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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 (IKv) in H9c2 cardiomyocytes. It was found that 4-AP inhibited the IKv by about 54%, while the inhibitory effect of AC on the IKv 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. Previous studies have confirmed that the generation of action potentials in cardiomyocytes is caused by the change in intracellular ion concentration, and the activation and inactivation of Na+, Ca2+, and K+ ion channels in human cardiomyocytes lead to the formation of action potentials in a certain sequence4,5,6. Disturbed voltage-gated potassium (Kv) channel currents (IKv) could change the normal heart rhythm, leading to arrhythmias, which are one of the leading causes of death. Therefore, recording the IKv is critical for understanding the mechanisms of drugs for treating life-threatening arrhythmias7.

The Kv channel is an important component of the potassium channel. The coordination function of the Kv channel plays an important role in the electrical activity and myocardial contractility of the mammalian heart8,9,10. In cardiomyocytes, the amplitude and duration of action potentials depend on the co-conduction of outward K+ currents by multiple Kv channel subtypes11. The regulation of the Kv channel function is very important for the normal repolarization of the cardiac action potential. Even the slightest change in Kv conductance greatly impacts cardiac repolarization and increases the possibility of arrhythmia12,13.

Representing a fundamental method in cellular electrophysiological research, a high-resistance seal between a small area of the cell membrane and a pipette tip for whole-cell patch-clamp recording can be established by applying a negative pressure. The continuous negative pressure makes the cell membrane come into contact with the pipette tip and stick onto the inner wall of the pipette. The resulting complete electrical circuit allows one to record any single ion channel current across the surface of the cell membrane14. This technique has a very high sensitivity for the cell membrane ion channel current and can be used to detect currents in all ion channels, and the applications are extremely broad15. Moreover, compared with fluorescent labeling and radioactive labeling, patch-clamp has higher authority and accuracy16. At present, the whole-cell patch-clamp technique has been used to detect the traditional Chinese medicine components acting on Kv channel currents17,18,19. For example, Wang et al. used the whole-cell patch-clamp technique and confirmed that the effective component of the lotus seed might achieve the inhibition of the Kv4.3 channel by blocking the activated state channels19. Aconitine (AC) is one of the effective and active ingredients of Aconitum species, such as Aconitum carmichaeli Debx and Aconitum pendulum Busch. Numerous studies have shown that overdoses of AC can cause arrhythmias and even cardiac arrest20. The interaction between AC and voltage-gated ion channels leads to the disruption of intracellular ion homeostasis, which is the key mechanism of cardiotoxicity21. Therefore, in this study, the whole-cell patch-clamp technique is used to determine the effects of AC on the IKv of cardiomyocytes.

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 IKv 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 Table of Materials).
  2. Prepare 1.5 M 4-AP solution by adding 14.1165 mg of 4-AP to 100 µL of DMSO solution (see Table of Materials). Dilute 1.5 M 4-AP to 1.5 mM by adding the extracellular solution (step 1.5).
  3. Prepare 400 mM AC by adding 5 mg of AC to 19.36 µL of DMSO solution (see Table of Materials).
    NOTE: All the above solutions were stored in a 4 °C refrigerator, and the final concentration of the DMSO must not exceed 0.1% (v/v).
  4. Prepare 50 mL of intracellular solution by adding 0.4100 g of KCl (110.0 mM), 0.0120 g of MgCl2 (1.2 mM), 0.1270 g of Na2 ATP (5.0 mM), 0.1190 g of HEPES (10.0 mM), and 0.1900 g of EGTA (10.0 mM) into 50 mL of double-distilled water (see Table 1 and Table of Materials).
    NOTE: The pH value of the intracellular solution was adjusted to 7.2 using 1 M KOH and was aliquoted into small volumes (1.5-2 mL) and stored at −20 °C.
  5. Prepare 50 mL of extracellular solution by adding 0.0185 g of KCl (5.0 mM), 0.3945 g of NaCl (135.0 mM), 0.0203 g of MgCl2·6H2O (2.0 mM), 0.0595 g of HEPES (5.0 mM), and 0.0990 g of D-glucose (10.0 mM) into 50 mL of double-distilled water (see Table 1 and Table of Materials).
    NOTE: The extracellular solution must be prepared for immediate use, and its pH value needs to be adjusted to 7.4 by using 1 M NaOH.

2. Cell culture

  1. Once the H9c2 culture dish becomes 80% confluent, digest the cells with 0.25% trypsin for 30 s.
  2. Culture 2 x 105 cells/mL with normal medium or drug-containing medium (25 µM, 50 µM, 100 µM, and 200 µM AC) in a 35 mm dish carpeted with glass plates for 24 h at 37 °C, with a 5% CO2 atmosphere and 70% to 80% relative humidity (see Table of Materials).

