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Functional Analysis of Recombinant Channels in Host Cells Using a Fast Agonist Application System

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NMDA Receptors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1677))

Abstract

A reduced recombinant system provides a unique opportunity to study the biophysical properties of NMDAR channels with known subunit compositions, by using a point mutation approach to analyze the structural determinants of receptor function (Wollmuth and Sobolevsky, Trends Neurosci 27:321–328, 2004). However, in addition to the well-developed repertoire of molecular biological techniques, these types of studies also require electrophysiological methods that allow a wide range of receptor activation protocols that can adequately assess desensitization, inactivation, ion permeability, and other properties of the channels. Currently, one of the most well-developed techniques suitable for addressing these issues is use of the fast agonist application system for rapid activation of ligand gated ion-channels (Colquhoun et al., J Physiol 458:261–287, 1992; Jonas and Sakmann, J Physiol 455:143–171, 1992).

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References

  1. Wollmuth LP, Sobolevsky AI (2004) Structure and gating of the glutamate receptor ion channel. Trends Neurosci 27:321–328

    Article  CAS  PubMed  Google Scholar 

  2. Colquhoun D, Jonas P, Sakmann B (1992) Action of brief pulses of glutamate on AMPA/kainate receptors in patches from different neurons of rat hippocampal slices. J Physiol 458:261–287

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Sabatini BL, Regehr WG (1999) Timing of synaptic transmission. Annu Rev Physiol 61:521–542

    Article  CAS  PubMed  Google Scholar 

  5. Ji D, Lape R, Dani JA (2001) Timing and location of nicotinic activity enhances or depresses hippocampal synaptic plasticity. Neuron 31:131–141

    Article  CAS  PubMed  Google Scholar 

  6. Klink R, de Kerchove d’Exaerde A, Zoli M, Changeux JP (2001) Molecular and physiological diversity of nicotinic acetylcholine receptors in the midbrain dopaminergic nuclei. J Neurosci 21:1452–1463

    CAS  PubMed  Google Scholar 

  7. Nashmi R, Dickinson ME, McKinney S et al (2003) Assembly of alpha4beta2 nicotinic acetylcholine receptors assessed with functional fluorescently labeled subunits: effects of localization, trafficking, and nicotine-induced upregulation in clonal mammalian cells and in cultured midbrain neurons. J Neurosci 23:11554–11567

    CAS  PubMed  Google Scholar 

  8. Franke C, Hatt H, Dudel J (1987) Liquid filament switch for ultra-fast exchanges of solutions at excised patches of synaptic membrane of crayfish muscle. Neurosci Lett 77:199–204

    Article  CAS  PubMed  Google Scholar 

  9. Komal P, Evans G, Nashmi R (2011) A rapid agonist application system for fast activation of ligand-gated ion channels. J Neurosci Methods 198:246–254

    Article  CAS  PubMed  Google Scholar 

  10. Brusa R, Zimmermann F, Koh DS et al (1995) Early-onset epilepsy and postnatal lethality associated with an editing-deficient GluR-B allele in mice. Science 270:1677–1680

    Article  CAS  PubMed  Google Scholar 

  11. Rozov A, Burnashev N (2016) Fast interaction between AMPA and NMDA receptors by intracellular calcium. Cell Calcium 60(6):407–414

    Article  CAS  PubMed  Google Scholar 

  12. Valiullina F, Zakharova Y, Mukhtarov M, Draguhn A, Burnashev N, Rozov A (2016) The relative contribution of NMDARs to excitatory postsynaptic currents is controlled by Ca(2+)-induced inactivation. Front Cell Neurosci 10:12. doi:10.3389/fncel.2016.00012

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

This work was supported by the program of competitive growth of Kazan Federal University and the subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities.

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Correspondence to Andrei Rozov .

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Rozov, A., Jappy, D. (2017). Functional Analysis of Recombinant Channels in Host Cells Using a Fast Agonist Application System. In: Burnashev, N., Szepetowski, P. (eds) NMDA Receptors. Methods in Molecular Biology, vol 1677. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7321-7_8

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  • DOI: https://doi.org/10.1007/978-1-4939-7321-7_8

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7320-0

  • Online ISBN: 978-1-4939-7321-7

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