Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells
Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells
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DOI：
10.1016/j.jneumeth.2006.10.005
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发表时间：
2007-03-30
影响因子：
3
通讯作者：
Kosmidis, E. K.
中科院分区：
文献类型：
--
作者：
Baker, B. J.;Lee, H.;Kosmidis, E. K.
在哺乳动物细胞中对三种第一代荧光蛋白电压敏感探针（FP 电压传感器）进行了表征。 Flare，FlaSh 的 Kv 1.4 变体 [Siegel MS，Isacoff EY。神经元1997； 1 9（十月（4））：735-41.1，SPARC [Ataka K，Pieribon VA。 Biophys J 2002；82（一月（I Pt 1））：509-16] 和 VSFP-1 [Sakai R、Repunte-Canonigo V、Raj CID、Knopfel T. Eur J Neurosci 200 1； 1 3(June (12)):2314-18] 在 HEK 293 细胞和分离的海马神经元中表达、成像和电压钳制。在对三种结构中的任何一种进行 16 次试验平均后，我们无法检测到响应膜电位变化的信号。使用疏水性电压敏感染料 di8-ANEPPS 作为表面标记，共聚焦分析表明 HEK 293 细胞和分离的海马神经元中 Flare、SPARC 和 VSFP-1 的质膜表达较差。几乎所有表达的 FP 电压传感器都存在于两种细胞类型的内膜中。这种内部表达产生背景荧光，增加光学测量中的噪声。 (c) 2006 Elsevier B.V. 保留所有权利。
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.