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. 1991 Nov;60(5):1267–1277. doi: 10.1016/S0006-3495(91)82160-9

Connexin32 gap junction channels in stably transfected cells. Equilibrium and kinetic properties.

A P Moreno 1, B Eghbali 1, D C Spray 1
PMCID: PMC1260180  PMID: 1722120

Abstract

Communication-deficient cells (the SKHep1 cell line) were stably transfected with a plasmid containing cDNA which encodes the major gap junction protein of rat liver, connexin32. Application of the dual whole-cell voltage clamp technique with patch electrodes to pairs of transfected SKHep1 cells revealed strong sensitivity of junctional conductance (gj) to transjunctional voltages (Vjs) of either polarity, with the ratio of minimal to maximal gj (gmin/gmax) being approximately 0.1 at the highest Vjs. Steady-state gj values as a function of voltages of either polarity were well fit by the Boltzmann equation. V0, the voltage at which gj was reduced by 50%, was approximately 25-30 mV; A, the Boltzmann parameter describing voltage dependence, was approximately 0.06 (corresponding to an energy difference between states of approximately 1 kCal/mol and to approximately 2 gating charges moving through the field). The kinetics of the transjunctional voltage dependence were slow (tau greater than 5 s at 20-40 mV, tau = 2 s at and beyond 70 mV). Voltage sensitivity of the opening rate constant (alpha) was approximately 30% lower than that of the closing rate constant (beta) over the Vj range 0-70 mV; at higher voltages, voltage sensitivity of alpha and beta saturated. The kinetic response of gj to a paradigm in which gj was first rendered low by a prepulse of opposite polarity indicated that the voltage sensors are likely to be arranged in series. Transitions between open and closed states in response to transjunctional voltages of either polarity are single order processes; transitions from one closed state to the other involve passage through the open state.

