Probing the Dynamics of Plasma Membrane Glutamate Transporters in Real Time by Total Internal Fluorescence Reflection Microscopy
Plasma membrane solute carriers (SLCs) mediate the transport of a variety of substrates across the plasma membranes. There is a great interest in understanding how they work and how their density at the plasma membrane is dynamically regulated, given their involvement in important physiological processes ranging from neurotransmission to nutrient and drug absorption. Genetically encoded fluorescent fusion proteins of transporters and total internal reflection fluorescence microscopy (TIRFM) provide the ideal methodological approaches to follow their dynamics in living cells. Owing to its ability to selectively excite a very thin fluorescent section (~100 nm) immediately above the glass cover on which cells are grown, TIRFM is becoming the technique of election for measuring in a quantitative manner events occurring at or near the plasma membrane with high temporal and spatial resolution. Here we provide a method for using TIRF microscopy to study the basal and regulated trafficking of the excitatory amino acid transporter 3 (EAAT3/EAAC1), a glutamate transporter of the solute carrier family 1 (SLC1). A detailed protocol of how carrying out image recording under TIRF microscopy and how to extract quantitative data on transporter density at the plasma membrane is presented. The method and the applied analyses can be extended to other plasma membrane proteins (solute and ion transporters, channels, and receptors) in different cellular contexts.
KeywordsCell signal transduction Constitutive and regulated trafficking Endocytosis Exocytosis Glutamate transporters Total internal reflection fluorescence microscopy
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