Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1992 Oct;63(4):942–953. doi: 10.1016/S0006-3495(92)81664-8

Limitations of the dual voltage clamp method in assaying conductance and kinetics of gap junction channels.

R Wilders 1, H J Jongsma 1
PMCID: PMC1262232  PMID: 1384745

Abstract

The electrical properties of gap junctions in cell pairs are usually studied by means of the dual voltage clamp method. The voltage across the junctional channels, however, cannot be controlled adequately due to an artificial resistance and a natural resistance, both connected in series with the gap junction. The access resistances to the cell interior of the recording pipettes make up the artificial resistance. The natural resistance consists of the cytoplasmic access resistances to the tightly packed gap junction channels in both cells. A mathematical model was constructed to calculate the actual voltage across each gap junction channel. The stochastic open-close kinetics of the individual channels were incorporated into this model. It is concluded that even in the ideal case of complete compensation of pipette series resistance, the number of channels comprised in the gap junction may be largely underestimated. Furthermore, normalized steady-state junctional conductance may be largely overestimated, so that transjunctional voltage dependence is easily masked. The model is used to discuss conclusions drawn from dual voltage clamp experiments and offers alternative explanations for various experimental observations.

Full text

PDF
942

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Burt J. M., Spray D. C. Single-channel events and gating behavior of the cardiac gap junction channel. Proc Natl Acad Sci U S A. 1988 May;85(10):3431–3434. doi: 10.1073/pnas.85.10.3431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chanson M., Bruzzone R., Spray D. C., Regazzi R., Meda P. Cell uncoupling and protein kinase C: correlation in a cell line but not in a differentiated tissue. Am J Physiol. 1988 Nov;255(5 Pt 1):C699–C704. doi: 10.1152/ajpcell.1988.255.5.C699. [DOI] [PubMed] [Google Scholar]
  3. Chapman R. A., Fry C. H. An analysis of the cable properties of frog ventricular myocardium. J Physiol. 1978 Oct;283:263–282. doi: 10.1113/jphysiol.1978.sp012499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clay J. R., DeFelice L. J. Relationship between membrane excitability and single channel open-close kinetics. Biophys J. 1983 May;42(2):151–157. doi: 10.1016/S0006-3495(83)84381-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. De Mazière A. M., van Ginneken A. C., Wilders R., Jongsma H. J., Bouman L. N. Spatial and functional relationship between myocytes and fibroblasts in the rabbit sinoatrial node. J Mol Cell Cardiol. 1992 Jun;24(6):567–578. doi: 10.1016/0022-2828(92)91041-3. [DOI] [PubMed] [Google Scholar]
  6. Fishman G. I., Moreno A. P., Spray D. C., Leinwand L. A. Functional analysis of human cardiac gap junction channel mutants. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3525–3529. doi: 10.1073/pnas.88.9.3525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hall J. E. Access resistance of a small circular pore. J Gen Physiol. 1975 Oct;66(4):531–532. doi: 10.1085/jgp.66.4.531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Harris A. L., Spray D. C., Bennett M. V. Kinetic properties of a voltage-dependent junctional conductance. J Gen Physiol. 1981 Jan;77(1):95–117. doi: 10.1085/jgp.77.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hille B. Pharmacological modifications of the sodium channels of frog nerve. J Gen Physiol. 1968 Feb;51(2):199–219. doi: 10.1085/jgp.51.2.199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Makowski L., Caspar D. L., Phillips W. C., Baker T. S., Goodenough D. A. Gap junction structures. VI. Variation and conservation in connexon conformation and packing. Biophys J. 1984 Jan;45(1):208–218. doi: 10.1016/S0006-3495(84)84149-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Manjunath C. K., Page E. Cell biology and protein composition of cardiac gap junctions. Am J Physiol. 1985 Jun;248(6 Pt 2):H783–H791. doi: 10.1152/ajpheart.1985.248.6.H783. [DOI] [PubMed] [Google Scholar]
  12. Maurer P., Weingart R. Cell pairs isolated from adult guinea pig and rat hearts: effects of [Ca2+]i on nexal membrane resistance. Pflugers Arch. 1987 Aug;409(4-5):394–402. doi: 10.1007/BF00583793. [DOI] [PubMed] [Google Scholar]
