Skip to main content
SpringerLink
Log in

A study of the outward background current conductance gK1, the pacemaker current conductance gf, and the gap junction conductance gj as determinants of biological pacing in single cells and in a two-cell syncytium using the dynamic clamp

  • Ion channels, receptors and transporters
  • Published:
Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

Abstract

We previously demonstrated that a two-cell syncytium, composed of a ventricular myocyte and an mHCN2 expressing cell, recapitulated most properties of in vivo biological pacing induced by mHCN2-transfected hMSCs in the canine ventricle. Here, we use the two-cell syncytium, employing dynamic clamp, to study the roles of gf (pacemaker conductance), gK1 (background K+ conductance), and gj (intercellular coupling conductance) in biological pacing. We studied gf and gK1 in single HEK293 cells expressing cardiac sodium current channel Nav1.5 (SCN5A). At fixed gf, increasing gK1 hyperpolarized the cell and initiated pacing. As gK1 increased, rate increased, then decreased, finally ceasing at membrane potentials near EK. At fixed gK1, increasing gf depolarized the cell and initiated pacing. With increasing gf, rate increased reaching a plateau, then decreased, ceasing at a depolarized membrane potential. We studied gj via virtual coupling with two non-adjacent cells, a driver (HEK293 cell) in which gK1 and gf were injected without SCN5A and a follower (HEK293 cell), expressing SCN5A. At the chosen values of gK1 and gf oscillations initiated in the driver, when gj was increased synchronized pacing began, which then decreased by about 35% as gj approached 20 nS. Virtual uncoupling yielded similar insights into gj. We also studied subthreshold oscillations in physically and virtually coupled cells. When coupling was insufficient to induce pacing, passive spread of the oscillations occurred in the follower. These results show a non-monotonic relationship between gK1, gf, gj, and pacing. Further, oscillations can be generated by gK1 and gf in the absence of SCN5A.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Beauchamp P, Choby C, Desplantez T, de Peyer K, Green K, Yamada KA, Weingart R, Saffitz JE, Kleber AG (2004) Electrical propagation in synthetic ventricular myocyte strands from germline connexin43 knockout mice. Circ Res 95:170–178

    Article  CAS  Google Scholar 

  2. Brink PR, Cronin K, Ramanan SV (1996) Gap junctions in excitable cells. J Bioenerg Biomembr 28:351–358

    Article  CAS  Google Scholar 

  3. Brown TR, Krogh-Madsen T, Christini DJ (2016) Illuminating myocyte-fibroblast homotypic and heterotypic gap junction dynamics using dynamic clamp. Biophys J 111:785–797. https://doi.org/10.1016/j.bpj.2016.06.042

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Choudhury M, Boyett MR, Morris GM (2015) Biology of the sinus node and its disease. Arrhythmia Electrophysiol Rev 4:28–34. https://doi.org/10.15420/aer.2015.4.1.28

    Article  Google Scholar 

  5. Cingolani E, Goldhaber JI, Marban E (2018) Next-generation pacemakers: from small devices to biological pacemakers. Nat Rev Cardiol 15:139–150. https://doi.org/10.1038/nrcardio.2017.165

    Article  PubMed  Google Scholar 

  6. Clausen C, Valiunas V, Brink PR, Cohen IS (2012) MATLAB implementation of a dynamic clamp with bandwidth of >125 kHz capable of generating I (Na) at 37 degrees C. Pflugers Arch - Eur J Physiol 465:497–507. https://doi.org/10.1007/s00424-012-1186-8

    Article  CAS  Google Scholar 

  7. Cole WC, Picone JB, Sperelakis N (1988) Gap junction uncoupling and discontinuous propagation in the heart. A comparison of experimental data with computer simulations. Biophys J 53:809–818

    Article  CAS  Google Scholar 

  8. Desplantez T, McCain ML, Beauchamp P, Rigoli G, Rothen-Rutishauser B, Parker KK, Kleber AG (2012) Connexin43 ablation in foetal atrial myocytes decreases electrical coupling, partner connexins, and sodium current. Cardiovasc Res 94:58–65. https://doi.org/10.1093/cvr/cvs025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Farrell B, Do Shope C, Brownell WE (2006) Voltage-dependent capacitance of human embryonic kidney cells. Phys Rev E Stat Nonlinear Soft Matter Phys 73:041930. https://doi.org/10.1103/PhysRevE.73.041930

    Article  CAS  Google Scholar 

  10. Gemel J, Valiunas V, Brink PR, Beyer EC (2004) Connexin43 and connexin26 form gap junctions, but not heteromeric channels in co-expressing cells. J Cell Sci 117:2469–2480

