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

Three first-generation fluorescent protein voltage sensitive probes (FP-voltage sensors) were characterized in mammalian cells. Flare, a Kv 1.4 variant of FlaSh [Siegel MS, Isacoff EY. Neuron 1997; 1 9(October (4)):735-41.1, SPARC [Ataka K, Pieribone VA. Biophys J 2002;82(January (I Pt 1)):509-16], and VSFP-1 [Sakai R, Repunte-Canonigo V, Raj CID, Knopfel T. Eur J Neurosci 200 1; 1 3(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 averaging 16 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. (c) 2006 Elsevier B.V. All rights reserved.