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Single channel recordings from synaptosomal AMPA receptors

Cell Biochemistry and Biophysics volume 42, pages 75–85 (2005)Cite this article

Abstract

Synaptic glutamate receptors play a prominent role in the excitatory neurotransmission in the vertebrate central nervous system. Although elucidation of the functional properties of glutamate receptors using electrophysiologic analyses has yielded important information, methodological and technological limitations have prevented direct measurement of single channel properties of synaptic receptors. Here, we have isolated murine mossy fiber synaptosomes and reconstituted them into small artificial lipid bilayers to characterize the single-channel properties of synaptic α amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-subtype glutamate receptors. The reconstituted synaptosomal receptors were activated by nanomolar concentrations of AMPA and blocked by a potent AMPA receptor antagonist. The synaptosomal AMPA receptors exhibited channel conductances of 14–56 pS and linear current-voltage relationship. The open and closed dwell time distributions of single channel currents were best described by three exponentials. These channels frequently exhibited burst behavior with long burst duration of approx 60 ms. Experiments with multichannel recordings revealed that steady state probabilities could not be fitted using a binomial distribution, indicating a cooperative channel gating behavior that would account for larger membrane currents. Our findings suggest that isolation, reconstitution into lipid bilayers, and subsequent single channel analysis of synaptosomal receptors is a useful method for investigation of synaptic AMPA receptors.

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References

  1. Dingledine, R., Borges, K., Bowie, D., and Traynelis, S.F. (1999) The glutamate receptor ion channel. Pharmacol. Rev. 51, 7–61.

    PubMed  CAS  Google Scholar 

  2. Malenka, R. C., and Nicoll, R. A. (1999) Long-term potentiation—a decade of progress?. Science 285, 1870–1874.

    Article  PubMed  CAS  Google Scholar 

  3. Benke, T. A., Luthi, A., Isaac, J. T. R., and Collingridge, G. L. (1998) Modulation of AMPA receptor unitary conductance by synaptic activity. Nature 393, 793–797.

    Article  PubMed  CAS  Google Scholar 

  4. Derkach, V., Barria A., and Soderling, T. R. (1999) Ca2+/calmodulin-kinase II enhances channel conductance of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate type glutamate receptors. Proc. Natl. Acad. Sci. U. S. A. 96, 3269–3274.

    Article  PubMed  CAS  Google Scholar 

  5. Ambros-Ingerson, J., and Lynch, G. (1993) Channel gating kinetics and synaptic efficacy: a hypothesis for expression of long-term potentiation. Proc. Natl. Acad. Sci. U S A 90, 7903–7907.

    Article  PubMed  CAS  Google Scholar 

  6. Hayashi, Y., Shi, S. H., Esteban, J. A., Piccini, A., Poncer, J. C., and Malinow, R. (2000) Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction. Science 287, 2262–2267.

    Article  PubMed  CAS  Google Scholar 

  7. Isaac, J. T., Nicoll, R. A., and Malenka, R. C. (1995) Evidence for silent synapses: implications for the expression of LTP. Neuron 15, 427–434.

    Article  PubMed  CAS  Google Scholar 

  8. Lledo, P. M., Zhang, X., Sudhof, T. C., Malenka, R. C., and Nicoll, R. A. (1998) Postsynaptic membrane fusion and long-term potentiation. Science 279, 399–403.

    Article  PubMed  CAS  Google Scholar 

  9. Liao, D., Hessler, N. A., and Malinow, R. (1995) Activation of postsynaptically silent synapses during pairing-induced LTP in CA1 region of hippocampal slice. Nature 375, 400–404.

    Article  PubMed  CAS  Google Scholar 

  10. Liao, D., Zhang, X., O'Brien, R., Ehlers, M. D., and Huganir, R. L. (1999) Regulation of morphological postsynaptic silent synapses in developing hippocampal neurons. Nat. Neurosci. 2, 37–43.

    Article  PubMed  CAS  Google Scholar 

  11. Braithwaite, S. P., Meyer, G., and Henley, J. M. (2000) Interactions between AMPA receptors and intracellular proteins. Neuropharmacology 39, 919–930.