    Article  CAS  Google Scholar 

  11. Gourdie RG (2019) The cardiac gap junction has discrete functions in electrotonic and ephaptic coupling. Anat Rec (Hoboken) 302:93–100. https://doi.org/10.1002/ar.24036

    Article  CAS  Google Scholar 

  12. Grant AO (2009) Cardiac ion channels. Circ Arrhythm Electrophysiol 2:185–194. https://doi.org/10.1161/CIRCEP.108.789081

    Article  PubMed  Google Scholar 

  13. Harris AL (2018) Electrical coupling and its channels. J General Physiol 150:1606–1639. https://doi.org/10.1085/jgp.201812203

    Article  Google Scholar 

  14. Jack JJB, Noble D, Tsien RW (1975) Electric current flow in excitable cells. Clarendon Press, Oxford

    Google Scholar 

  15. Kleber AG, Rudy Y (2004) Basic mechanisms of cardiac impulse propagation and associated arrhythmias. Physiol Rev 84:431–488. https://doi.org/10.1152/physrev.00025.2003

    Article  CAS  PubMed  Google Scholar 

  16. Miake J, Marban E, Nuss HB (2002) Functional role of inward rectifier current porbed by Kir2.1 overexpression and dominant-negative suppression 19. Biophys J 82:587A–587A

    Google Scholar 

  17. Miake J, Marban E, Nuss HB (2002) Gene therapy - biological pacemaker created by gene transfer. Nature 419:132–133

    Article  CAS  Google Scholar 

  18. Miake J, Marban E, Nuss HB (2003) Functional role of inward rectifier current in heart probed by Kir2.1 overexpression and dominant-negative suppression. J Clin Invest 111:1529–1536. https://doi.org/10.1172/JCI17959

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. Plotnikov AN, Bucchi A, Shlapakova I, Danflo P, Brink PR, Robinson RB, Cohen IS, Rosen MR (2008) HCN212-channel biological pacemakers manifesting ventricular tachyarrhythmias are responsive to treatment with I-f blockade 7. Heart Rhythm 5:282–288

    Article  Google Scholar 

  21. Plotnikov AN, Sosunov EA, Qu JH, Shlapakova IN, Anyukhovsky EP, Liu LL, Janse MJ, Brink PR, Cohen IS, Robinson RB, Danilo P, Rosen MR (2004) Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates. Circulation 109:506–512

    Article  Google Scholar 

  22. Potapova I, Plotnikov A, Lu Z, Danilo P Jr, Valiunas V, Qu J, Doronin S, Zuckerman J, Shlapakova IN, Gao J, Pan Z, Herron AJ, Robinson RB, Brink PR, Rosen MR, Cohen IS (2004) Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers. CircRes 94:952–959

    CAS  Google Scholar 

  23. Qu J, Plotnikov AN, Danilo P Jr, Shlapakova I, Cohen IS, Robinson RB, Rosen MR (2003) Expression and function of a biological pacemaker in canine heart. Circulation 107:1106–1109. https://doi.org/10.1161/01.cir.0000059939.97249.2c

    Article  PubMed  Google Scholar 

  24. Rhett JM, Ongstad EL, Jourdan J, Gourdie RG (2012) Cx43 associates with Na(v)1.5 in the cardiomyocyte perinexus. J Membr Biol 245:411–422. https://doi.org/10.1007/s00232-012-9465-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Rohr S, Kucera JP, Kleber AG (1998) Slow conduction in cardiac tissue, I: effects of a reduction of excitability versus a reduction of electrical coupling on microconduction. CircRes 83:781–794

    CAS  Google Scholar 

  26. Rosen MR, Robinson RB, Brink PR, Cohen IS (2011) The road to biological pacing. Nat Rev Cardiol 8:656–666. https://doi.org/10.1038/nrcardio.2011.120

    Article  PubMed  Google Scholar 

  27. Saito Y, Nakamura K, Yoshida M, Sugiyama H, Ohe T, Kurokawa J, Furukawa T, Takano M, Nagase S, Morita H, Kusano KF, Ito H (2015) Enhancement of spontaneous activity by HCN4 overexpression in mouse embryonic stem cell-derived cardiomyocytes - a possible biological pacemaker. PLoS One 10:e0138193. https://doi.org/10.1371/journal.pone.0138193

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Sakmann B, Trube G (1984) Conductance properties of single inwardly rectifying potassium channels in ventricular cells from guinea-pig heart 1. J Physiol 347:641

    Article  CAS  Google Scholar 

  29. Santos-Sacchi J, Navarrete E (2002) Voltage-dependent changes in specific membrane capacitance caused by prestin, the outer hair cell lateral membrane motor. Pflugers Arch - Eur J Physiol 444:99–106. https://doi.org/10.1007/s00424-002-0804-2

