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. 1993 Aug 1;90(15):7280–7284. doi: 10.1073/pnas.90.15.7280

Atomic force microscopy of cloned nicotinic acetylcholine receptor expressed in Xenopus oocytes.

R Lal 1, L Yu 1
PMCID: PMC47120  PMID: 7688475

Abstract

The nicotinic acetylcholine receptor (AChR) was expressed in Xenopus oocytes from in vitro transcribed mRNA and was imaged by atomic force microscopy. A characteristic pentameric structure of AChR was readily observed on the extracellular face of the cell membrane, with a central pore surrounded by protruding AChR subunits. These structures were seen only in mRNA-injected oocytes that also gave acetylcholine-induced membrane currents. The size of individual AChR channels, the angles between subunits, and the interchannel spacing were all compatible with the current model of AChR. In addition, localized patches of microscopic AChR clustering were observed, with packing density approaching that at the neuromuscular junction. These findings show the potential of studying cloned membrane proteins in oocytes for both their surface topography and their structure-function relationship in native membrane without the need for crystallization.

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Selected References

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

  1. Bertazzon A., Conti-Tronconi B. M., Raftery M. A. Scanning tunneling microscopy imaging of Torpedo acetylcholine receptor. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9632–9636. doi: 10.1073/pnas.89.20.9632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Binnig G, Quate CF, Gerber C. Atomic force microscope. Phys Rev Lett. 1986 Mar 3;56(9):930–933. doi: 10.1103/PhysRevLett.56.930. [DOI] [PubMed] [Google Scholar]
  3. Bon F., Lebrun E., Gomel J., Van Rapenbusch R., Cartaud J., Popot J. L., Changeux J. P. Image analysis of the heavy form of the acetylcholine receptor from Torpedo marmorata. J Mol Biol. 1984 Jun 25;176(2):205–237. doi: 10.1016/0022-2836(84)90421-2. [DOI] [PubMed] [Google Scholar]
  4. Brisson A., Unwin P. N. Quaternary structure of the acetylcholine receptor. Nature. 1985 Jun 6;315(6019):474–477. doi: 10.1038/315474a0. [DOI] [PubMed] [Google Scholar]
  5. Butt H. J., Downing K. H., Hansma P. K. Imaging the membrane protein bacteriorhodopsin with the atomic force microscope. Biophys J. 1990 Dec;58(6):1473–1480. doi: 10.1016/S0006-3495(90)82492-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clemmer C. R., Beebe T. P., Jr Graphite: a mimic for DNA and other biomolecules in scanning tunneling microscope studies. Science. 1991 Feb 8;251(4994):640–642. doi: 10.1126/science.1992517. [DOI] [PubMed] [Google Scholar]
  7. Dascal N. The use of Xenopus oocytes for the study of ion channels. CRC Crit Rev Biochem. 1987;22(4):317–387. doi: 10.3109/10409238709086960. [DOI] [PubMed] [Google Scholar]
  8. Drake B., Prater C. B., Weisenhorn A. L., Gould S. A., Albrecht T. R., Quate C. F., Cannell D. S., Hansma H. G., Hansma P. K. Imaging crystals, polymers, and processes in water with the atomic force microscope. Science. 1989 Mar 24;243(4898):1586–1589. doi: 10.1126/science.2928794. [DOI] [PubMed] [Google Scholar]
  9. Edstrom R. D., Meinke M. H., Yang X. R., Yang R., Elings V., Evans D. F. Direct visualization of phosphorylase-phosphorylase kinase complexes by scanning tunneling and atomic force microscopy. Biophys J. 1990 Dec;58(6):1437–1448. doi: 10.1016/S0006-3495(90)82489-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fertuck H. C., Salpeter M. M. Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after 125I-alpha-bungarotoxin binding at mouse neuromuscular junctions. J Cell Biol. 1976 Apr;69(1):144–158. doi: 10.1083/jcb.69.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Flucher B. E., Daniels M. P. Distribution of Na+ channels and ankyrin in neuromuscular junctions is complementary to that of acetylcholine receptors and the 43 kd protein. Neuron. 1989 Aug;3(2):163–175. doi: 10.1016/0896-6273(89)90029-9. [DOI] [PubMed] [Google Scholar]
  12. Froehner S. C., Luetje C. W., Scotland P. B., Patrick J. The postsynaptic 43K protein clusters muscle nicotinic acetylcholine receptors in Xenopus oocytes. Neuron. 1990 Oct;5(4):403–410. doi: 10.1016/0896-6273(90)90079-u. [DOI] [PubMed] [Google Scholar]
