Skip to main content
SpringerLink
Log in

Establishment of the Dual Whole Cell Recording Patch Clamp Configuration for the Measurement of Gap Junction Conductance

  • Protocol
  • First Online:
Book cover Gap Junction Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1437))

Abstract

The development of the patch clamp technique has enabled investigators to directly measure gap junction conductance between isolated pairs of small cells with resolution to the single channel level. The dual patch clamp recording technique requires specialized equipment and the acquired skill to reliably establish gigaohm seals and the whole cell recording configuration with high efficiency. This chapter describes the equipment needed and methods required to achieve accurate measurement of macroscopic and single gap junction channel conductances. Inherent limitations with the dual whole cell recording technique and methods to correct for series access resistance errors are defined as well as basic procedures to determine the essential electrical parameters necessary to evaluate the accuracy of gap junction conductance measurements using this approach.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Auerbach AA, Bennett MVL (1969) A rectifying electrical synapse in the central nervous system of a vertebrate. J Gen Physiol 53:211–237

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Spray DC, Harris AL, Bennett MVL (1979) Voltage dependence of junctional conductance in early amphibian embryos. Science 204:432–434

    Article  CAS  PubMed  Google Scholar 

  3. Spray DC, Harris AL, Bennett MVL (1981) Equilibrium properties of a voltage-dependent junctional conductance. J Gen Physiol 77:77–93

    Article  CAS  PubMed  Google Scholar 

  4. Harris AL, Spray DC, Bennett MVL (1981) Kinetic properties of a voltage-dependent junctional conductance. J Gen Physiol 77:95–117

    Google Scholar 

  5. Hamill OP, Marty A, Neher E et al (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391:85–100

    Article  CAS  PubMed  Google Scholar 

  6. Neyton J, Trautmann A (1985) Single channel currents of an intercellular junction. Nature 317:331–335

    Article  CAS  PubMed  Google Scholar 

  7. Veenstra RD, DeHaan RL (1986) Measurement of single channel currents from cardiac gap junctions. Science 233:972–974

    Article  CAS  PubMed  Google Scholar 

  8. Paul DL (1985) Molecular cloning of cDNA for rat liver gap junction protein. J Cell Biol 103:123–134

    Article  Google Scholar 

  9. Beyer EC, Paul DL, Goodenough DA (1987) Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol 105:2621–2629

    Article  CAS  PubMed  Google Scholar 

  10. Dahl G, Miller T, Paul D et al (1987) Expression of functional cell-cell channels from cloned rat liver gap junction complementary DNA. Science 236:1290–1293

    Article  CAS  PubMed  Google Scholar 

  11. Barrio LC, Suchyna T, Bargiello T et al (1991) Gap junctions formed by connexins 26 and 32 alone and in combination are differently affected by applied voltage. Proc Natl Acad Sci U S A 88:8410–8414

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hennemann H, Suchyna T, Lichtenberg-Fraté H et al (1992) Molecular cloning and functional expression of mouse connexin40, a second gap junction gene preferentially expressed in lung. J Cell Biol 117:1299–1310

    Article  CAS  PubMed  Google Scholar 

  13. Veenstra RD, Wang HZ, Westphale EM et al (1992) Multiple connexins confer distinct regulatory and conductance properties of gap junctions in developing heart. Circ Res 71:1277–1283

    Article  CAS  PubMed  Google Scholar 

  14. Bers DM, Patton CW, Nuccitelli R (2010) A practical guide to the preparation of Ca2+ buffers. Methods Cell Biol 99:1–26

    Article  CAS  PubMed  Google Scholar 

  15. Flaming DG, Brown KT (1982) Micropipette puller design: form of the heating filament and effects of filament width on tip length and diameter. J Neurosci Methods 6:91–102

    Article  CAS  PubMed  Google Scholar 

  16. Rae JL, Levis RA (2004) Fabrication of patch pipets. Curr Protoc Neurosci 26:1–32

    Google Scholar 

  17. Sutter Instrument Company (2015) Pipette cookbook 2015. P-97 and P-1000 micropipette pullers. Rev E. http://www.sutter.com/PDFs/pipette_cookbook.pdf

  18. Colquhoun D, Sigworth FJ (1983) Fitting and statistical analysis of single-channel records. In: Sakmann B, Neher E (eds) Single-channel recording. Plenum Press, New York, pp 191–263

    Chapter  Google Scholar 

  19. Trube G (1983) Enzymatic dispersion of heart and other tissues. In: Sakmann B, Neher E (eds) Single-channel recording. Plenum Press, New York, pp 69–76

