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

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.