TY - JOUR
T1 - Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells
AU - Baker, B. J.
AU - Lee, H.
AU - Pieribone, V. A.
AU - Cohen, L. B.
AU - Isacoff, E. Y.
AU - Knopfel, T.
AU - Kosmidis, E. K.
PY - 2007/3/30
Y1 - 2007/3/30
N2 - 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.
AB - 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.
KW - di8-ANEPPS
KW - Fluorescent protein
KW - Plasma membrane expression
KW - Voltage sensor
UR - http://www.scopus.com/inward/record.url?scp=33847351663&partnerID=8YFLogxK
U2 - 10.1016/j.jneumeth.2006.10.005
DO - 10.1016/j.jneumeth.2006.10.005
M3 - Journal article
C2 - 17126911
AN - SCOPUS:33847351663
SN - 0165-0270
VL - 161
SP - 32
EP - 38
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
IS - 1
ER -
