Skip to main content
Log in

Heptanol-induced decrease in cardiac gap junctional conductance is mediated by a decrease in the fluidity of membranous cholesterol-rich domains

The Journal of Membrane Biology Aims and scope Submit manuscript

Abstract

To assess whether alterations in membrane fluidity of neonatal rat heart cells modulate gap junctional conductance (g j), we compared the effects of 2mm 1-heptanol and 20 μm 2-(methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)-octanoate (A2C) in a combined fluorescence anisotropy and electrophysiological study. Both substances decreased fluorescence steady-state anisotropy (rss), as assessed with the fluorescent probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) by 9.6±1.1% (mean ±sem,n=5) and 9.8±0.6% (n=5), respectively, i.e., both substances increased bulk membrane fluidity. Double whole-cell voltage-clamp experiments showed that 2mm heptanol uncoupled cell pairs completely (n=6), whereas 20 μm A2C, which increased bulk membrane fluidity to the same extent, did not affect coupling at all (n=5).

Since gap junction channels are embedded in relatively cholesterol-rich domains of the membrane, we specifically assessed the fluidity of the cholesterol-rich domains with dehydroergosterol (DHE). Using DHE, heptanol increased rss by 14.9±3.0% (n=5), i.e., decreased cholesterol domain fluidity, whereas A2C had no effect on rss (−0.4±6.7%,n=5).

Following an increase of cellular “cholesterol” content (by loading the cells with DHE), 2mm heptanol did not uncouple cell pairs completely:g j decreased by 80±20% (range 41–95%,n=5). The decrease ing j was most probably due to a decrease in the open probability of the gap junction channels, because the unitary conductances of the channels were not changed nor was the number of channels comprising the gap junction. The sensitivity of non-junctional membrane channels to heptanol was unaltered in cholesterol-enriched myocytes.

These results indicate that the fluidity of cholesterol-rich domains is of importance to gap junctional coupling, and that heptanol decreasesg j by decreasing the fluidity of cholesterol-rich domains, rather than by increasing the bulk membrane fluidity.

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

Access this article

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

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Beeler, G.W., McGuigan, J.A.S. 1978. Voltage clamping of multicellular myocardial preparations: Capabilities and limitations of existing methods.Prog. Biophys. Mol. Biol. 34:219–254

    Article  CAS  Google Scholar 

  • Beyer, E.C., Paul, D.L., Goodenough, D.A. 1987. Connexin43: A protein from rat heart homologous to a gap junction protein from liver.J. Cell Biol. 105:2621–2629

    Article  CAS  Google Scholar 

  • Burt, J.M., Spray, D.C. 1988. Single-channel events and gating behavior of the cardiac gap junction channel.Proc. Natl. Acad. Sci. USA 85:3431–3434

    Article  CAS  Google Scholar 

  • Burt, J.M. 1989. Uncoupling of cardiac cells by doxyl stearic acids: Specificity and mechanism of action.Am. J. Physiol. 256:C913-C924

    Article  CAS  Google Scholar 

  • Burt, J.M., Spray, D.C. 1989. Volatile anesthetics block intercellular communication between neonatal rat myocardial cells.Circ. Res. 65:829–837

    Article  CAS  Google Scholar 

  • Chanson, M., Bruzzone, R., Bosco, D., Meda, P. 1989. Effects ofn-alcohols on junctional coupling and amylase secretion of pancreatic acinar cells.J. Cell. Physiol. 139:147–156

    Article  CAS  Google Scholar 

  • De Bruijne, J., Jongsma, H.J. 1980. Membrane properties of aggregates of collagenase-dissociated rat heart cells.In: Advances in Myocardiology. M. Tajuddin, P.K. Das, M. Tariq, N.S. Dhalla, editors. pp. 231–243. University Park, Baltimore

    Google Scholar 

  • Délèze, J., Hervé, J.C. 1983. Effect of several uncouplers of cell-to-cell communication on gap junction morphology in mammalian heart.J. Membrane Biol. 74:203–215

    Article  Google Scholar 

  • De Mazière, A., Analbers, L., Jongsma, H.J., Gros, D. 1992. Immunoelectron microscopoc visualization of the gap junction protein connexin40 in the mammalian heart.Eur. J. Morph. 30:305–308