    Article  PubMed  CAS  Google Scholar 

  12. Yamada, K. A. (2000) Therapeutic potential of positive AMPA receptor modulators in the treatment of neurological disease. Expert Opin. Investig. Drugs 9, 765–778.

    Article  PubMed  CAS  Google Scholar 

  13. Lissin, D. V., Gomperts, S. N., Carrollm R. C., Christine, C. W., Kalmanm, D., and Kitamuram, M. (1998) Activity differentially regulates the surface expression of synaptic AMPA and NMDA glutamate receptors. Proc. Natl. Acad. Sci. USA 95, 7097–7102.

    Article  PubMed  CAS  Google Scholar 

  14. Friedman, H. V., Bresle, T., Garner, C. C., and Ziv, N. E. (2000) Assembly of new individual excitatory synapses: time course and temporal order of synaptic molecule recruitment. Neuron 27, 57–69.

    Article  PubMed  CAS  Google Scholar 

  15. Sheng, M., and Kim, M. J. (2002) Postsynaptic signaling and plasticity mechanisms. Science 298, 776–780.

    Article  PubMed  CAS  Google Scholar 

  16. Song, I., and Huganir, R. L. (2002) Regulation of AMPA receptors during synaptic plasticity. Trends Neurosci. 11, 578–588.

    Article  Google Scholar 

  17. Livsey, C. T., Costa, E., and Vicini, S. (1993) Glutamate-activated currents in outside-out patches from spiny versus aspiny hilar neurons of rat hippocampal slices. J. Neurosci. 13, 5324–5333.

    PubMed  CAS  Google Scholar 

  18. Taupin, P., Zini, S., Cesselin, F., Ben-Ari, Y., and Roisin, M. P. (1994) Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: morphological and biochemical characterization in control and degranulated rat hippocampus. J. Neurochem. 62, 1586–1595.

    Article  PubMed  CAS  Google Scholar 

  19. Suppiramaniam, V., Bahr, B. A., Sinnarajah, S., Owens, K., Rogers, G., and Yilma, S. (2001) Member of the Ampakine class of memory enhancers prolongs the single channel open time of reconstituted AMPA receptors. Synapse 40, 154–158.

    Article  PubMed  CAS  Google Scholar 

  20. White, W. F., and Snodgrass, S. R. (1983) Isoguvacine binding, uptake, and release: relation to the GABA system. J. Neurochem. 40, 1701–1708.

    Article  PubMed  CAS  Google Scholar 

  21. Davis, L. G., Brunngraber, E. G., and Routtenberg, A. (1979) A regional study of electrophoretically separated proteins from the crude synaptosomal fractions of the developing rat brain. J. Neurosci. Res. 4, 25–34.

    Article  PubMed  CAS  Google Scholar 

  22. Pickard, L., Noel, J., Henley, J. M., Collingridge, G. L., and Molnar, E. (2000) Developmental changes in synaptic AMPA and NMDA receptor distribution and AMPA receptor subunit composition in living hippocampal neurons. J. Neurosci. 20, 7922–7931.

    PubMed  CAS  Google Scholar 

  23. Coronado, R. and Lattore, R. (1983) Phospholipid bilayers made from monolayers on patch-clamp pipettes. Biophys. J. 43, 231–236.

    PubMed  CAS  Google Scholar 

  24. Suarez-Isla, B. A., Wan, K., Lindstrom, J., and Montal, M. (1983) Single channel recordings from purified acetylcholine receptors reconstituted in bilayers formed at the tip of patch pipettes. Biochemistry 22, 2319–2323.

    Article  PubMed  CAS  Google Scholar 

  25. Wilmsen, U., Methfesselm, F., Hanke, W., and Boheim, G. (1983) Channel current fluctuation studies with solvent free lipid bilayers using Neher-Sakmann pipettes, in Physical Chemistry of Transmembrane Ion Motion (Spach, G., ed.) Elsevier, Amsterdam, pp. 479–485.

