David-Watine B, Shorte S L, Fucile S, de Saint Jan D, Korn H, Bregestovski P
INSERM U-261, Institut Pasteur, Paris, France.
Neuropharmacology. 1999 Jun;38(6):785-92. doi: 10.1016/s0028-3908(99)00015-5.
The alpha subunit (alphaZ1) of the zebrafish glycine receptor (GlyR) has been N-terminus fused with green fluorescent protein (GFP). We found that both pharmacological and electrophysiological properties of this chimeric alphaZ1-GFP are indistinguishable from those of the wild-type receptor when expressed in Xenopus oocytes and cell lines. The apparent affinities of this receptor for agonists (glycine, taurine and GABA), and the antagonist (strychnine) are unchanged, and single channel kinetics are not altered. In the same expression systems, alphaZ1-GFP was visualized using fluorescence microscopy. Fluorescence was distributed anisotropically across cellular membranes. In addition to the Golgi apparatus and endoplasmic reticulum, its presence was also detected on the plasmalemma, localized at discrete hot-spots which were identified as sites of high membrane turnover. Overall, the preservation in alphaZ1-GFPs of the wild type receptor functional properties makes it a promising new tool for further in situ investigations of GlyR expression, distribution and function.
斑马鱼甘氨酸受体（GlyR）的α亚基（αZ1）在N端与绿色荧光蛋白（GFP）融合。我们发现，当在非洲爪蟾卵母细胞和细胞系中表达时，这种嵌合的αZ1-GFP的药理学和电生理学特性与野生型受体无异。该受体对激动剂（甘氨酸、牛磺酸和GABA）以及拮抗剂（士的宁）的表观亲和力未变，单通道动力学也未改变。在相同的表达系统中，利用荧光显微镜观察到了αZ1-GFP。荧光各向异性地分布在细胞膜上。除了高尔基体和内质网外，在质膜上也检测到了它的存在，其定位于离散的热点区域，这些区域被确定为高膜更新位点。总体而言，αZ1-GFP保留了野生型受体的功能特性，这使其成为进一步原位研究GlyR表达、分布和功能的有前景的新工具。
