Biochemistry
Biochemistry
5 years ago

Generation and Characterization of Anti-VGLUT Nanobodies Acting as Inhibitors of Transport

Generation and Characterization of Anti-VGLUT Nanobodies Acting as Inhibitors of Transport
Toshiharu Suzuki, Andrea Volterra, Saša Štefanić, Yvonne Neldner, Els Pardon, Stephan Schenck, Laura Kunz, Eric R. Geertsma, Iaroslav Savtchouk, Daniela Sahlender, Jan Steyaert, Raimund Dutzler
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.

Publisher URL: http://dx.doi.org/10.1021/acs.biochem.7b00436

DOI: 10.1021/acs.biochem.7b00436

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