    Google Scholar 

  26. Vodyanoy, V. (1989) Cyclic nucleotide-gated electrical activity in olfactory receptors, in Receptor and Transduction Mechanisms in Taste and Olfaction (Brand, J. G. and Teeter, J. H., eds.), Marcel Dekker, New York, pp. 319–345.

    Google Scholar 

  27. Vodyanoy, V. (1989) Molecular sensor based on olfactory transduction, in Molecular Electronics: Biosensors and Biocomputers (Hong, F. T., ed.), Plenum Publishing Corp., New York, pp. 317–328.

    Google Scholar 

  28. Sinnarajah, S., Suppiramaniam, V., Kumar, K. P., Hall, R. A., Bahr, B. A., and Vodyanoy, V. (1999) Heparin modulates the single channel kinetics of reconstituted AMPA receptors from rat brain. Synapse 31, 203–209.

    Article  PubMed  CAS  Google Scholar 

  29. Sigurdson, W. J., Morris, C. E., Brezden B. L., and Gardner, D. R. (1987) Stretch activation of a K+ channel in molluscan heart cells. J. Exp. Biol. 127, 191–209.

    Google Scholar 

  30. Manivannan, K., Ramanan, S. V., Mathias, R. T., and Brink, P. R. (1992) Multichannel recordings from membranes which contain gap junctions. Biophys. J. 1, 216–227.

    Google Scholar 

  31. Manivannan, K., Gudowska-Nowak, E., and Mathias, R. T. (1996) Description of interacting channel gating using a stochastic Markovian model. Bull. Math. Biol. 58, 141–174.

    Article  PubMed  CAS  Google Scholar 

  32. Smith, T. C., Wang, L. Y., and Howe, J. R. (2000) Heterogeneous conductance levels of native AMPA receptors. J. Neurosci. 20, 2073–2085.

    PubMed  CAS  Google Scholar 

  33. Jonas, P., and Sakmann, B. (1992) Glutamate receptor channels in isolated patches from CA1 and CA3 pyramidal cells of rat hippocampal slices. J. Physiol. 455, 143–171.

    PubMed  CAS  Google Scholar 

  34. Vodyanoy, V., Bahr, B. A., Suppiramaniam, V., Hall, R. A., Baudry, M., and Lynch, G. (1993) Single channel recording of reconstituted AMPA receptors reveal low and high conductance states. Neurosci. Lett. 50, 80–84.

    Article  Google Scholar 

  35. Vyklicky, L. Jr., Patneau, D. K., and Mayer, M. L. (1991) Modulation of excitatory synaptic transmission by drugs that reduce desensitization at AMPA/kainate receptors. Neuron 7, 971–984.

    Article  PubMed  CAS  Google Scholar 

  36. Hjelmstad, G. O., Isaac, J. T., Nicoll, R. A., and Malenka, R. C. (1999) Lack of AMPA receptor desensitization during basal synaptic transmission in the hippocampal slice. J. Neurophysiol. 81, 3096–3099.

    PubMed  CAS  Google Scholar 

  37. Jonas, P., Major, G., and Sakmann, B. (1993). Quantal components of unitary EPSCs at the mossy fiber synapse on CA3 pyramidal cells of rat hippocampus. J. Physiol. 472, 615–663.

    PubMed  CAS  Google Scholar 

  38. Wyneken, U., Riquelme, G., Villanueva, S., and Orrego, F. (1997) Effect of glutamate receptor phosphorylation by endogenous protein kinases on electrical activity of isolated postsynaptic densities of rat cortex and hippocampus. Neurosci. Lett. 224, 131–135.

    Article  PubMed  CAS  Google Scholar 

  39. Traynelis, S. F., Silver, R. A., and Cull-Candy, S. G. (1993) Estimated conductance of glutamate receptor channels activated during EPSCs at the cerebellar mossy fiber-granule cell synapse. Neuron 11, 279–289.

    Article  PubMed  CAS  Google Scholar 

  40. Wyllie, D. J., Traynelis, S. F., and Cull-Candy, S. G. (1993) Evidence for more than one type of non-NMDA receptor in outside-out patches from cerebellar granule cells of the rat. J. Physiol. 463, 193–226.