  13. Metzger P., Weingart R. Electric current flow in cell pairs isolated from adult rat hearts. J Physiol. 1985 Sep;366:177–195. doi: 10.1113/jphysiol.1985.sp015791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Moreno A. P., Eghbali B., Spray D. C. Connexin32 gap junction channels in stably transfected cells. Equilibrium and kinetic properties. Biophys J. 1991 Nov;60(5):1267–1277. doi: 10.1016/S0006-3495(91)82160-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Neyton J., Trautmann A. Single-channel currents of an intercellular junction. 1985 Sep 26-Oct 2Nature. 317(6035):331–335. doi: 10.1038/317331a0. [DOI] [PubMed] [Google Scholar]
  16. Noma A., Tsuboi N. Dependence of junctional conductance on proton, calcium and magnesium ions in cardiac paired cells of guinea-pig. J Physiol. 1987 Jan;382:193–211. doi: 10.1113/jphysiol.1987.sp016363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Revel J. P., Karnovsky M. J. Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J Cell Biol. 1967 Jun;33(3):C7–C12. doi: 10.1083/jcb.33.3.c7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rook M. B., Jongsma H. J., van Ginneken A. C. Properties of single gap junctional channels between isolated neonatal rat heart cells. Am J Physiol. 1988 Oct;255(4 Pt 2):H770–H782. doi: 10.1152/ajpheart.1988.255.4.H770. [DOI] [PubMed] [Google Scholar]
  19. Rook M. B., de Jonge B., Jongsma H. J., Masson-Pévet M. A. Gap junction formation and functional interaction between neonatal rat cardiocytes in culture: a correlative physiological and ultrastructural study. J Membr Biol. 1990 Nov;118(2):179–192. doi: 10.1007/BF01868475. [DOI] [PubMed] [Google Scholar]
  20. Rüdisüli A., Weingart R. Electrical properties of gap junction channels in guinea-pig ventricular cell pairs revealed by exposure to heptanol. Pflugers Arch. 1989 Oct;415(1):12–21. doi: 10.1007/BF00373136. [DOI] [PubMed] [Google Scholar]
  21. Schwarzmann G., Wiegandt H., Rose B., Zimmerman A., Ben-Haim D., Loewenstein W. R. Diameter of the cell-to-cell junctional membrane channels as probed with neutral molecules. Science. 1981 Jul 31;213(4507):551–553. doi: 10.1126/science.7244653. [DOI] [PubMed] [Google Scholar]
  22. Shibata Y., Yamamoto T. Freeze-fractrue studies of gap junctions in vertebrate cardiac muscle cells. J Ultrastruct Res. 1979 Apr;67(1):79–88. doi: 10.1016/s0022-5320(79)80020-9. [DOI] [PubMed] [Google Scholar]
  23. Spray D. C., Burt J. M. Structure-activity relations of the cardiac gap junction channel. Am J Physiol. 1990 Feb;258(2 Pt 1):C195–C205. doi: 10.1152/ajpcell.1990.258.2.C195. [DOI] [PubMed] [Google Scholar]
  24. Spray D. C., Harris A. L., Bennett M. V. Equilibrium properties of a voltage-dependent junctional conductance. J Gen Physiol. 1981 Jan;77(1):77–93. doi: 10.1085/jgp.77.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Takens-Kwak B. R., Jongsma H. J., Rook M. B., Van Ginneken A. C. Mechanism of heptanol-induced uncoupling of cardiac gap junctions: a perforated patch-clamp study. Am J Physiol. 1992 Jun;262(6 Pt 1):C1531–C1538. doi: 10.1152/ajpcell.1992.262.6.C1531. [DOI] [PubMed] [Google Scholar]
  26. Veenstra R. D., DeHaan R. L. Cardiac gap junction channel activity in embryonic chick ventricle cells. Am J Physiol. 1988 Jan;254(1 Pt 2):H170–H180. doi: 10.1152/ajpheart.1988.254.1.H170. [DOI] [PubMed] [Google Scholar]
  27. Veenstra R. D. Developmental changes in regulation of embryonic chick heart gap junctions. J Membr Biol. 1991 Feb;119(3):253–265. doi: 10.1007/BF01868730. [DOI] [PubMed] [Google Scholar]
  28. Veenstra R. D. Voltage-dependent gating of gap junction channels in embryonic chick ventricular cell pairs. Am J Physiol. 1990 Apr;258(4 Pt 1):C662–C672. doi: 10.1152/ajpcell.1990.258.4.C662. [DOI] [PubMed] [Google Scholar]
  29. Weingart R. Electrical properties of the nexal membrane studied in rat ventricular cell pairs. J Physiol. 1986 Jan;370:267–284. doi: 10.1113/jphysiol.1986.sp015934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. White R. L., Spray D. C., Campos de Carvalho A. C., Wittenberg B. A., Bennett M. V. Some electrical and pharmacological properties of gap junctions between adult ventricular myocytes. Am J Physiol. 1985 Nov;249(5 Pt 1):C447–C455. doi: 10.1152/ajpcell.1985.249.5.C447. [DOI] [PubMed] [Google Scholar]
  31. Wittenberg B. A., White R. L., Ginzberg R. D., Spray D. C. Effect of calcium on the dissociation of the mature rat heart into individual and paired myocytes: electrical properties of cell pairs. Circ Res. 1986 Aug;59(2):143–150. doi: 10.1161/01.res.59.2.143. [DOI] [PubMed] [Google Scholar]
  32. Zampighi G., Unwin P. N. Two forms of isolated gap junctions. J Mol Biol. 1979 Dec 5;135(2):451–464. doi: 10.1016/0022-2836(79)90446-7. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES