GABA transporter function, oligomerization state, and anchoring: correlates with subcellularly resolved FRET.

The Journal of General Physiology
Fraser J MossHenry A Lester

Abstract

The mouse gamma-aminobutyric acid (GABA) transporter mGAT1 was expressed in neuroblastoma 2a cells. 19 mGAT1 designs incorporating fluorescent proteins were functionally characterized by [(3)H]GABA uptake in assays that responded to several experimental variables, including the mutations and pharmacological manipulation of the cytoskeleton. Oligomerization and subsequent trafficking of mGAT1 were studied in several subcellular regions of live cells using localized fluorescence, acceptor photobleach Förster resonance energy transfer (FRET), and pixel-by-pixel analysis of normalized FRET (NFRET) images. Nine constructs were functionally indistinguishable from wild-type mGAT1 and provided information about normal mGAT1 assembly and trafficking. The remainder had compromised [(3)H]GABA uptake due to observable oligomerization and/or trafficking deficits; the data help to determine regions of mGAT1 sequence involved in these processes. Acceptor photobleach FRET detected mGAT1 oligomerization, but richer information was obtained from analyzing the distribution of all-pixel NFRET amplitudes. We also analyzed such distributions restricted to cellular subregions. Distributions were fit to either two or three Gaussian components. Two of ...Continue Reading

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Citations

Dec 29, 2010·The Journal of General Physiology·Rahul SrinivasanHenry A Lester
Jul 27, 2011·The Journal of General Physiology·Edward N Pugh
Jan 1, 2014·The Journal of General Physiology·Brandon J HendersonHenry A Lester
Mar 16, 2011·Experimental Cell Research·Nicole M I NivillacImogen R Coe
Aug 8, 2014·Biophysical Journal·Omer DushekVahid Shahrezaei
Feb 11, 2015·Neuropharmacology·Brandon J Henderson, Henry A Lester
May 27, 2014·Frontiers in Cellular Neuroscience·Annalisa Scimemi
Jul 6, 2014·Frontiers in Cellular Neuroscience·Annalisa Scimemi
Sep 18, 2015·Nanoscale·Felipe T Lee-MontielP I Imoukhuede
Jul 25, 2014·The Journal of Biological Chemistry·Ying WangHenry A Lester
Sep 7, 2014·The Journal of Biological Chemistry·Weston A NicholsJulie M Miwa
Aug 9, 2020·Journal of Neurochemistry·Kumaresan JayaramanThomas Stockner
Feb 6, 2019·The Journal of General Physiology·Amol V ShivangeHenry A Lester
Apr 8, 2015·The Biochemical Journal·Susanne MilatzDorothee Günzel
May 1, 2021·Frontiers in Cellular Neuroscience·Renae M RyanAnnalisa Scimemi
May 16, 2019·Analytical Chemistry·Si Chen, P I Imoukhuede

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Methods Mentioned

BETA
FRET
transfections
transfection
transfect
PCR
fluorescence microscopy
fluorescence
electrophoresis
from
FRETing

Software Mentioned

FRET
NFRET
Pix
C1
ImageJ
Origin
Ensembl
MATLAB
EZ

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