Sons Ltd. England: West Sussex.

简介

该方案用于测量异源转染的细胞系如HEK293细胞的钠电流。标准全细胞膜片钳技术用于评估离子通道功能。该方案已经用于研究心脏型Na v 1.5钠通道,特别是用于比较与Brugada综合征(BrS)相关的野生型和突变型通道。与BrS相关的突变引起心脏钠通道功能的丧失。这通过钠电流密度的降低和/或活化或失活动力学参数的改变,例如从失活缓慢恢复来证明。这些效应可以解释导致在Brugada综合征患者的心电图中观察到的特征性ST升高的心脏动作电位的改变。该方案具有一些变化,适合于研究与心脏类型钠通道功能的改变相关的其它心律失常以及用于研究其它电压依赖性钠通道。

关键字:钠电流, 膜片钳, 外源转染, 离子通道, 全细胞电流

材料和试剂

  1. 人胚胎肾HEK293细胞(Health Protection Agency Culture Collections,目录号:96121229)
  2. 载体:含有感兴趣的cDNA(即pcDNA3 + SCN5A)的哺乳动物表达载体
  3. Dulbecco's Modified Eagle's Medium(DMEM)(Sigma-Aldrich,目录号:D6546)
  4. 胎牛血清(Sigma-Aldrich,目录号:F4135)
  5. 青霉素 - 链霉亲和素(Life Technologies,Gibco ,目录号:15140-122)
  6. GlutaMAX TM (Life Technologies,Gibco ,目录号:35050-038)
  7. 0.05%胰蛋白酶-EDTA(Life Technologies,Gibco ,目录号:25300-062)
  8. Dulbecco's磷酸盐缓冲盐水(Sigma-Aldrich,目录号:D8662)
  9. GeneCellin TM 转染试剂(BioCellChallenge,目录号:GC-1000)
  10. Opti-MEM Reduced Serum Media + GlutaMAX TM (Life Technologies,Gibco ,目录号:31985-062)
  11. 酒吧粘蜡(Cera de Reus,目录号:25005001)
  12. 氯化钠(NaCl)(Sigma-Aldrich)
  13. 氯化钾(KCl)(Sigma-Aldrich)
  14. 氯化铯(CsCl)(Sigma-Aldrich)
  15. 氯化钙(CaCl 2)(Sigma-Aldrich)
  16. 氯化镁(MgCl 2)(Sigma-Aldrich)
  17. N-2-羟乙基哌嗪-N'-2-乙磺酸(HEPES)(Sigma-Aldrich)
  18. 乙二醇 - 双(2-氨基乙基醚)-N,N,N',N'-四乙酸(EGTA)(Sigma-Aldrich)
  19. 腺苷5'-三磷酸镁盐(ATP-Mg 2+)(Sigma-Aldrich)
  20. 氢氧化钠(NaOH)(Sigma-Aldrich)
  21. 氢氧化铯(CsOH)(Sigma-Aldrich)
  22. 葡萄糖(Sigma-Aldrich)
  23. 浴溶液(见配方)
  24. 移液器溶液(见配方)

设备

  1. 用于落射荧光的倒置显微镜Nikon Eclipse Ti(Nikon Instruments Inc.)
  2. 玻璃毛细管PC-10 Puller(Narishige International USA Inc.)
  3. 微型操纵器MP-285(Sutter Instrument Co.)
  4. 蒸气压渗透压计VAPOR 5520(Wescor Inc.)
  5. Axopatch 200B电容反馈贴片钳放大器(Molecular Devices)
  6. Axon Digidata 1440A数据采集系统(Molecular Devices)
  7. CO 2 2培养箱
  8. Nunclon 细胞培养皿35×10mm(Sigma-Aldrich,目录号:D7804)
  9. 玻璃毛细管(Brand GmbH + CO KG,目录号:7493-21)

软件

  1. pCLAMP 10.2电生理学数据采集和分析软件(Molecular Devices)
  2. OriginPro 8软件(OriginLab公司)

程序

  1. 细胞培养和转染
    1. 在转染前一天,在35mm皿中平板HEK293细胞,其密度将在24小时后导致70%汇合(这是2.1×10 5个细胞,但其可以根据细胞的多快而变化 线生长),并在37℃下在补充有10%胎牛血清,1%青霉素 - 链霉素和1%GlutaMAX TM的DMEM中的CO 2培养箱中维持。
    2. 使用200μlOpti-MEM 培养基,3μg(总共)感兴趣的载体和4μlGeneCellin TM混合物对每个35mm培养皿的HEK293细胞进行转染 转染 试剂。包含携带编码荧光蛋白的cDNA的载体将允许鉴定转染的细胞。
      注意:其他转染试剂和方法已成功用于转染钠通道。
    3. 转染后24小时,用1.5ml DPBS洗涤每个皿,然后加入0.2ml 0.05%胰蛋白酶-EDTA,并在37℃孵育2分钟,细胞应该看起来彼此分离,加入1.5ml的DMEM到盘,用细尖移液管分散,并且在具有1.5ml培养基的新培养皿中培养板200-300μl。
      注意:您应该获取单个单元格进行录制。

