title: Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells.
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.
authors.affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520, USA. Bradley.baker@yale.edu
authors.forename: B J
authors.initials: BJ
authors.lastname: Baker
authors.forename: H
authors.initials: H
authors.lastname: Lee
authors.forename: V A
authors.initials: VA
authors.lastname: Pieribone
authors.forename: L B
authors.initials: LB
authors.lastname: Cohen
authors.forename: E Y
authors.initials: EY
authors.lastname: Isacoff
authors.forename: T
authors.initials: T
authors.lastname: Knopfel
authors.forename: E K
authors.initials: EK
authors.lastname: Kosmidis
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D018920:Coculture Techniques; D004558:Electric Stimulation / Q000379:methods; D004622:Embryo, Mammalian; D005456:Fluorescent Dyes / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology*; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology; D051665:Kv1.4 Potassium Channel / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology / Q000528:radiation effects; D051379:Mice; D008822:Mice, Transgenic; D009474:Neurons / Q000648:ultrastructure*; D018408:Patch-Clamp Techniques / Q000379:methods; D014162:Transfection / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D051665:Kv1.4 Potassium Channel; D008164:Luminescent Proteins