Dynamic visualization of membrane-inserted fraction of pHluorin-tagged channels using repetitive acidification technique.

BMC Neuroscience
Serguei S KhirougLeonard Khiroug

Abstract

Changes in neuronal excitability, synaptic efficacy and generally in cell signaling often result from insertion of key molecules into plasma membrane (PM). Many of the techniques used for monitoring PM insertion lack either spatial or temporal resolution. We improved the imaging method based on time-lapse total internal reflection fluorescence (TIRF) microscopy and pHluorin tagging by supplementing it with a repetitive extracellular acidification protocol. We illustrate the applicability of this method by showing that brief activation of NMDA receptors ("chemical LTP") in cultured hippocampal neurons induced a persistent PM insertion of glutamate receptors containing the pHluorin-tagged GluR-A(flip) subunits. The repetitive acidification technique provides a more accurate way of monitoring the PM-inserted fraction of fluorescently tagged molecules and offers a good temporal and spatial resolution.

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Citations

Aug 21, 2013·Nature Structural & Molecular Biology·G Brent DaweDerek Bowie
Dec 29, 2010·The Journal of General Physiology·Rahul SrinivasanHenry A Lester
Sep 23, 2011·Molecular Biology of the Cell·Kyung Hee KimJeffrey R Bender
Sep 7, 2014·The Journal of Biological Chemistry·Weston A NicholsJulie M Miwa
Aug 14, 2015·The Journal of Biological Chemistry·Ashley M FoxChristopher I Richards
Oct 30, 2016·Proceedings of the National Academy of Sciences of the United States of America·Yi Gu, Richard L Huganir
Feb 6, 2017·PloS One·Lotta von OssowskiKari Keinänen
Jul 13, 2017·Scientific Reports·Roman M LazarenkoQi Zhang

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Methods Mentioned

BETA
transfection

Software Mentioned

Origin
Olympus Biosystems AnalySIS
PowerPoint

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