Fluorophore assisted light inactivation (FALI) of recombinant 5-HT(3)A receptor constitutive internalization and function

Russell A. Morton, Guoxiang Luo, Margaret I. Davis, Tim G. Hales, David M. Lovinger

    Research output: Contribution to journalArticlepeer-review

    6 Citations (Scopus)

    Abstract

    Fluorescent proteins and molecules are now widely used to tag and visualize proteins resulting in an improved understanding of protein trafficking, localization, and function. In addition, fluorescent tags have also been used to inactivate protein function in a spatially and temporally-defined manner, using a technique known as fluorophore-assisted light inactivation (FALI) or chromophore-assisted light inactivation (CALI). In this study we tagged the serotonin(3) A subunit with the alpha-bungarotoxin binding sequence (BBS) and subsequently labeled 5-HT(3)A/BBS receptors with fluorescently conjugated alpha-bungarotoxin in live cells. We show that 5-HT(3)A/BBS receptors are constitutively internalized in the absence of an agonist and internalization as well as receptor function are inhibited by fluorescence. The fluorescence-induced disruption of function and internalization was reduced with oxygen radical scavengers suggesting the involvement of reactive oxygen species, implicating the FALI process. Furthermore, these data suggest that intense illumination during live-cell microscopy may result in inadvertent FALI and inhibition of protein trafficking. (C) 2011 Published by Elsevier Inc.

    Original languageEnglish
    Pages (from-to)79-92
    Number of pages14
    JournalMolecular and Cellular Neuroscience
    Volume47
    Issue number2
    DOIs
    Publication statusPublished - Jun 2011

    Keywords

    • 5-Hydroxytryptamine type 3 (5-HT3) receptors
    • Fluorophore assisted light inactivation
    • Constitutive internalization
    • Electrophysiology
    • GREEN FLUORESCENT PROTEIN
    • EGFP-FUSION PROTEINS
    • LASER INACTIVATION
    • SINGLET-OXYGEN
    • 5-HT3 RECEPTORS
    • MODULATION
    • SUBUNIT
    • CALI
    • ENDOCYTOSIS
    • ANTAGONIST

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