    Chapter  Google Scholar 

  20. Spector I (1983) A primer in cell culture for pathologists. In: Sakmann B, Neher E (eds) Single-channel recording. Plenum Press, New York, pp 77–90

    Chapter  Google Scholar 

  21. Lin X, Gemel J, Beyer EC et al (2005) A dynamic model for ventricular junctional conductance during the cardiac action potential. Am J Physiol Heart Circ Physiol 288:H1113–H1123

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Marty A, Neher E (1983) Whole-cell recording. In: Sakmann B, Neher E (eds) Single-channel recording. Plenum Press, New York, pp 107–122

    Chapter  Google Scholar 

  23. Lin X, Zemlin C, Hennan J et al (2008) Enhancement of ventricular gap junction coupling by rotigaptide. Cardiovasc Res 79:416–426

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Valiunas V, Beyer EC, Brink PR (2002) Cardiac gap junction channels show quantitative differences in selectivity. Circ Res 91:104–111

    Article  CAS  PubMed  Google Scholar 

  25. Veenstra RD (2001) Voltage clamp limitations of dual whole cell recordings of gap junction current and voltage recordings. I. Conductance measurements. Biophys J 80:2231–2247

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Woodhull A (1973) Ionic blockage of sodium channels in nerve. J Gen Physiol 61:687–708

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Musa H, Veenstra RD (2003) Voltage-dependent blockade of connexin40 gap junctions by spermine. Biophys J 84:205–219

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Veenstra RD (2001) Determining ionic permeabilities of gap junction channels. In: Bruzzone R, Giaume C (eds) Methods in molecular biology, vol 154, Connexin methods and protocols. Humana Press, New Jersey, pp 293–311

    Google Scholar 

  29. Warner Instruments. (2004) Chloriding Ag/AgCl electrodes. http://www.warneronline.com/Documents/uploader/ChloridingAgAgClelectrodes(2004.02.02).pdf

  30. Stong BC, Chang Q, Ahmad S et al (2006) A novel mechanism for connexin26 mutation linked deafness: cell death caused by leaky gap junction hemichannels. Laryngoscope 116:2205–2210

    Article  CAS  PubMed  Google Scholar 

  31. McSpadden LC, Kirkton RD, Bursac N (2009) Electrotonic loading of anisotropic cardiac monolayers by unexcitable cells depends on connexin type and expression level. Am J Physiol Cell Physiol 297:C339–C351

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Sroka J, Czyz J, Wojewoda M et al (2008) The inhibitory effect of diphenyltin on gap junctional intercellular communication in HEK293 cells is reduced by thioredoxin reductase 1. Toxicol Lett 183:45–51

    Article  CAS  PubMed  Google Scholar 

  33. Patel D, Zhang X, Veenstra RD (2014) Connexin hemichannel and pannexin channel electrophysiology: how do they differ? FEBS Lett 588:1372–1378

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Horn R, Marty A (1988) Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol 92:145–159

    Article  CAS  PubMed  Google Scholar 

  35. Rae J, Cooper K, Gates P et al (1991) Low access resistance perforated patch recordings using amphotericin B. J Neurosci Methods 37:15–26

    Article  CAS  PubMed  Google Scholar 

  36. Fan JS, Palade P (1998) Perforated patch recording with beta-escin. Pflugers Arch 436:1021–1023

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by NIH grant HL-042220 and a Hendricks Fund grant to R.D.V. Xian Zhang and Dakshesh Patel proofread the manuscript.

Author information

Authors and Affiliations

  1. Department of Pharmacology, SUNY Upstate Medical University, 3162 Weiskotten Hall, 766 Irving Ave., Syracuse, NY, 13210, USA

    Richard D. Veenstra

  2. Department Cell and Developmental Biology, SUNY Upstate Medical University, 3162 Weiskotten Hall, 766 Irving Ave., Syracuse, NY, 13210, USA

    Richard D. Veenstra

Authors
  1. Richard D. Veenstra
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Richard D. Veenstra .

Editor information

Editors and Affiliations

  1. Department of in Vitro Toxicology and D, Vrije Universiteit Brussel, Brussels, Belgium

    Mathieu Vinken

  2. BHF Glasgow Cardiovascular Research, University of Glasgow, Glasgow, United Kingdom

    Scott R. Johnstone

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Veenstra, R.D. (2016). Establishment of the Dual Whole Cell Recording Patch Clamp Configuration for the Measurement of Gap Junction Conductance. In: Vinken, M., Johnstone, S. (eds) Gap Junction Protocols. Methods in Molecular Biology, vol 1437. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3664-9_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3664-9_16

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3662-5

  • Online ISBN: 978-1-4939-3664-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation