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Pflügers Archiv

, Volume 407, Issue 6, pp 577–588 | Cite as

Patch clamp measurements onXenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels

  • C. Methfessel
  • V. Witzemann
  • T. Takahashi
  • M. Mishina
  • S. Numa
  • B. Sakmann
Excitable Tissues and Central Nervous Physiology

Abstract

  1. 1.

    Functional acetylcholine receptor (AChR) and sodium channels were expressed in the membrane ofXenopus laevis oocytes following injection with poly(A)+-mRNA extracted from denervated rat leg muscle. Wholecell currents, activated by acetylcholine or by depolarizing voltage steps had properties comparable to those observed in rat muscle. Oocytes injected with specific mRNA, transcribed from cDNA templates and coding for the AChR ofTorpedo electric organ, expressed functional AChR channels at a much higher density.

     
  2. 2.

    Single-channel currents were recorded from the oocyte plasma membrane following removal of the follicle cell layer and the vitelline membrane from the oocyte. The follicle cell layer was removed enzymatically with collagenase. The vitelline membrane was removed either mechanically after briefly exposing the oocyte to a hypertonic solution, or by enzyme treatment with pronase.

     
  3. 3.

    Stretch activated (s.a.) currents were observed in most recordings from cell-attached patches obtained with standard patch pipettes. S.a.-currents were evoked by negative or positive pressure (≥5 mbar) applied to the inside of the pipette, and were observed in both normal and mRNA injected oocytes indicating that they are endogenous to the oocyte membrane.

     
  4. 4.

    The s.a.-channels are cation selective and their conductance is 28 pS in normal frog Ringer's solution (20±1°C). Their gating is voltage dependent, and their open probability increases toward more positive membrane potentials.

     
  5. 5.

    The density of s.a.-channels is estimated to be 0.5–2 channels per μm2 of oocyte plasma membrane. In cell-attached patches s.a.-currents are observed much less frequently when current measurement is restricted to smaller patches of 3–5 μm2 area using thick-walled pipettes with narrow tips. In outside-out patches s.a.-currents occur much less frequently than in cell-attached or inside-out patches.

     
  6. 6.

    AChR-channel and sodium channel currents were observed only in a minority of patches from oocytes injected with poly(A)+-mRNA from rat muscle. AChR-channel currents were seen in all patches of oocytes injected with specific mRNA coding forTorpedo AChR. In normal frog Ringer's solution (20±2°C) the conductance of implanted rat muscle AChR-channels was 38 pS and that of sodium channels 20 pS. The conductance of implantedTorpedo AChR channels was 40 pS. The conductance of implanted channels was similar in cell-attached and in cell-free patches.

     
  7. 7.

    The conductances of rat muscle AChR and sodium channels implanted into the oocyte membrane were similar to those of channels in their native muscle membrane, suggesting that important functional properties of these channels are determined by their primary amino acid sequence.

     