    PubMed  CAS  Google Scholar 

  41. Wyllie, D. J. A., and Cull-Candy, S. G. (1994) A comparison of non-NMDA receptor channels in type-2 astrocytes and granule cells from rat cerebellum. J. Physiol. 475, 95–114.

    PubMed  CAS  Google Scholar 

  42. Ramanan, S. V., and Brink, P. R. (1993) Multichannel recordings from membranes which contain gap junctions. II. Substates and conductance shifts. Biophys. J. 4, 1387–1395.

    Google Scholar 

  43. Kaleshian, A. M., Edeson, R. O., Liu, G., and Madsen, B. W. (2000) Evidence for cooperativity between nicotinic acetylcholine receptors in patch clamp records. Biophys. J. 78, 1–12.

    Google Scholar 

  44. Yeramian, E., Trautmann A., and Claverie, P. (1986) Acetylcholine receptors are not functionally independent. Biophys. J. 50, 253–263.

    Article  PubMed  CAS  Google Scholar 

  45. Liu, Y., and Dilger, J. P. (1993) Application of the one- and two-dimensional Ising model to studies of cooperativity between ion channels. Biophys. J. 64, 26–35.

    PubMed  CAS  Google Scholar 

  46. Neumcke, B., and Stampfli, R. (1983) Alteration of the conductance of Na+ channels in the nodal membrane of frog nerve by holding potential and tetrodotoxin. Biochem. Biophys. Acta 727, 177–184.

    Article  PubMed  CAS  Google Scholar 

  47. Kiss, T., and Nagy, K. (1985) Interaction between sodium channels in mouse neuroblastoma cells. Eur. Biophys. J. 12, 13–18.

    Article  PubMed  CAS  Google Scholar 

  48. Iwasa, K., Ehrenstein, G., Moran, N., and Jia, M. (1986) Evidence for interactions between batrachotoxin-modified channels in hybrid neuroblastoma cells. Biophys. J. 50, 531–537.

    PubMed  CAS  Google Scholar 

  49. Draber, S., Schultze, R., and Hansen, U. P. (1993) Cooperative behavior of K+ channels in the tonoplast of Chara corallina. Biophys. J. 65, 1553–1559.

    PubMed  CAS  Google Scholar 

  50. Ding, S., and Sachs, F. (2002) Evidence for non-independent gating of P2X2 receptors expressed in Xenopus oocytes. BMC Neurosci. 3, 3–17.

    Article  Google Scholar 

  51. Tardin, C., Cognet, L., Bats, C., Lounis, B., and Choquet, D. (2003) Direct imaging of lateral movement of AMPA receptors inside synapses. Neuron 22, 4656–4665.

    CAS  Google Scholar 

  52. Rao, A., Kim, E., Sheng, M., and Craig, A. M. (1998) Heterogeneity in the molecular composition of excitatory postsynaptic sites during development of hippocampal neurons in culture. J. Neurosci. 18, 1217–1229.

    PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Pharmacal Sciences, Harrison School of Pharmacy, Auburn University, 401 Walker Building, 36849, Auburn, AL

    Thirumalini Vaithianathan, Markus P. Dey & Vishnu Suppiramaniam

  2. Department of Physics, Astronomy, and Material Science, Southwest Missouri State University, 901 South National Avenue, 65804, Springfield, MO

    Kandiah Manivannan

  3. Zentrum für Molekulare Neurobiologie, Universität Hamburg, Martinistr. 52, 20246, Hamburg, Germany

    Ralf Kleene & Alexander Dityatev

  4. Department of Pharmaceutical Sciences, University of Connecticut, 06269, Storrs, CT

    Ben A. Bahr

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  1. Thirumalini Vaithianathan
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Correspondence to Alexander Dityatev or Vishnu Suppiramaniam.

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Vaithianathan, T., Manivannan, K., Kleene, R. et al. Single channel recordings from synaptosomal AMPA receptors. Cell Biochem Biophys 42, 75–85 (2005). https://doi.org/10.1385/CBB:42:1:075

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Index Entries

  • AMPA receptor
  • single channel
  • cooperativity
  • synaptosomes
  • patch clamp