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Whole-cell and Perforated Patch-clamp Recordings from Acutely-isolated Murine Sino-atrial Node Cells

小鼠窦房结细胞快速分离过程中全细胞和穿孔膜片钳记录

QA Qadeer Aziz *
MN Muriel Nobles *
AT Andrew Tinker

 (*contributed equally to this work) 发布: 2020年01月05日第10卷第1期 DOI: 10.21769/BioProtoc.3478 浏览次数: 6144

评审: Gal HaimovichAkira KarasawaAnonymous reviewer(s)

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Abstract

Cardiac pacemaker cells of the sino-atrial node are responsible for the initiation of the heart beat and express an array of ion channels. The patch-clamp technique is the gold standard method for investigating the function of ion channels expressed in electrically active cells. Conventional whole-cell and perforated patch-clamp techniques can be used to investigate ionic currents in the voltage-clamp mode and changes in membrane potential (e.g., action potential) in the current-clamp mode. Here, we provide details of protocols used to measure spontaneous and triggered action potentials and whole-cell funny current If (HCN4) in single cardiomyocytes isolated from the mouse sino-atrial node (SAN).

Keywords: Cardiomyocytes (心肌细胞) search Sinoatrial node (窦房结) search Cardiac pacemaker (心脏起搏器) search Funny current (起搏电流) search Action potential (动作电位) search KATP current (KATP电流) search

Background

The patch-clamp technique allows for the real-time measurement of ionic currents and membrane potential from electrically active cells (Hamill et al., 1981). Electrically active SAN cells are also known as pacemaker cells as they initiate the beating of the heart. The automaticity of the SAN is in part due to the slow diastolic depolarization of the membrane initiated by the opening of If (HCN4) allowing the membrane potential to reach threshold and leading to firing of the SAN action potential. The If current (also known as the pacemaker current) is a non-selective sodium/potassium current activated at hyperpolarized membrane potentials and by intracellular cyclic adenosine monophosphate. Whole-cell voltage-clamp and current-clamp recordings allow for understanding of the role of various ionic currents underlying the SAN action potential in more details (Aziz et al., 2018; Nobles et al., 2018). Patch-clamp of isolated SAN cells can be challenging, here we provide detailed protocols for the measurement of ionic currents and action potentials using conventional and perforated whole-cell patch-clamp techniques.

Materials and Reagents

  1. 13 mm glass coverslips (VWR, catalog number: 631-0148)
  2. Thin filamented borosilicate glass capillaries (Harvard Apparatus, catalog number: 30-0066)
  3. Thin borosilicate glass capillaries (Harvard Apparatus, catalog number: 30-0094)
  4. 12-well cell culture plates, CellStar (Greiner Bio-One, catalog number: 665 180)
  5. Microloader pipette tips (Eppendorf, Thermo Fisher Scientific, catalog number: 10289651)
  6. Adult mice, 6-12 weeks, e.g., C57/Bl6 (The Jackson Laboratory, catalog number: 000664)
  7. Laminin (Sigma-Aldrich, catalog number: L2020)
  8. Deionized, filtered water (dH2O) (Milli-Q, Merck Millipore, catalog number: ZRXQ003WW)
  9. Sodium Chloride (NaCl) (Sigma-Aldrich, catalog number: S7653)
  10. Potassium Chloride (KCl) (Sigma-Aldrich, catalog number: P9333)
  11. Potassium Hydroxide pellets (KOH) (Sigma-Aldrich, catalog number: P6310)
  12. Sodium Hydroxide pellets (NaOH) (Thermo Fisher Scientific, catalog number: S/4920/53)
  13. HEPES (Sigma-Aldrich, catalog number: H3375)
  14. D-Glucose (Sigma-Aldrich, catalog number: G5767)
  15. L-aspartic acid (C4H6KNO4) (Sigma-Aldrich, catalog number: A6558)
  16. Potassium D-Gluconate (C6H11KO7) (Sigma-Aldrich, catalog number: G4500)
  17. Adenosine 5′-triphosphate (ATP) disodium salt (Sigma-Aldrich, catalog number: A7669)
  18. Adenosine 5′-triphosphate (ATP) magnesium salt (Sigma-Aldrich, catalog number: A9187)
  19. Ethylene glycol-bis (2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889)
  20. Phosphocreatine di(tris) salt (Sigma-Aldrich, catalog number: P1937)
  21. Guanosine 5′-triphosphate (GTP) Sodium salt hydrate (Sigma-Aldrich, catalog number: G8877)
  22. Amphotericin B (Sigma-Aldrich, catalog number: A4888)
  23. DMSO (Sigma-Aldrich, catalog number: D4540)
  24. Potassium D-Gluconate (C6H11KO7) (Sigma-Aldrich, catalog number: G4500)
  25. Calcium Chloride (CaCl2) 1 M solution (Fluka, catalog number: 21114)
  26. Magnesium Chloride (MgCl2) 1 M solution (Fluka, catalog number: 63026)
  27. Tyrode Extracellular solution (see Recipes)
  28. Perforated Patch-Clamp intracellular solution (see Recipes)
  29. Whole-Cell Current-Clamp solution (see Recipes)
  30. Amphotericin stock solution (see Recipes)
  31. Laminin-coated coverslips (see Recipes)

Equipment

  1. Digidata 1440A data acquisition system (Molecular Devices)
  2. Axopatch 200B patch-clamp amplifier (Molecular Devices)
  3. Patch-clamp recording chamber (Warner Instruments, RC-26 Chamber)
  4. Inverted Microscope Nikon TE200 Eclipse (Nikon)
  5. Micromanipulator (Patch Star) (Scientifica)
  6. Pipette Puller PP-830 (Narashige)
  7. Microforge MF-830 (Narashige)

Software

  1. pCLAMP 10 data acquisition and analysis software (Molecular Devices)
  2. Origin 6.0 analysis software (OriginLab Corporation)
  3. GraphPad Prism v6

Procedure

English
中文翻译

文章信息

版权信息

© 2020 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Aziz, Q., Nobles, M. and Tinker, A. (2020). Whole-cell and Perforated Patch-clamp Recordings from Acutely-isolated Murine Sino-atrial Node Cells. Bio-protocol 10(1): e3478. DOI: 10.21769/BioProtoc.3478.
  2. Aziz, Q., Finlay, M., Montaigne, D., Ojake, L., Li, Y., Anderson, N., Ludwig, A. and Tinker, A. (2018). ATP-sensitive potassium channels in the sinoatrial node contribute to heart rate control and adaptation to hypoxia. J Biol Chem 293(23): 8912-8921.

    3. ris ris Download Citation in RIS Format

分类

生物物理学 > 电生理 > 膜片钳技术

细胞生物学 > 基于细胞的分析方法 > 电生理技术

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