The pentameric ligand-gated ion channel (pLGIC) family can be loosely divided into excitatory cation-selective channels and inhibitory anion-selective channels. Agonists bind to the interface of principal and complentary subunits, where excitatory and inhibitory agonists alike rely on an amine moiety being coordinated by an aromatic box within the principal face. In contrast to the excitatory pLGIC agonists acetylcholine and serotonin, however, the inhibitory pLGIC agonists GABA, glutamate and glycine also possess a carboxylate moiety. Recognition of this carboxylate is facilitated by a positively charged arginine residue that is highly conserved in Loop D of inhibitory pLGICs and whose mutation to alanine greatly impairs agonist recognition. Scant, if any, evidence exists as to why arginine is preferred over other amino acids, at this position, and we therefore sought to establish if the positive charge of this side chain or other electrostatic interactions are required for recognition of the agonist carboxylate. As conventional mutagenesis is insufficient for dissecting the requirements of agonist recognition on chemical properties of the arginine side chain, we replaced this Loop D arginine with isosteric, isoelectric analogues in glutamate-gated chloride channels (GluCls) and glycine receptors (GlyRs) expressed in Xenopus laevis oocytes. Expression of these mutants was achieved by co-injecting oocytes with mutant channel cRNA containing the amber stop codon and analogues ligated to tRNA containing the appropriate anticodon. Agonist-gated currents were then measured with two-electrode voltage clamp. In GluCls, the presence of uncharged arginine analogues at the Loop D position was far less detrimental to glutamate sensitivity than substitution with conventional amino acids such as lysine and alanine. This suggests that although the positive charge of arginine may make some contribution to the recognition of carboxylate agonists, it must be other electrostatic interactions that make arginine so well-suited to this role in agonist recognition.