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

• Published in: Journal of neuroscience methods Vol. 161; no. 1; pp. 32 - 38
• Main Authors: Baker, B.J., Lee, H., Pieribone, V.A., Cohen, L.B., Isacoff, E.Y., Knopfel, T., Kosmidis, E.K.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 30.03.2007
• Subjects: Animals; Cell Membrane - metabolism; Cells, Cultured; Coculture Techniques; di8-ANEPPS; Electric Stimulation - methods; Embryo, Mammalian; Fluorescent Dyes - metabolism; Fluorescent protein; Gene Expression - physiology; Hippocampus - cytology; Humans; Ion Channel Gating - physiology; Kv1.4 Potassium Channel - genetics; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Membrane Potentials - physiology; Membrane Potentials - radiation effects; Mice; Mice, Transgenic; Neurons - ultrastructure; Patch-Clamp Techniques - methods; Plasma membrane expression; Transfection - methods; Voltage sensor; Voltage sensor; Fluorescent protein; Plasma membrane expression; di8-ANEPPS
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/j.jneumeth.2006.10.005

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.