  2. 膜片钳方法(转染后48小时)
    1. 从玻璃毛细管吸取吸管,并用熔化的牙科蜡精细涂覆尖端(小心不要堵塞尖端孔)。填充移液管溶液时,其电阻应在2至4MΩ之间
    2. 从您将使用的转染细胞培养皿中取出DMEM,用Bath溶液(总共约3ml)冲洗细胞两次后,用相同溶液填充约三分之一的高度。
    3. 选择您将从单个荧光细胞记录的单元格。
    4. 用移液管溶液填充移液器,并将其放在电极夹中。降低移液管将其放入Bath溶液中。补偿偏移后,在远程显微操纵器的帮助下,将移液器靠近选定的细胞,形成高电阻细胞附着密封。
    5. 一旦形成密封并且建立了整个电池构造,就补偿80-90%的串联电阻。
    6. 等待五分钟后开始记录。这允许细胞内容物与移液管溶液平衡
    7. 要进行采集,请将滤波器设置为5 kHz,采样率设置为20-25 kHz
  3. 钠测量方案
    1. 宏观钠电流:从-120mV的保持电位通过50ms去极化步骤(以5mV的增量从-80至+ 80mV)引发电流。

    2. 稳态失活(h∞):电流以-20 mV脉冲(20 ms),50 ms前脉冲至不同电位(-140至+5 mV,以5 mV为增量)测量。

    3. 从失活中恢复:电流由-20mV,20ms脉冲(P2)引发,之前是从-120mV的保持电位去极化预脉冲至-20mV(P1)的50ms,随后是超极化脉冲到-120mV的增加持续时间(1-100ms)。

    4. 慢速灭活:双脉冲方案1:在20ms(P2)期间使用测试脉冲测量电流至-20mV,在去极化脉冲之前是从-120mV至-20mV(P1)的持续时间增加(10-100ms以10ms的增量),随后在20ms期间超极化脉冲至-120mV,以去除快速失活。双脉冲协议2:该协议与前一个协议相同,除了第一去极化脉冲到-20(P1)的持续时间以100ms为增量从100到2,000ms。


  4. 钠测量分析(pClamp 10.2和OriginPro 8软件)
    1. 电流密度 - 电压(pA/pF):在施加的不同电压下的测量峰值电流由单元电容归一化。
    2. 活化曲线:通过将在每个电位获得的峰值电流除以驱动力( IV),从电流 - 密度关系( IV )获得全细胞电导> VE ion )。这些值标准化为最大电导( G ),并对每个电压作图。数据拟合到形式为 G = G max /(1 + exp [ em> 是施加的电位, 1/2 是半个通道的电压 激活,并且 k 是斜率因子。
    3. 停用时间常数:时间常数( t ), t 通过将用宏观钠电流方案引出的电流拟合为二阶指数函数,获得快速 。在某些电压下,当电流太快或太小时,不可能拟合二阶指数。在这种情况下,使用一阶指数。被分析以获得时间常数的区域包括从电流的峰值到电流已经达到接近零的平台期的点。 和 t 快速
    4. 稳态失活曲线:峰值电流幅度( I )被归一化为最大峰值电流幅度( max )。来自测试脉冲的 I / I max 值与预脉冲期间的电压作图。实验数据被拟合到形式为的发射波尔兹曼方程中:I = I max /(1 + exp [ V )/ k ]),其中是施加的电压,/em> 1/2是在一半的通道被去激活时的电压,并且 k 是玻尔兹曼常数。
    5. 从失活中恢复:恢复电流值通过将P2的峰值电流除以P1处的峰值电流获得。 P2/P1比率对恢复间隔时间作图。从失活曲线恢复拟合单指数函数以获得时间常数( t )。
    6. 慢失活:在P2和P1测量峰值电流。将P2/P1比值对去极化脉冲间隔时间作图。将慢失活曲线拟合为单指数函数 获得慢灭活常数( t )。

食谱

  1. 浴溶液
    140mM NaCl 3 mM KCl
    10 mM HEPES
    1.8mM CaCl 2 v/v 1.2mM MgCl 2·h/v 用NaOH将pH调节至7.4 重量摩尔渗透压浓度通过将葡萄糖加入到约325mOsm来调节。
  2. 移液器溶液
    130 mM CsCl
    1 mM EGTA
    10 mM HEPES
    10mM NaCl 2mM ATP-Mg 2 +
    用CsOH将pH调节至7.2 重量摩尔渗透压浓度通过加入葡萄糖至约308mOsm来调节 注意:移液器和浴溶液之间渗透压的差异应该在5%左右。

致谢

该协议改编自Riuro等人(2013)和Tarradas等人(2013)。

参考文献

  1. Riuró,H.,Beltran-Alvarez,P.,Tarradas,A.,Selga,E.,Campuzano,O.,Vergés,M.,Pagans,S.,Iglesias,A.,Brugada,J.and Brugada,P 。(2013)。钠通道β2亚基中的错义突变显示SCN2B < em>作为Brugada综合征的新候选基因。 Hum Mutat 34(7):961-966。
  2. Tarradas,A.,Selga,E.,Beltran-Alvarez,P.,Perez-Serra,A.,Riuro,H.,Pico,F.,Iglesias,A.,Campuzano,O.,Castro-Urda, ,Fernandez-Lozano,I.,Perez,GJ,Scornik,FS和Brugada,R。(2013)。 心脏钠通道孔隙区域的新型错义突变I890T引起Brugada综合征。 a> PLoS One 8(1):e53220。 

附加建议书目

  1. Sakmann,Bert和Neher,Erwin。 (1995)。单通道记录。 Springer-Verlag,2 nd Edition。纽约。
  2. Molleman,Areles(2008年重印)。 补丁夹。 膜片钳电生理入门指南。 John Wiley and Sons Ltd. England:West Sussex。
  • English
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Riuró, H., Tarradas, A., Selga, E., Brugada, R., Scornik, F. and Pérez, G. (2013). Sodium Current Measurements in HEK293 Cells. Bio-protocol 3(16): e858. DOI: 10.21769/BioProtoc.858.
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