title: Fluorophore assisted light inactivation (FALI) of recombinant 5-HT₃A receptor constitutive internalization and function.
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 serotonin₃ A subunit with the α-bungarotoxin binding sequence (BBS) and subsequently labeled 5-HT₃A/BBS receptors with fluorescently conjugated α-bungarotoxin in live cells. We show that 5-HT₃A/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.
authors.affiliation: Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD 20852, USA.
authors.forename: Russell A
authors.initials: RA
authors.lastname: Morton
authors.forename: Guoxiang
authors.initials: G
authors.lastname: Luo
authors.forename: Margaret I
authors.initials: MI
authors.lastname: Davis
authors.forename: Tim G
authors.initials: TG
authors.lastname: Hales
authors.forename: David M
authors.initials: DM
authors.lastname: Lovinger
mesh_terms: D002038:Bungarotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008027:Light; D008856:Microscopy, Fluorescence / Q000379:methods*; D018408:Patch-Clamp Techniques; D017382:Reactive Oxygen Species / Q000378:metabolism; D044406:Receptors, Serotonin, 5-HT3 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D002038:Bungarotoxins; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D044406:Receptors, Serotonin, 5-HT3; D011993:Recombinant Fusion Proteins