3. Fabrication of micropipettes

  1. Turn on the micropipette puller (see Table of Materials), and preheat for 30 min.
  2. Put a borosilicate glass capillary with a filament (OD: 1.5 mm, ID: 1.10 mm, 10 cm length) on the micropipette puller. Select the program set in step 3.3, and click on Enter on the control panel. Click on the Ramp program in the upper-right corner to determine the "Heat" value of the glass capillary.
  3. Write the program for pulling the electrode according to the value determined by the "Ramp" test, and use the following steps: "Heat" value = "Ramp" value, Pull = 0, Vel = pulling rod moving speed, Time = 200-250. Click on Pull to start fabricating the pipettes.
    NOTE: Observe the color of the desiccants in the sealed container of the micropipette puller before using it. If the color changes from blue to pink, the desiccants must be replaced. To prevent the contamination of the electrode tip, try not to touch the middle part of the glass capillary during the pipette preparation. Subsequently, carefully remove the produced pipettes, and place them in a closed and sealed container.

4. Instrument setup

  1. Turn on the corresponding instruments in the following order: the digital-analog converter, the signal amplifier, the micromanipulator, the microscope, and the camera (see Table of Materials).
  2. Open the imaging application, the signal-amplifier software, and the data acquisition software in sequence (see Table of Materials).
    NOTE: The instrument must be turned on sequentially; otherwise, the "Demo Digitizer" state will appear after opening the software.

5. IKv parameter setting

  1. Edit the protocol for recording the IKv following the steps below.
    1. Click on Edit, and select Edit Protocol in the data acquisition software (see Table of Materials).
    2. Edit the program in the "Waveform" interface: Epoch A (First level = −60 mV, Delta level = 0 mV, First duration = 20 ms, and Delta duration = 0 ms); Epoch B (First level = −40 mV, Delta level = 10 mV, First duration = 150 ms, and Delta duration = 0 ms), and Epoch C (First level = −60 mV, Delta level = 0 mV, First duration = 30 ms, and Delta duration = 0 ms).
    3. Then, click on the Mode/Rate interface, and set the "Trial Hierarchy" data: Trial delay = 0 s, Runs = 1, Sweeps = 11, and Sweep duration = 0.22 s22,23.
      NOTE: Acquire the IKv channel currents by applying 150 ms depolarizing steps from −40 mV to +60 mV with an increment of 10 mV at a holding potential of −60 mV under control conditions. The total time of the protocol needs to be greater than the time set in the "Waveform" program.
  2. Edit the P/N Leak Subtraction program: click on Edit Protocol to select the Stimulus button, and set the leak subtraction program in the P/N Leak Subtraction dialog box: number of Subsweeps = 2-8 (4 in this study), Settling time = 100-1,000 ms (200 in this study), "Polarity" = "Opposite to waveform", holding level = −80 mV.
    NOTE: The P/N Leak subtraction must have a holding level lower than the "First holding" (−60 mV).

6. Whole-cell patch-clamp recording of the I Kv in voltage-clamp mode

  1. Establish the data storage path: click on File, and select Set Data File Names in the data acquisition software. Open the established IKv protocol (step 5.1) by selecting Edit and clicking on Open Protocol in the data acquisition software. Finally, click on the Tools option, and select Membrane Test to start running the protocol.
  2. Add the extracellular solution (step 1.5) to the cell bath on the whole-cell patch-clamp apparatus (see Table of Materials), and place the coverslip upward with the H9c2 cells (cultured in step 2.1) in the bath.
  3. Fill 30% of the pipette (fabricated in step 3) with the extracellular solution, and install it on the recording electrode holder integrated into the patch-clamp apparatus. Tighten the pipette with the o-ring rubber gasket and plastic washer. Then, drop the pipette into the bath with the micromanipulator. Click on the Pipette offset interface of the signal-amplifier software (see Table of Materials) to maintain the IKv current baseline at 0 pA.
    NOTE: The intracellular solution (step 1.4) must be in contact with the AgCl/Ag portion of the silver wire, and the silver wire must not be close to the inner wall of the pipette. The resistance of the pipette needs to be 2-6 MΩ. To avoid the occlusion of the pipette tip, a continuous positive pressure must be delivered to the pipette using a 1.0 mL syringe connected to the recording electrode holder via plastic tubing24.
  4. Deliver an appropriate positive pressure manually with a 1.0 mL syringe connected to the recording electrode holder through a plastic tube. Move the pipette slightly by manipulating the micromanipulator in three dimensions to contact the cell.
  5. Once the membrane test square wave drops by 1/3 to 1/2 after the pipette touches the cell membrane, remove the positive pressure, and manually deliver an appropriate negative pressure. Then, click on the Patch interface of the data acquisition software to form the GΩ seal. Use the "Cp Fast" and "Cp Slow" signal-amplifier software during cell sealing to compensate for the fast and slow capacitance.
    NOTE: When the membrane test is ≥1 GΩ, a cell-attached configuration is formed between the pipette tip and the cell.
  6. Apply brief pulses of negative pressure to rupture a patch of the cell membrane.
    NOTE: A precipitous reduction in membrane resistance characterizes a successful whole-cell recording pattern. If the access resistance (Ra) ≥30 MΩ, the cells must be reselected for steps 6.3-6.6. In order to ensure the accuracy of the recorded IKv data, the negative pressure must be removed after the cell membrane is broken.
  7. Execute whole-cell membrane capacitance compensation by clicking on the Whole-cell button of the signal-amplifier software. Finally, save and record the data by clicking on the Data recording button.
  8. Open the saved IKv data with the data analysis software. Save the current-voltage (I−V) relationships: click on Analyze to select Statistics to do the following: select the Range as Cousors 1,2 for data analysis; click on the Peak Amplitude and Mean buttons, and then click on OK to view the data in the Results page. Finally, copy the column of IKv mean data into the function drawing software (see Table of Materials) for further analysis.
  9. Save the representative IKv current traces: click on Edit to select Transfer Traces; then, select the Full trace in the Region to transfer; next, click on Select in Trace Selection, and select IN 0 (pA) in Signals; finally, click on OK to view the data in the "Results" page, and copy the total data into the function drawing software to draw the current traces.
  10. Save the IKv protocol: click on Edit to select Create Stimulus Waveform Signal, and select OK. Then, redo step 6.9 except for selecting A0 # 0 (mA) in "Signals".