  13. Hansma H. G., Vesenka J., Siegerist C., Kelderman G., Morrett H., Sinsheimer R. L., Elings V., Bustamante C., Hansma P. K. Reproducible imaging and dissection of plasmid DNA under liquid with the atomic force microscope. Science. 1992 May 22;256(5060):1180–1184. doi: 10.1126/science.256.5060.1180. [DOI] [PubMed] [Google Scholar]
  14. Hansma P. K., Elings V. B., Marti O., Bracker C. E. Scanning tunneling microscopy and atomic force microscopy: application to biology and technology. Science. 1988 Oct 14;242(4876):209–216. doi: 10.1126/science.3051380. [DOI] [PubMed] [Google Scholar]
  15. Henderson E., Haydon P. G., Sakaguchi D. S. Actin filament dynamics in living glial cells imaged by atomic force microscopy. Science. 1992 Sep 25;257(5078):1944–1946. doi: 10.1126/science.1411511. [DOI] [PubMed] [Google Scholar]
  16. Hoh J. H., Lal R., John S. A., Revel J. P., Arnsdorf M. F. Atomic force microscopy and dissection of gap junctions. Science. 1991 Sep 20;253(5026):1405–1408. doi: 10.1126/science.1910206. [DOI] [PubMed] [Google Scholar]
  17. Holtzman E., Wise D., Wall J., Karlin A. Electron microscopy of complexes of isolated acetylcholine receptor, biotinyl-toxin, and avidin. Proc Natl Acad Sci U S A. 1982 Jan;79(2):310–314. doi: 10.1073/pnas.79.2.310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Karlin A., Holtzman E., Yodh N., Lobel P., Wall J., Hainfeld J. The arrangement of the subunits of the acetylcholine receptor of Torpedo californica. J Biol Chem. 1983 Jun 10;258(11):6678–6681. [PubMed] [Google Scholar]
  19. Kistler J., Stroud R. M., Klymkowsky M. W., Lalancette R. A., Fairclough R. H. Structure and function of an acetylcholine receptor. Biophys J. 1982 Jan;37(1):371–383. doi: 10.1016/S0006-3495(82)84685-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Krafte D. S., Lester H. A. Expression of functional sodium channels in stage II-III Xenopus oocytes. J Neurosci Methods. 1989 Jan;26(3):211–215. doi: 10.1016/0165-0270(89)90118-0. [DOI] [PubMed] [Google Scholar]
  21. Mitra A. K., McCarthy M. P., Stroud R. M. Three-dimensional structure of the nicotinic acetylcholine receptor and location of the major associated 43-kD cytoskeletal protein, determined at 22 A by low dose electron microscopy and x-ray diffraction to 12.5 A. J Cell Biol. 1989 Aug;109(2):755–774. doi: 10.1083/jcb.109.2.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Radmacher M., Tillamnn R. W., Fritz M., Gaub H. E. From molecules to cells: imaging soft samples with the atomic force microscope. Science. 1992 Sep 25;257(5078):1900–1905. doi: 10.1126/science.1411505. [DOI] [PubMed] [Google Scholar]
  23. Unwin N., Toyoshima C., Kubalek E. Arrangement of the acetylcholine receptor subunits in the resting and desensitized states, determined by cryoelectron microscopy of crystallized Torpedo postsynaptic membranes. J Cell Biol. 1988 Sep;107(3):1123–1138. doi: 10.1083/jcb.107.3.1123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Weisenhorn A. L., Drake B., Prater C. B., Gould S. A., Hansma P. K., Ohnesorge F., Egger M., Heyn S. P., Gaub H. E. Immobilized proteins in buffer imaged at molecular resolution by atomic force microscopy. Biophys J. 1990 Nov;58(5):1251–1258. doi: 10.1016/S0006-3495(90)82465-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Yang J., Tamm L. K., Tillack T. W., Shao Z. New approach for atomic force microscopy of membrane proteins. The imaging of cholera toxin. J Mol Biol. 1993 Jan 20;229(2):286–290. doi: 10.1006/jmbi.1993.1033. [DOI] [PubMed] [Google Scholar]
  26. Yoshii K., Yu L., Mayne K. M., Davidson N., Lester H. A. Equilibrium properties of mouse-Torpedo acetylcholine receptor hybrids expressed in Xenopus oocytes. J Gen Physiol. 1987 Oct;90(4):553–573. doi: 10.1085/jgp.90.4.553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yu L., Leonard R. J., Davidson N., Lester H. A. Single-channel properties of mouse-Torpedo acetylcholine receptor hybrids expressed in Xenopus oocytes. Brain Res Mol Brain Res. 1991 Jun;10(3):203–211. doi: 10.1016/0169-328x(91)90062-3. [DOI] [PubMed] [Google Scholar]

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