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Médecine

通过全细胞膜片钳技术 记录 H9c2心肌细胞上的电压依赖性钾电流

Published: November 11, 2022
doi:
10.3791/64805
, , , , , , ,
1State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy,Chengdu University of Traditional Chinese Medicine, 2Chengdu Techman Instrument Co., Ltd., 3Innovative Institute of Chinese Medicine and Pharmacy,Chengdu University of Traditional Chinese Medicine, 4Research Institute of Integrated TCM & Western Medicine,Chengdu University of Traditional Chinese Medicine, 5School of Ethnic Medicine,Chengdu University of Traditional Chinese Medicine

Summary

本协议描述了一种使用全细胞膜片钳技术实时和动态采集H9c2心肌细胞中的电压门控钾（Kv）通道电流的有效方法。

Abstract

心肌细胞膜上的钾通道在调节细胞电生理活动中起重要作用。作为主要的离子通道之一，电压门控钾（Kv）通道与一些严重的心脏病密切相关，例如药物引起的心肌损伤和心肌梗塞。本研究采用全细胞膜片钳技术测定1.5 mM 4-氨基吡啶（4-AP，一种广谱钾通道抑制剂）和乌头碱（AC，25 μM、50 μM、100 μM 和 200 μM）对 H9c2 心肌细胞中 Kv 通道电流 （IKv） 的影响。结果表明，4-AP抑制I Kv约54%，而AC对IKv的抑制作用呈剂量依赖趋势（25 μM无影响，50 μM抑制率为30%，100 μM抑制率为46%，200 μM抑制率为54%）。由于具有较高的灵敏度和精密度的特点，该技术将促进民族医学靶向离子通道的心脏毒性和药理作用的探索。

Introduction

离子通道是嵌入细胞膜脂质双层的特殊整合蛋白。在活化剂存在下，这种特殊整合蛋白的中心形成高度选择性的亲水孔，允许适当大小和电荷的离子以被动运输方式通过1。离子通道是细胞兴奋性和生物电的基础，在各种细胞活动中起着关键作用2。心脏通过由动作电位启动的激发-收缩耦合过程引起的规律收缩向其他器官供血3.已有研究证实，心肌细胞动作电位的产生是由细胞内离子浓度的变化引起的，人心肌细胞中Na+、Ca2+和K+离子通道的活化和失活导致动作电位按一定序列4，5，6形成。电压门控钾（Kv）通道电流（IKv）紊乱可能会改变正常的心律，导致心律失常，这是导致死亡的主要原因之一。因此，记录IKv对于了解治疗危及生命的心律失常的药物机制至关重要7。

Kv通道是钾通道的重要组成部分。Kv通道的协调功能在哺乳动物心脏的电活动和心肌收缩力中起重要作用8，9，10。在心肌细胞中，动作电位的幅度和持续时间取决于多个 Kv 通道亚型向外 K+ 电流的共同传导11。Kv通道功能的调节对于心脏动作电位的正常复极化非常重要。即使是Kv电导的最轻微变化也会极大地影响心脏复极化并增加心律失常的可能性12，13。

代表细胞电生理研究的基本方法，可以通过施加负压在细胞膜的小区域和用于全细胞膜片钳记录的移液器吸头之间建立高电阻密封。持续的负压使细胞膜与移液器吸头接触并粘在移液器的内壁上。由此产生的完整电路允许人们记录穿过细胞膜14表面的任何单个离子通道电流。该技术对细胞膜离子通道电流具有非常高的灵敏度，可用于检测所有离子通道中的电流，应用极其广泛15。此外，与荧光标记和放射性标记相比，膜片钳具有更高的权威性和准确性16。目前，全细胞膜片钳技术已被用于检测作用于Kv通道电流17，18，19的中药成分。例如，Wang等人使用全细胞膜片钳技术，证实莲子的有效成分可能通过阻断活化状态通道19来实现对Kv4.3通道的抑制。乌头碱（AC）是乌头属的有效活性成分之一，如乌头和乌头摆布希。大量研究表明，过量服用AC会导致心律失常甚至心脏骤停20。AC和电压门控离子通道之间的相互作用导致细胞内离子稳态的破坏，这是心脏毒性的关键机制21。因此，本研究采用全细胞膜片钳技术测定AC对心肌细胞IKv的影响。

Protocol

将商业获得的H9c2大鼠心肌细胞（见 材料表）在含有10%热灭活胎牛血清（FBS）和1%青霉素 – 链霉素的DMEM中在37°C的5%CO2加湿气氛中孵育。然后采用全细胞膜片钳技术检测正常H9c2细胞和4-AP或AC处理细胞中IKv 的变化（图1 和 图2）。 1. 溶液制备 制备含有10S和1%青霉素-链霉素的DMEM细胞培…