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Selected References

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

  1. Auerbach A. A., Bennett M. V. A rectifying electrotonic synapse in the central nervous system of a vertebrate. J Gen Physiol. 1969 Feb;53(2):211–237. doi: 10.1085/jgp.53.2.211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baylor D. A., Nicholls J. G. Chemical and electrical synaptic connexions between cutaneous mechanoreceptor neurones in the central nervous system of the leech. J Physiol. 1969 Aug;203(3):591–609. doi: 10.1113/jphysiol.1969.sp008881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bennett M. V., Barrio L. C., Bargiello T. A., Spray D. C., Hertzberg E., Sáez J. C. Gap junctions: new tools, new answers, new questions. Neuron. 1991 Mar;6(3):305–320. doi: 10.1016/0896-6273(91)90241-q. [DOI] [PubMed] [Google Scholar]
  4. Catterall W. A. Structure and function of voltage-sensitive ion channels. Science. 1988 Oct 7;242(4875):50–61. doi: 10.1126/science.2459775. [DOI] [PubMed] [Google Scholar]
  5. Cota G., Armstrong C. M. Sodium channel gating in clonal pituitary cells. The inactivation step is not voltage dependent. J Gen Physiol. 1989 Aug;94(2):213–232. doi: 10.1085/jgp.94.2.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Doerr R., Zvibel I., Chiuten D., D'Olimpio J., Reid L. M. Clonal growth of tumors on tissue-specific biomatrices and correlation with organ site specificity of metastases. Cancer Res. 1989 Jan 15;49(2):384–392. [PubMed] [Google Scholar]
  7. Ebihara L., Beyer E. C., Swenson K. I., Paul D. L., Goodenough D. A. Cloning and expression of a Xenopus embryonic gap junction protein. Science. 1989 Mar 3;243(4895):1194–1195. doi: 10.1126/science.2466337. [DOI] [PubMed] [Google Scholar]
  8. Eghbali B., Kessler J. A., Spray D. C. Expression of gap junction channels in communication-incompetent cells after stable transfection with cDNA encoding connexin 32. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1328–1331. doi: 10.1073/pnas.87.4.1328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. FURSHPAN E. J., POTTER D. D. Transmission at the giant motor synapses of the crayfish. J Physiol. 1959 Mar 3;145(2):289–325. doi: 10.1113/jphysiol.1959.sp006143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Giaume C., Korn H. Bidirectional transmission at the rectifying electrotonic synapse: a voltage-dependent process. Science. 1983 Apr 1;220(4592):84–87. doi: 10.1126/science.6298940. [DOI] [PubMed] [Google Scholar]
  12. HODGKIN A. L., HUXLEY A. F. A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol. 1952 Aug;117(4):500–544. doi: 10.1113/jphysiol.1952.sp004764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Jaslove S. W., Brink P. R. The mechanism of rectification at the electrotonic motor giant synapse of the crayfish. Nature. 1986 Sep 4;323(6083):63–65. doi: 10.1038/323063a0. [DOI] [PubMed] [Google Scholar]
  15. Moreno A. P., Eghbali B., Spray D. C. Connexin32 gap junction channels in stably transfected cells: unitary conductance. Biophys J. 1991 Nov;60(5):1254–1266. doi: 10.1016/S0006-3495(91)82159-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Moreno A. P., de Carvalho A. C., Verselis V., Eghbali B., Spray D. C. Voltage-dependent gap junction channels are formed by connexin32, the major gap junction protein of rat liver. Biophys J. 1991 Apr;59(4):920–925. doi: 10.1016/S0006-3495(91)82305-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Obaid A. L., Socolar S. J., Rose B. Cell-to-cell channels with two independently regulated gates in series: analysis of junctional conductance modulation by membrane potential, calcium, and pH. J Membr Biol. 1983;73(1):69–89. doi: 10.1007/BF01870342. [DOI] [PubMed] [Google Scholar]
  19. Reverdin E. C., Weingart R. Electrical properties of the gap junctional membrane studied in rat liver cell pairs. Am J Physiol. 1988 Feb;254(2 Pt 1):C226–C234. doi: 10.1152/ajpcell.1988.254.2.C226. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Spray D. C., Bennett M. V. Physiology and pharmacology of gap junctions. Annu Rev Physiol. 1985;47:281–303. doi: 10.1146/annurev.ph.47.030185.001433. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Spray D. C., Ginzberg R. D., Morales E. A., Gatmaitan Z., Arias I. M. Electrophysiological properties of gap junctions between dissociated pairs of rat hepatocytes. J Cell Biol. 1986 Jul;103(1):135–144. doi: 10.1083/jcb.103.1.135. [DOI] [PMC free article] [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. Spray D. C., Harris A. L., Bennett M. V. Voltage dependence of junctional conductance in early amphibian embryos. Science. 1979 Apr 27;204(4391):432–434. doi: 10.1126/science.312530. [DOI] [PubMed] [Google Scholar]
  26. Spray D. C., White R. L., de Carvalho A. C., Harris A. L., Bennett M. V. Gating of gap junction channels. Biophys J. 1984 Jan;45(1):219–230. doi: 10.1016/S0006-3495(84)84150-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Unwin N. The structure of ion channels in membranes of excitable cells. Neuron. 1989 Dec;3(6):665–676. doi: 10.1016/0896-6273(89)90235-3. [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. Verselis V. K., Bennett M. V., Bargiello T. A. A voltage-dependent gap junction in Drosophila melanogaster. Biophys J. 1991 Jan;59(1):114–126. doi: 10.1016/S0006-3495(91)82204-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Young J. D., Cohn Z. A., Gilula N. B. Functional assembly of gap junction conductance in lipid bilayers: demonstration that the major 27 kd protein forms the junctional channel. Cell. 1987 Mar 13;48(5):733–743. doi: 10.1016/0092-8674(87)90071-7. [DOI] [PubMed] [Google Scholar]

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