Schenck Stephan, Kunz Laura, Sahlender Daniela, Pardon Els, Geertsma Eric R, Savtchouk Iaroslav, Suzuki Toshiharu, Neldner Yvonne, Štefanić Saša, Steyaert Jan, Volterra Andrea, Dutzler Raimund
Department of Biochemistry, University of Zurich , Winterthurerstrasse 190, 8057 Zurich, Switzerland.
Department of Fundamental Neurosciences, University of Lausanne , Rue du Bugnon 9, 1005 Lausanne, Switzerland.
Biochemistry. 2017 Aug 1;56(30):3962-3971. doi: 10.1021/acs.biochem.7b00436. Epub 2017 Jul 21.
The uptake of glutamate by synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). The central role of these transporters in excitatory neurotransmission underpins their importance as pharmacological targets. Although several compounds inhibit VGLUTs, highly specific inhibitors were so far unavailable, thus limiting applications to in vitro experiments. Besides their potential in pharmacology, specific inhibitors would also be beneficial for the elucidation of transport mechanisms. To overcome this shortage, we generated nanobodies (Nbs) by immunization of a llama with purified rat VGLUT1 and subsequent selection of binders from a phage display library. All identified Nbs recognize cytosolic epitopes, and two of the binders greatly reduced the rate of uptake of glutamate by reconstituted liposomes and subcellular fractions enriched with synaptic vesicles. These Nbs can be expressed as functional green fluorescent protein fusion proteins in the cytosol of HEK cells for intracellular applications as immunocytochemical and biochemical agents. The selected binders thus provide valuable tools for cell biology and neuroscience.
突触囊泡对谷氨酸的摄取由囊泡谷氨酸转运体（VGLUTs）介导。这些转运体在兴奋性神经传递中的核心作用奠定了它们作为药理学靶点的重要性。尽管有几种化合物能抑制VGLUTs，但迄今为止还没有高度特异性的抑制剂，因此限制了其在体外实验中的应用。除了在药理学方面的潜力外，特异性抑制剂对阐明转运机制也将是有益的。为了克服这一短缺，我们通过用纯化的大鼠VGLUT1免疫骆驼并随后从噬菌体展示文库中筛选结合物来生成纳米抗体（Nbs）。所有鉴定出的Nbs都识别胞质表位，其中两种结合物极大地降低了重组脂质体和富含突触囊泡的亚细胞组分对谷氨酸的摄取速率。这些Nbs可以作为功能性绿色荧光蛋白融合蛋白在HEK细胞的胞质中表达，用于细胞内应用，如免疫细胞化学和生化试剂。因此，所选择的结合物为细胞生物学和神经科学提供了有价值的工具。