Representative Results

该协议允许根据全细胞膜片钳技术中设置的参数记录IKv。IKv 由 −40 至 +60 mV 的 150 ms 去极化脉冲激励触发，保持电位为 −60 mV（图 3A）。H9c2大鼠心肌细胞的IKv首先出现在-20 mV左右，然后随着进一步去极化而振幅增加。根据测得的电流幅度计算IKv与膜电位之间的平均关系。结果表明，与对照组相比，用1.5 mM 4-AP处理5 min后，IKv振幅明显?…

Discussion

膜片钳电生理技术主要用于记录和反映细胞膜25上离子通道的电活动和功能特性。目前，膜片钳技术的主要记录方法包括单通道记录和全细胞记录26。对于全细胞模式，玻璃微电极和负压用于在细胞膜的小面积和移液器吸头27之间形成高电阻密封。一旦持续的负压导致移液器的尖端破裂细胞膜并且膜附着在移液器的内壁上，移液器和细胞之间形?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

感谢国家自然科学基金（82130113）和青海省科技厅重点研发转化计划（2020-SF-C33）的资助。

Materials

4-Aminopyridine Sigma MKCJ2184
Aconitine Chengdu Lemetian Medical Technology Co., Ltd DSTDW000602
Amplifier Axon Instrument MultiClamp 700B
Analytical Balance Sartorius 124S-CW
ATP Na2 Solarbio 416O022
Borosilicate glass with filament (O.D.: 1.5 mm, I.D.: 1.10 mm, 10 cm length)  Sutter Instrument 163225-5
Cell culture dish (100 mm) Zhejiang Sorfa Life Science Research Co., Ltd 1192022
Cell culture dish (35 mm) Zhejiang Sorfa Life Science Research Co., Ltd 3012022
Clampex software Molecular Devices, LLC. Version 10. 5
Clampfit software Molecular Devices, LLC. Version 10. 6. 0. 13 data acqusition software
D-(+)-glucose Rhawn RH289133
Digital camera Hamamatsu C11440
Digitizer Axon Instrument Axon digidata 1550B
DMSO Boster Biological Technology Co., Ltd PYG0040
Dulbecco's modified eagle medium (1x) Gibco 8121587
EGTA Biofroxx EZ6789D115
Fetal bovine serum Gibco 2166090RP
Flaming/brown micropipette puller Sutter Instrument Model P-1000
H9c2 cells Hunan Fenghui Biotechnology Co., Ltd CL0111
HCImageLive Hamamatsu 4.5.0.0
HCl Sichuan Xilong Scientific Co., Ltd 2106081
HEPES Xiya Chemical Technology (Shandong) Co., Ltd 20210221
KCl Chengdu Colon Chemical Co., Ltd 2020082501
KOH Chengdu Colon Chemical Co., Ltd 2020112601
MgCl2 Tianjin Guangfu Fine Chemical Research Institute 20160408
MgCl2·6H2O Chengdu Colon Chemical Co., Ltd 2021020101
Micromanipulator Sutter Instrument MP-285A
Microscope Olympus IX73
Microscope cover glass (20 × 20 mm) Jiangsu Citotest Experimental Equipment Co. Ltd 80340-0630
Milli-Q Chengdu Bioscience Technology Co., Ltd Milli-Q IQ 7005
MultiClamp 700B commander Axon Instrument MultiClamp commander 2.0 signal-amplifier software 
OriginPro 8 software OriginLab Corporation v8.0724(B724)
Penicillin-Streptomycin (100x) Boster Biological Technology Co., Ltd 17C18B16
PH meter  Mettler Toledo S201K
Phosphate buffered saline (1x) Gibco 8120485
Trypsin 0.25% (1x) HyClone J210045
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Tags

Keywords: Voltage-dependent Potassium CurrentWhole-cell Patch-clamp TechniqueH9C2 CardiomyocytesIon Channel ResearchPharmacological MechanismsCardiotoxicityMicropipette PullerBorosilicate Glass CapillaryDigital Analog ConverterSignal AmplifierMicro ManipulatorData Acquisition SoftwareLeak SubtractionMembrane Test
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Jiang, H., Zhang, Y., Hou, Y., Li, L., Zhang, S., Zhang, Y., Meng, X., Wang, X. Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique. J. Vis. Exp. (189), e64805, doi:10.3791/64805 (2022).
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