Unique Contributions of an Arginine Side Chain to Ligand Recognition in a Glutamate-gated Chloride Channel

J Biol Chem. 2017 Mar 3;292(9):3940-3946. doi: 10.1074/jbc.M116.772939. Epub 2017 Jan 17.


Glutamate recognition by neurotransmitter receptors often relies on Arg residues in the binding site, leading to the assumption that charge-charge interactions underlie ligand recognition. However, assessing the precise chemical contribution of Arg side chains to protein function and pharmacology has proven to be exceedingly difficult in such large and complex proteins. Using the in vivo nonsense suppression approach, we report the first successful incorporation of the isosteric, titratable Arg analog, canavanine, into a neurotransmitter receptor in a living cell, utilizing a glutamate-gated chloride channel from the nematode Haemonchus contortus Our data unveil a surprisingly small contribution of charge at a conserved arginine side chain previously suggested to form a salt bridge with the ligand, glutamate. Instead, our data show that Arg contributes crucially to ligand sensitivity via a hydrogen bond network, where Arg interacts both with agonist and with a conserved Thr side chain within the receptor. Together, the data provide a new explanation for the reliance of neurotransmitter receptors on Arg side chains and highlight the exceptional capacity of unnatural amino acid incorporation for increasing our understanding of ligand recognition.

Keywords: Unnatural amino acids; arginine; biophysics; channel activation; glutamate; ion channel; ligand recognition; membrane protein; neurotransmitter.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arginine / chemistry*
  • Binding Sites
  • Canavanine / chemistry
  • Chloride Channels / chemistry*
  • Drosophila melanogaster
  • Glutamic Acid / chemistry
  • Haemonchus / metabolism
  • Humans
  • Hydrogen Bonding
  • Hydrogen-Ion Concentration
  • Ligands
  • Mutagenesis
  • Mutation
  • Neurotransmitter Agents / metabolism
  • Oocytes / cytology
  • Salts / chemistry
  • Xenopus laevis


  • Chloride Channels
  • Ligands
  • Neurotransmitter Agents
  • Salts
  • glutamate-gated chloride channels
  • Canavanine
  • Glutamic Acid
  • Arginine

Associated data

  • PDB/3RIF