    Google Scholar 

  • Gamble W., Vaughan, M., Kruth, H.S., Avigan, J. 1978. Procedure for determination of free and total cholesterol in micro- or nanogram amounts suitable for studies with cultured cells.J. Lipid Res. 19:1068–1070

    CAS  PubMed  Google Scholar 

  • Giaume, C., Fromaget, C., El Aoumari, A., Cordier, J., Glowinski, J., Gros, D. 1991. Gap junctions in cultured astrocytes: Single-channel currents and characterization of channel-forming protein.Neuron 6:133–143

    Article  CAS  Google Scholar 

  • Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J. 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.Pfluegers Arch. 391:85–100

    Article  CAS  Google Scholar 

  • Havel R.J., Eder, H.A., Bragdon, J. 1955. The distribution and chemical preparation of ultracentrifugically separated lipoproteins in human serum.J. Clin. Invest. 34:1345–1353

    Article  CAS  Google Scholar 

  • Johnston, M.F., Simon, S.A., Ramón, F. 1980. Interaction of anaesthetics with electrical synapses.Nature 286:498–500

    Article  CAS  Google Scholar 

  • Kanter, H.L., Saffitz, J.E., Beyer, E.C. 1992. Cardiac myocytes express multiple gap junction proteins.Circ. Res. 70:438–444

    Article  CAS  Google Scholar 

  • Kosower E.M., Kosower, N.S., Faltin, F. 1974. Membrane mobility agents: A new class of biologically active molecules.Biochim. Biophys. Acta 363:261–266

    Article  CAS  Google Scholar 

  • Kuhry, J.-G., Fonteneau, P., Duportail, G., Maechling, C., Laustriat, G. 1983. TMA-DPH: A suitable fluorescence polarization probe for specific plasma membrane fluidity studies in intact living cells.Cell Biophys. 5:129–140

    Article  CAS  Google Scholar 

  • Lakowicz, J.R., ed. 1983. Principles of Fluorescence Spectroscopy. Plenum, New York

    Google Scholar 

  • Malewicz, B., Kumar, V.V., Johnson, R.G., Baumann, W.J., 1990. Lipids in gap junction assembly and function.Lipids 25:419–427

    Article  CAS  Google Scholar 

  • Meda, P., Bruzzone, R., Knodel, S., Orci, L. 1986. Blockage of cell-to-cell communication within pancreatic acini is associated with increased basal release of amylase.J. Cell Biol. 103:475–483

    Article  CAS  Google Scholar 

  • Meyer, R., Malewicz, B., Baumann, W.J., Johnson, R.G. 1990. Increased gap junction assembly between cultured cells upon cholesterol supplementation.J. Cell Sci. 96:231–238

    CAS  PubMed  Google Scholar 

  • Niggli, E., Rüdisüli, A., Maurer, P., Weingart, R. 1989. Effects of general anesthetics on current flow across membranes in guinea pig myocytes.Am. J. Physiol. 256:C273-C281

    Article  CAS  Google Scholar 

  • Page, E. 1992. Cardiac gap junctions.In: The Heart and the Cardiovascular System. H.A. Fozzard, E. Haber, R.B. Jennings, A.M. Katz, H.E. Morgan, editors. pp. 1003–1047. Raven, New York

    Google Scholar 

  • Pérez-Armendariz, M., Roy, C., Spray, D.C., Bennett, M.V.L. 1991. Biophysical properties of gap junctions between freshly dispersed pairs of mouse pancreatic beta cells.Biophys. J. 59:76–92

    Article  Google Scholar 

  • Robenek, H., Jung, W., Gebhardt, R. 1982. The topography of filipin-cholesterol complexes in the plasma membrane of cultured hepatocytes and their relation to cell junction formation.J. Ultrastr. Res. 78:95–106

    Article  CAS  Google Scholar 

  • Rook, M.B., Jongsma, H.J., van Ginneken, A.C.G. 1988. Properties of single gap junctional channels between isolated neonatal rat heart cells.Am. J. Physiol. 255:H770-H782

    CAS  PubMed  Google Scholar 

  • Rüdisüli, A., Weingart, R. 1989. Electrical properties of gap junction channels in guinea-pig ventricular cell pairs revealed by exposure to heptanol.Fluegers Arch. 415:12–21

    Article  Google Scholar 

  • Schroeder, F., Jefferson, J.R., Kier, A.B., Knittel, J., Scallen, T.J., Gibson Wood, W., Hapala, I. 1991. Membrane cholesterol dynamics: Cholesterol domains and kinetic pools.Proc. Soc. Exp. Biol. Med. 196:235–252

