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J Neurosci Methods. 2007 Mar 30;161(1):32-8. Epub 2006 Nov 28.

Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells.

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  • 1Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520, USA. Bradley.baker@yale.edu

Abstract

Three first-generation fluorescent protein voltage sensitive probes (FP-voltage sensors) were characterized in mammalian cells. Flare, a Kv1.4 variant of FlaSh [Siegel MS, Isacoff EY. Neuron 1997;19(October (4)):735-41], SPARC [Ataka K, Pieribone VA. Biophys J 2002;82(January (1 Pt 1)):509-16], and VSFP-1 [Sakai R, Repunte-Canonigo V, Raj CD, Knopfel T. Eur J Neurosci 2001;13(June (12)):2314-18] were expressed, imaged and voltage clamped in HEK 293 cells and in dissociated hippocampal neurons. We were unable to detect a signal in response to changes in membrane potential after averaging16 trials with any of the three constructs. Using the hydrophobic voltage sensitive dye, di8-ANEPPS, as a surface marker, confocal analyses demonstrated poor plasma membrane expression for Flare, SPARC and VSFP-1 in both HEK 293 cells and dissociated hippocampal neurons. Almost all of the expressed FP-voltage sensors reside in internal membranes in both cell types. This internal expression generates a background fluorescence that increases the noise in the optical measurement.

PMID:
17126911
DOI:
10.1016/j.jneumeth.2006.10.005
[PubMed - indexed for MEDLINE]
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