Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells.
| Title | Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells. |
| Publication Type | Journal Article |
| Year of Publication | 2007 |
| Authors | Baker, B. J., Lee H., Pieribone V. A., Cohen L. B., Isacoff E. Y., Knopfel T., & Kosmidis E. K. |
| Journal | J Neurosci Methods |
| Volume | 161 |
| Issue | 1 |
| Pagination | 32-8 |
| Date Published | 2007 Mar 30 |
| ISSN | 0165-0270 |
| Keywords | Animals, Cell Membrane, Cells, Cultured, Coculture Techniques, Electric Stimulation, Embryo, Mammalian, Fluorescent Dyes, Gene Expression, Hippocampus, Humans, Ion Channel Gating, Kv1.4 Potassium Channel, Luminescent Proteins, Membrane Potentials, Mice, Mice, Transgenic, Neurons, Patch-Clamp Techniques, Transfection |
| 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. |
| DOI | 10.1016/j.jneumeth.2006.10.005 |
| Alternate Journal | J. Neurosci. Methods |
| PubMed ID | 17126911 |
| Grant List | DC05259 / DC / NIDCD NIH HHS / United States NS050833 / NS / NINDS NIH HHS / United States R21MH064214 / MH / NIMH NIH HHS / United States |