    Article  CAS  Google Scholar 

  • Severs, N.J. 1981. Plasma membrane cholesterol in myocardial muscle and capillary endothelial cells. Distribution of filipin-induced deformations in freeze-fracture.Eur. J. Cell Biol. 25:289–299

    CAS  PubMed  Google Scholar 

  • Sheridan N.P., Block, E.R. 1988. Plasma membrane fluidity measurements in intact endothelial cells: Effect of hyperoxia on fluorescence anisotropies of 1-[4-(trimethylamino)phenyl]-6-phenyl hexatriene.J. Cell Physiol. 134:117–123

    Article  CAS  Google Scholar 

  • Shinitzky, M. 1984. Membrane fluidity and cellular functions.In: Physiology of Membrane Fluidity. M. Shinitzky, editor. Vol. 1, pp. 1–51. CRC, Boca Raton, FL

    Google Scholar 

  • Sklar, L.A., Miljanich, G.P., Bursten, L.S., Dratz, E.A. 1979. Thermal lateral phase separations in bovine retinal rod outer segment membranes and phospholipids as evidenced by parinaric acid fluorescence polarization and energy transfer.J. Biol. Chem. 254:9583–9597.

    CAS  PubMed  Google Scholar 

  • Sklar, L.A. 1980. The partition of cis-parinaric acid and transparinaric acid among aqueous, fluid lipid and solid lipid phase.Mol. Cell. Biochem. 32:169–177

    Article  CAS  Google Scholar 

  • Sorisky A., Kucera, G.L., Rittenhouse, S.E. 1990. Stimulated cholesterol-enriched platelets display increased cytosolic Ca2+ and phospholipase A activity independent of changes in inositol triphosphates and agonist/receptor binding.Biochem. J. 265:747–754

    Article  CAS  Google Scholar 

  • Spray, D.C., Burt, J.M. 1990. Structure-activity relations of the cardiac gap junction channel.Am. J. Physiol. 258:C195-C205

    Article  CAS  Google Scholar 

  • Spray, D.C., Moreno, A.P., Kessler, J.A., Dermietzel, R. 1991. Characterization of gap junctions between cultured leptomeningeal cells.Brain Res. 568:1–14

    Article  CAS  Google Scholar 

  • Sumbilla, C., Lakowicz, J.R. 1983. Evidence for normal fibroblast cell membranes from individuals with Huntington's disease.J. Neurol. Sci. 62:23–40

    Article  CAS  Google Scholar 

  • Takens-Kwak, B.R., Jongsma, H.J., Rook, M.B., van Ginneken, A.C.G. 1992. Mechanism of heptanol-induced uncoupling of cardiac gap junctions: A perforated patch-clamp study.Am. J. Physiol. 262:C1531-C1538

    Article  CAS  Google Scholar 

  • Van der Meer, B.W. 1988. Biomembrane structure and dynamics viewed by fluorescence.In: Subcellular Biochemistry. Hilderson et al., editors, Vol. 13, pp. 1–53. Plenum, New York

    Google Scholar 

  • Veenstra, R.D., De Haan, R.L. 1986. Measurement of single channel currents from cardiac gap junctions.Science 233:972–974

    Article  CAS  Google Scholar 

  • Wilders, R., Jongsma, H.J. 1992. Limitations of the dual voltage clamp method in assaying conductance and kinetics of gap junction channels.Biophys. J. 63:942–953

    Article  CAS  Google Scholar 

  • Yeagle, P.L. 1985. Cholesterol and the cell membrane.Biochim. Biophys. Acta 822:267–287

    Article  CAS  Google Scholar 

  • Zwijsen R.M.L., Oudenhoven, I.M.J., de Haan, L.H.J. 1992. Effects of cholesterol and oxysterols on gap junctional communication between human smooth muscle cells.Eur. J. Pharmacol. 228:115–120

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bastiaanse, E.M.L., Jongsma, H.J., van der Laarse, A. et al. Heptanol-induced decrease in cardiac gap junctional conductance is mediated by a decrease in the fluidity of membranous cholesterol-rich domains. J. Membrain Biol. 136, 135–145 (1993). https://doi.org/10.1007/BF02505758

Download citation

  • Received:

  • Revised:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02505758

Key words

Navigation