Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique

Summary

The present protocol describes an efficient method for the real-time and dynamic acquisition of voltage-gated potassium (Kv) channel currents in H9c2 cardiomyocytes using the whole-cell patch-clamp technique.

Abstract

Potassium channels on the myocardial cell membrane play an important role in the regulation of cell electrophysiological activities. Being one of the main ion channels, voltage-gated potassium (Kv) channels are closely associated with some serious heart diseases, such as drug-induced myocardial damage and myocardial infarction. In the present study, the whole-cell patch-clamp technique was employed to determine the effects of 1.5 mM 4-aminopyridine (4-AP, a broad-spectrum potassium channel inhibitor) and aconitine (AC, 25 µM, 50 µM, 100 µM, and 200 µM) on the Kv channel current (I_Kv) in H9c2 cardiomyocytes. It was found that 4-AP inhibited the I_Kv by about 54%, while the inhibitory effect of AC on the I_Kv showed a dose-dependent trend (no effect for 25 µM, 30% inhibitory rate for 50 µM, 46% inhibitory rate for 100 µM and 54% inhibitory rate for 200 µM). Due to the characteristics of higher sensitivity and precision, this technique will promote the exploration of cardiotoxicity and the pharmacological effects of ethnomedicine targeting ion channels.

Introduction

Ion channels are special integrated proteins embedded in the lipid bilayer of the cell membrane. In the presence of activators, the centers of such special integrated proteins form highly selective hydrophilic pores, allowing ions of an appropriate size and charge to pass through in a passive transport manner¹. Ion channels are the basis of cell excitability and bioelectricity and play a key role in a variety of cellular activities². The heart supplies blood to other organs through regular contractions resulting from an excitation-contraction-coupled process initiated by action potentials³. Previo....

Protocol

The commercially obtained H9c2 rat cardiomyocytes (see the Table of Materials) were incubated in DMEM containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in a 5% CO₂ humidified atmosphere. The whole-cell patch-clamp technique was then employed to detect the changes in I_Kv in normal H9c2 cells and 4-AP- or AC-treated cells (Figure 1 and Figure 2).

1. Solution preparation

1. Prepare the DMEM cell culture medium containing 10% FBS and 1% penicillin-streptomycin (see ....

Results

This protocol allowed the recording of the I_Kv according to the parameters set in the whole-cell patch-clamp technique. The I_Kv was triggered by 150 ms of depolarizing pulse stimulus from −40 to +60 mV at a holding potential of −60 mV (Figure 3A). The I_Kv of the H9c2 rat cardiomyocytes first appeared around −20 mV, and then the amplitude increased with further depolarization. The mean relationship between the I_Kv and membrane pote.......

Discussion

The patch-clamp electrophysiological technique is mainly used to record and reflect the electrical activity and functional characteristics of ion channels on the cell membrane²⁵. At present, the main recording methods of the patch-clamp technique include single-channel recording and whole-cell recording²⁶. For the whole-cell mode, the glass microelectrode and negative pressure are used to form a high-resistance seal between a small area of the cell membrane and a pipette ti.......

Materials

Name	Company	Catalog Number	Comments
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