Key words

Patch clamp recording Oocyte membrane Ion channels 

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References

  1. Almers W, Roberts WM, Ruff RL (1984) Voltage clamp of rat and human skeletal muscle: measurements with an improved loosepatch technique. J Physiol 347:751–768Google Scholar
  2. Armstrong CM, Bezanilla F (1974) Charge movement associated with the opening and closing of the activation gates of the Na channels. J Gen Physiol 63:533–552Google Scholar
  3. Aviv H, Leder P (1972) Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci USA 69:1408–1412Google Scholar
  4. Barnard EA, Miledi R, Sumikawa K (1982) Translation of exogenous messenger RNA coding for nicotinic acetylcholine receptors produces functional receptors inXenopus oocytes. Proc R Soc Lond B 215:241–246Google Scholar
  5. Colquhoun D, Sakmann B (1985) Fast events in single-channel currents activated by acetylcholine and its analogues at the frog muscle end-plate. J Physiol 369:501–557Google Scholar
  6. Dumont JN (1972) Oogenesis inXenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J Morphol 136:153–180Google Scholar
  7. Dumont J, Brummett AR (1978) Oogenesis inXenopus laevis (Daudin). V. Relationship between developing oocytes and their investing follicular tissues. J Morphol 155:73–98Google Scholar
  8. Glisin V, Crkvenjakov R, Byus C (1974) Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13:2633–2637Google Scholar
  9. Guharay F, Sachs F (1984) Stretch-activated single ion channel currents in tissue-cultured embryonic chick skeletal muscle. J Physiol 352:685–701Google Scholar
  10. Gundersen CB, Miledi R, Parker I (1983) Voltage-operated channels induced by foreign messenger RNA inXenopus oocytes. Proc R Soc Lond B 220:131–140Google Scholar
  11. Gundersen CB, Miledi R, Parker I (1984) Messenger RNA from human brain induces drug- and voltage-operated channels inXenopus oocytes. Nature 308:421–424Google Scholar
  12. Gurdon JB (1974) The control of gene expression in animal development. Harvard University Press, Cambridge, MAGoogle Scholar
  13. Gurdon JB, Lane CD, Woodland HR, Marbaix G (1971) Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells. Nature 233:177–182Google Scholar
  14. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391:85–100Google Scholar
  15. Kado RT, Marcher K, Ozon R (1981) Electrical membrane properties of theXenopus laevis oocyte during progesterone-induced meiotic maturation. Dev Biol 84:471–476Google Scholar
  16. Kusano K, Miledi R, Stinnakre J (1982) Cholinergic and catecholaminergic receptors in theXenopus oocyte membrane. J Physiol 328:143–170Google Scholar
  17. Miledi R, Parker I, Sumikawa K (1983) Recording of single γ-aminobutyrate-and acetylcholine-activated receptor channels translated by exogenous mRNA inXenopus oocytes. Proc R Soc Lond B 218:481–484Google Scholar
  18. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  19. Mishina M, Kurosaki T, Tobimatsu T, Morimoto Y, Noda M, Yamamoto T, Terao M, Lindstrom J, Takahashi T, Kuno M, Numa S (1984) Expression of functional acetylcholine receptor from cloned cDNAs. Nature 307:604–608Google Scholar
  20. Mishina M, Tobimatsu T, Imoto K, Tanaka K, Fujita Y, Fukuda K, Kurasaki M, Takahashi H, Morimoto Y, Hirose T, Inayama S, Takahashi T, Kuno M, Numa S (1985) Location of functional regions of acetylcholine receptor α-subunit by site-directed mutagenesis. Nature 313:364–369Google Scholar
  21. Mishina M, Takai T, Imoto K, Noda M, Takahashi T, Numa S, Methfessel C, Sakmann B (1986) Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature 321:406–411Google Scholar
  22. Ncher E, Sakmann B (1975) Voltage-dependence of drug-induced conductance in frog neuromuscular junction. Proc Natl Acad Sci USA 72:2140–2144Google Scholar
  23. Neher E, Sakmann B (1976) Noise analysis of drug induced voltage clamp currents in denervated frog muscle fibres. J Physiol 258:705–729Google Scholar
  24. Pappone PA (1980) Voltage-clamp experiments in normal and denervated mammalian skeletal muscle fibres. J Physiol 306:377–410Google Scholar
  25. Pelham HRB, Jackson RJ (1976) An efficient mRNA-dependent translation system for reticulocyte lysates. Eur J Biochem 67:247–256Google Scholar
  26. Sakmann B, Neher E (1983) Geometric parameters of pipettes and membrane patches. In: Sakmann B, Neher E (eds) Single channel recording. Plenum Press, New York, pp 37–51Google Scholar
  27. Sakmann B, Patlak J, Neher E (1980) Single acetylcholine-activated channels show burst-kinetics in presence of desensitizing concentrations of agonist. Nature 286:71–73Google Scholar
  28. Sakmann B, Bormann J, Hamill OP (1983) Ion transport by single receptor channels. In: Cold Spring Harbor Symposia on Quantitative Biology, vol XLVIII. Cold Spring Harbor Laboratory, pp 247–257Google Scholar
  29. Sakmann B, Methfessel C, Mishina M, Takahashi T, Takai T, Kurasaki M, Fukuda K, Numa S (1985) Role of acetylcholine receptor subunits in gating of the channel. Nature 318:538–543Google Scholar
  30. Sigworth FJ (1980) The variance of sodium current fluctuations at the node of Ranvier. J Physiol 307:97–129Google Scholar
  31. Sigworth FJ, Neher E (1980) Single Na+ channel currents observed in cultured rat muscle cells. Nature 287:447–449Google Scholar
  32. Smart TG, Constanti A, Bilbe G, Brown DA, Barnard EA (1983) Synthesis of functional chick brain GABA-benzodiazepine-barbiturate/receptor complexes in mRNA-injectedXenopus oocytes. Neurosci Lett 40:55–59Google Scholar
  33. Sumikawa K, Houghton M, Emtage JS, Richards BM, Barnard EA (1981) Active multi-subunit ACh receptor assembled by translation of heterologous mRNA inXenopus oocytes. Nature 292:862–864Google Scholar

Copyright information

© Springer-Verlag 1986

Authors and Affiliations

  • C. Methfessel
    • 1
  • V. Witzemann
    • 2
  • T. Takahashi
    • 3
  • M. Mishina
    • 4
  • S. Numa
    • 4
  • B. Sakmann
    • 1
  1. 1.Abt. ZellphysiologieMax-Planck-Institut für biophysikalische ChemieGöttingenFederal Republic of Germany
  2. 2.Abt. NeurochemieMax-Planck-Institut für biophysikalische ChemieGöttingenFederal Republic of Germany
  3. 3.Dep. of PhysiologyKyoto University Faculty of MedicineKyotoJapan
  4. 4.Dept. of Medical Chemistry and Molecular GeneticsKyoto University Faculty of MedicineKyotoJapan

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