Dynamic visualization of membrane-inserted fraction of pHluorin-tagged channels using repetitive acidification technique
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BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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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TIRFM enables the visualization of fluorescently labeled intracellular molecules within ∼300 nm of the cell-coverslip interface.
“TIRFM enables the visualization of fluorescently labeled intracellular molecules within ∼300 nm of the cell–coverslip interface (Oancea et al., 2006; Fish, 2009; Khiroug et al., 2009).”
From Nicotine up-regulates α4β2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning (2011) · cites this paper as Khiroug et al., 2009
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- Authors
- Khiroug, S. S., Pryazhnikov, E., Coleman, S. K., Jeromin, A., Keinänen, K., & Khiroug, L.
- Affiliations
- University of Helsinki (FI)
- Allen Institute for Brain Science (US)
- Corresponding author
- Khiroug, Leonard
- Venue
- BMC Neuroscience
- Published
- 2009-11-30
- Volume
- 10
- Issue
- 1
- Pages
- 141-141
- ISSN
- 1471-2202 (ISSN-L)
- PMC
- PMC2794868
- OpenAlex
- W2028397158
- Cited by
- 19
- References
- 21
- Citations by year
- 2025: 1 · 2024: 1 · 2017: 4 · 2016: 3 · 2015: 1 · 2014: 1 · 2013: 4
- FWCI
- 0.55
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- BMC Neuroscience (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- WestminsterResearch (University of Westminster) (submittedVersion, cc-by)
- Funders
- Academy of Finland
- Kansainvälisen Liikkuvuuden ja Yhteistyön Keskus
- Topics
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
- Keywords
- membrane insertion, pHluorin, NMDA receptor activation, AMPA receptor subunits, hippocampal neurons, glutamate receptors, dynamic visualization
- MeSH terms
- Cell Membrane, Cells, Cultured, Hippocampus, Microscopy, Fluorescence, Synaptic Transmission, Neuronal Plasticity, Neurons, Receptors, N-Methyl-D-Aspartate, Green Fluorescent Proteins
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