    Article  CAS  Google Scholar 

  30. Seifert R, Scholten A, Gauss R, Mincheva A, Lichter P, Kaupp UB (1999) Molecular characterization of a slowly gating human hyperpolarization-activated channel predominantly expressed in thalamus, heart, and testis. Proc Natl Acad Sci U S A 96:9391–9396. https://doi.org/10.1073/pnas.96.16.9391

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Shah A, Cohen IS, Rosen MR (1988) Stimulation of cardiac alpha receptors increases Na/K pump current and decreases gK via a pertussis toxin-sensitive pathway. Biophys J 54:219–225. https://doi.org/10.1016/S0006-3495(88)82950-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Shaw RM, Rudy Y (1997) Ionic mechanisms of propagation in cardiac tissue. Roles of the sodium and L-type calcium currents during reduced excitability and decreased gap junction coupling. CircRes 81:727–741

    CAS  Google Scholar 

  33. Shi W, Wymore R, Yu H, Wu J, Wymore RT, Pan Z, Robinson RB, Dixon JE, McKinnon D, Cohen IS (1999) Distribution and prevalence of hyperpolarization-activated cation channel (HCN) mRNA expression in cardiac tissues. Circ Res 85:e1–e6. https://doi.org/10.1161/01.res.85.1.e1

    Article  CAS  PubMed  Google Scholar 

  34. Shi W, Yu H, Wu J, Zuckerman J, Wymore R, Dixon JE, Robinson RB, McKinnon D, Cohen IS (2000) The distribution and prevalence of HCN isoforms in the canine heartand their relation to the voltage dependence of If. Biophys J 78(1):353A

    Google Scholar 

  35. Valiunas V, Brink PR (2018) Biophysical properties of gap junctions. In: Zipes DP, Jalife J, Stevenson BR (eds) Cardiac electrophysiolgy: from cell to bedside, 7th edn. Saunders Elsevier, Philadelphia

    Google Scholar 

  36. Valiunas V, Gemel J, Brink PR, Beyer EC (2001) Gap junction channels formed by coexpressed connexin40 and connexin43. Am J Phys Heart Circ Phys 281:H1675–H1689

    CAS  Google Scholar 

  37. Valiunas V, Kanaporis G, Valiuniene L, Gordon C, Wang HZ, Li L, Robinson RB, Rosen MR, Cohen IS, Brink PR (2009) Coupling an HCN2-expressing cell to a myocyte creates a two-cell pacing unit. J Physiol 587:5211–5226. https://doi.org/10.1113/jphysiol.2009.180505

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Valiunas V, Weingart R, Brink PR (2000) Formation of heterotypic gap junction channels by connexins 40 and 43. Circ Res 86:E42–E49

    Article  CAS  Google Scholar 

  39. Wit AL, Peters NS (2012) The role of gap junctions in the arrhythmias of ischemia and infarction. Heart Rhythm 9:308–311

    Article  Google Scholar 

  40. Yu H, Chang F, Cohen IS (1995) Pacemaker current i(f) in adult canine cardiac ventricular myocytes. J Physiol 485(Pt 2):469–483. https://doi.org/10.1113/jphysiol.1995.sp020743

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Yu H, Wu J, Potapova I, Wymore RT, Holmes B, Zuckerman J, Pan Z, Wang H, Shi W, Robinson RB, El-Maghrabi MR, Benjamin W, Dixon J, McKinnon D, Cohen IS, Wymore R (2001) MinK-related peptide 1: a beta subunit for the HCN ion channel subunit family enhances expression and speeds activation. Circ Res 88:E84–E87. https://doi.org/10.1161/hh1201.093511

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This study was supported by National Institutes of General Medical Sciences grant R01GM088181 (to VV).

Author information

Authors and Affiliations

  1. Department of Physiology and Biophysics and the Institute for Molecular Cardiology, Stony Brook University, Stony Brook, NY, 11794-8661, USA

    Virginijus Valiunas, Ira S. Cohen, Peter R. Brink & Chris Clausen

Authors
  1. Virginijus Valiunas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ira S. Cohen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Peter R. Brink
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chris Clausen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Virginijus Valiunas.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Chris Clausen deceased

A Commentary to this article is available online at https://doi.org/10.1007/s00424-020-02392-3

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Valiunas, V., Cohen, I.S., Brink, P.R. et al. A study of the outward background current conductance gK1, the pacemaker current conductance gf, and the gap junction conductance gj as determinants of biological pacing in single cells and in a two-cell syncytium using the dynamic clamp. Pflugers Arch - Eur J Physiol 472, 561–570 (2020). https://doi.org/10.1007/s00424-020-02378-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00424-020-02378-1

Keywords

Search

Navigation