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

Biochemistry. 2017 Aug 1;56(30):3962-3971. doi: 10.1021/acs.biochem.7b00436. Epub 2017 Jul 21.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biological Transport / drug effects
  • Camelids, New World
  • Cells, Cultured
  • Central Nervous System Depressants / chemistry
  • Central Nervous System Depressants / metabolism
  • Central Nervous System Depressants / pharmacology*
  • Cerebral Cortex / cytology
  • Cerebral Cortex / drug effects*
  • Cerebral Cortex / metabolism
  • Embryo, Mammalian / cytology
  • Glutamic Acid / metabolism
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Membrane Transport Modulators / chemistry
  • Membrane Transport Modulators / metabolism
  • Membrane Transport Modulators / pharmacology*
  • Mice
  • Models, Molecular*
  • Nerve Tissue Proteins / antagonists & inhibitors*
  • Nerve Tissue Proteins / chemistry
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism
  • Neurons / cytology
  • Neurons / drug effects*
  • Neurons / metabolism
  • Peptide Library
  • Rats
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Single-Domain Antibodies / chemistry
  • Single-Domain Antibodies / genetics
  • Single-Domain Antibodies / metabolism
  • Single-Domain Antibodies / pharmacology*
  • Synaptic Transmission / drug effects
  • Synaptic Vesicles / drug effects
  • Synaptic Vesicles / metabolism
  • Vesicular Glutamate Transport Protein 1 / antagonists & inhibitors*
  • Vesicular Glutamate Transport Protein 1 / chemistry
  • Vesicular Glutamate Transport Protein 1 / genetics
  • Vesicular Glutamate Transport Protein 1 / metabolism

Substances

  • Central Nervous System Depressants
  • Membrane Transport Modulators
  • Nerve Tissue Proteins
  • Peptide Library
  • Recombinant Fusion Proteins
  • Single-Domain Antibodies
  • Slc17a7 protein, rat
  • Vesicular Glutamate Transport Protein 1
  • Green Fluorescent Proteins
  • Glutamic Acid