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Quantitative Analysis of Cell Surface Expressed, Intracellular, and Internalized Neurotransmitter Receptor Populations in Brain Slices Using Biotinylation

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Receptor and Ion Channel Detection in the Brain

Part of the book series: Neuromethods ((NM,volume 169))

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Abstract

Cell surface trafficking and endocytosis of neurotransmitter receptors are important regulatory mechanisms of neurotransmission. Biotinylation of plasma membrane (PM) proteins in brain slices allows their separation from those present in intracellular organelles. An extension of this approach also enables the selective retrieval of proteins endocytosed from the plasma membrane into intracellular compartments. Membrane-impermeable, thiol-cleavable amine-reactive biotinylation reagents (e.g., EZ-link sulfo-NH-SS-biotin) form a stable covalent linkage with primary amino groups of surface-exposed proteins. Following homogenization of brain slices and solubilization of membranes, biotin-labeled proteins can be isolated with avidin (or streptavidin or NeutrAvidin) linked to a solid matrix. Bound biotinylated proteins are released from avidin in the presence of reducing agents (e.g., glutathione or β-mercaptoethanol). Quantitative differences in the molecular composition of biotin-labeled (surface or internalized) and unlabeled (intracellular) protein fractions can be analyzed separately using immunoblotting with target protein-specific antibodies. While many variations of this procedure exist in the literature, in this chapter we describe the biotinylation protocol that we have applied for the investigation of quantitative changes in the cell surface expression and internalization of ionotropic glutamate receptors in acute brain slices.

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Acknowledgments

I would like to thank Dr. Clare M. Gladding for adapting the cell surface biotinylation technique for the investigation of tryrosine dephosporylation of AMPA receptors in mGluR-LTD and NMDAR-LTD using hippocampal slices [21]. This research was supported by an Innovate UK (UK Research and Innovation) Knowledge Transfer Partnership grant (Ref No KTP 12333, 2020 - 5146) and the Biotechnology and Biological Sciences Research Council, UK (Grant BB/J015938/1).

Conflict of Interest

E.M. is a Scientific Advisory Board member of Hello Bio [http://www.hellobio.com].

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Authors and Affiliations

  1. School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, UK

    Elek Molnár

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  1. Elek Molnár
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Correspondence to Elek Molnár .

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Editors and Affiliations

  1. Instituto de Investigación en Discapacidades Neurológicas (IDINE), Departamento de Ciencias Médicas, Facultad de Medicina, Universidad Castilla-La Mancha, Albacete, Spain

    Rafael Lujan

  2. Pharmacology Unit, Department of Pathology and Experimental Therapeutics, School of Medicine and Health Sciences, IDIBELL, Universitat de Barcelona, L’Hospitalet de Llobregat, Barcelona, Spain

    Francisco Ciruela

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Molnár, E. (2021). Quantitative Analysis of Cell Surface Expressed, Intracellular, and Internalized Neurotransmitter Receptor Populations in Brain Slices Using Biotinylation. In: Lujan, R., Ciruela, F. (eds) Receptor and Ion Channel Detection in the Brain. Neuromethods, vol 169. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1522-5_4

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  • DOI: https://doi.org/10.1007/978-1-0716-1522-5_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1521-8

  • Online ISBN: 978-1-0716-1522-5

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