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

J Neurosci Methods. 2007 Mar 30;161(1):32-8. doi: 10.1016/j.jneumeth.2006.10.005. Epub 2006 Nov 28.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Membrane / metabolism*
  • Cells, Cultured
  • Coculture Techniques
  • Electric Stimulation / methods
  • Embryo, Mammalian
  • Fluorescent Dyes / metabolism
  • 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
  • Transfection / methods


  • Fluorescent Dyes
  • Kv1.4 Potassium Channel
  • Luminescent Proteins