Results

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

Disclosures

The authors have nothing to disclose.

Acknowledgements

We appreciate the financial support from the National Natural Science Foundation of China (82130113) and the Key R&D and Transformation Program of the Science & Technology Department of Qinghai Province (2020-SF-C33).

Materials

NameCompanyCatalog NumberComments
4-AminopyridineSigmaMKCJ2184
AconitineChengdu Lemetian Medical Technology Co., LtdDSTDW000602
AmplifierAxon InstrumentMultiClamp 700B
Analytical BalanceSartorius124S-CW
ATP Na2Solarbio416O022
Borosilicate glass with filament (O.D.: 1.5 mm, I.D.: 1.10 mm, 10 cm length) Sutter Instrument163225-5
Cell culture dish (100 mm)Zhejiang Sorfa Life Science Research Co., Ltd1192022
Cell culture dish (35 mm)Zhejiang Sorfa Life Science Research Co., Ltd3012022
Clampex softwareMolecular Devices, LLC.Version 10. 5
Clampfit softwareMolecular Devices, LLC.Version 10. 6. 0. 13data acqusition software
D-(+)-glucoseRhawnRH289133
Digital cameraHamamatsuC11440
DigitizerAxon InstrumentAxon digidata 1550B
DMSOBoster Biological Technology Co., LtdPYG0040
Dulbecco's modified eagle medium (1x)Gibco8121587
EGTABiofroxxEZ6789D115
Fetal bovine serumGibco2166090RP
Flaming/brown micropipette pullerSutter InstrumentModel P-1000
H9c2 cellsHunan Fenghui Biotechnology Co., LtdCL0111
HCImageLiveHamamatsu4.5.0.0
HClSichuan Xilong Scientific Co., Ltd2106081
HEPESXiya Chemical Technology (Shandong) Co., Ltd20210221
KClChengdu Colon Chemical Co., Ltd2020082501
KOHChengdu Colon Chemical Co., Ltd2020112601
MgCl2Tianjin Guangfu Fine Chemical Research Institute20160408
MgCl2·6H2OChengdu Colon Chemical Co., Ltd2021020101
MicromanipulatorSutter InstrumentMP-285A
MicroscopeOlympusIX73
Microscope cover glass (20 × 20 mm)Jiangsu Citotest Experimental Equipment Co. Ltd80340-0630
Milli-QChengdu Bioscience Technology Co., LtdMilli-Q IQ 7005
MultiClamp 700B commanderAxon InstrumentMultiClamp commander 2.0signal-amplifier software 
OriginPro 8 softwareOriginLab Corporationv8.0724(B724)
Penicillin-Streptomycin (100x)Boster Biological Technology Co., Ltd17C18B16
PH meter Mettler ToledoS201K
Phosphate buffered saline (1x)Gibco8120485
Trypsin 0.25% (1x)HyCloneJ210045

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Voltage dependent Potassium CurrentWhole cell Patch clampH9c2 CardiomyocytesAconitine EffectsIon ChannelsCardiotoxicityPharmacological MechanismsCell CultureTrypsin DigestionMicropipette PullerData Acquisition SoftwareElectrode FabricationSignal AmplificationLeak Subtraction ProgramExtracellular Environment

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