Lynagh T , Komnatnyy VV , Pless SA .

	FIGURE 1. Arg-Lys substitutions drastically reduce glutamate recognition. A, X-ray structure of GLC-1 GluCl (PDB 3RIF; gray shading, notional cell membrane). Magnified view shows glutamate binding site and selected amino acid side chains. These include GLC-1 arginine residues 37 and 56, which are labeled Arg76 and Arg95 to describe the equivalent residues from the AVR-14B GluCl used in the present study. B, left, example recordings of glutamate (Glu) and ivermectin (IVM, 1 μm) responses at oocytes expressing mutant AVR-14B GluCls (scale bars: x, 5 s; y, 2 μA). Activation by IVM, which binds elsewhere on the receptor, confirms cell surface expression in the absence of Glu-gated currents. Right, mean ± S.E. (n = 4–8) peak current responses to increasing concentrations of Glu, normalized to maximum Glu-gated current (WT) or maximum IVM-gated current (mutants).
	FIGURE 2. Incorporation of titratable arginine analog, canavanine. A, l-lysine (Lys), l-arginine (Arg), and l-canavanine (Can). B, graphic illustrating nonsense suppression of Arg76UAG mRNA by co-injection of Can-ligated tRNA into Xenopus laevis oocytes (yellow/brown spheres). C, mean peak current amplitude (± S.E.) in response to 10 mm glutamate at oocytes injected with mRNA and tRNA ± Can (n = 8–15; *, p < 0.05; n.s. not significant, ANOVA). Inset shows example responses to 10 mm glutamate at oocytes (pH = 7) injected with indicated RNA combinations (scale bars: x, 5 s; y, 20 nA). D, example recordings of current responses to increasing concentrations of glutamate and mean ± S.E. peak current responses (n = 5–8; normalized to maximum glutamate-gated current) at Can76 GluCls (Can76). Scale bars: x, 10 s; y, 100 nA. E, left, glutamate-gated currents at oocytes expressing wild-type or Can76 GluCls continuously perfused at pH 7.0 or 5.8, as indicated (scale bars: x, 30 s; y, 100 nA). Right, mean ± S.E. peak current responses to 30 μm glutamate, normalized to maximum glutamate-gated current (I30 μm/Imax; n = 5–8; *, p < 0.05, ***, p < 0.001, Student's t test). Gray arrows illustrate inhibition of 30 μm glutamate-gated current. Can76 pH 7.0 recording in E is repeated from D.
	FIGURE 3. Conventional mutagenesis shows the importance of Thr93 in glutamate recognition. A, magnified view of GLC-1 crystal structure illustrating proximity of Thr93 hydroxyl oxygen to Arg76 ϵ nitrogen (2.9 Å; orange dashed line; numbers refer to equivalent residues in AVR-14B). Dashed lines indicate inter-atomic distances ≤3.5 Å. Amino acid sequence alignment shows selected Loop G and Loop D residues from ecdysozoan GluCls (dark font), lophotrochozoan GluCls and vertebrate and invertebrate GABA and glycine receptors (light font). B, example recordings of glutamate (Glu) and ivermectin (IVM) responses at oocytes expressing mutant AVR-14B GluCls (scale bars: x, 5 s; y, 2 μA). C, mean ± S.E. (n = 6–8) peak current responses to increasing concentrations of glutamate, normalized to maximum glutamate-gated current (WT and T93S) or maximum IVM-gated current (T93V).
	FIGURE 4. Double mutant cycle analysis. A, glutamate-gated currents at oocytes expressing double mutant T93S/R95K or R76K/T93S receptors and mean (± S.E.) data. Robust activation by IVM indicates successful expression of R76K/T93S despite small responses to glutamate. WT and single mutant data are repeated from earlier figures for comparison. B, principles of double mutant cycle analysis and analysis of Arg76–Thr93 and Arg95–Thr93 coupling. For two residues “A” and “B,” EC50 values of WT (AB), single mutant A′B, single mutant AB′, and double mutant A′B′ are used to calculate the coupling coefficient, Ω, from which the coupling energy, ΔΔG, can be calculated (22). Our final values for Ω and ΔΔG are only estimates because the EC50 values of certain single and double mutants could not be calculated: here the EC50 values have been estimated from A.

Akahoshi, Site-specific incorporation of arginine analogs into proteins using arginyl-tRNA synthetase. 2011, Pubmed

Akahoshi, Site-specific incorporation of arginine analogs into proteins using arginyl-tRNA synthetase. 2011, Pubmed
Althoff, X-ray structures of GluCl in apo states reveal a gating mechanism of Cys-loop receptors. 2014, Pubmed
Armstrong, Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: crystal structures of the GluR2 ligand binding core. 2000, Pubmed
Borders, A structural role for arginine in proteins: multiple hydrogen bonds to backbone carbonyl oxygens. 1994, Pubmed
Chen, Structural features of the glutamate binding site in recombinant NR1/NR2A N-methyl-D-aspartate receptors determined by site-directed mutagenesis and molecular modeling. 2005, Pubmed , Xenbase
Chen, Molecular determinants of proton modulation of glycine receptors. 2004, Pubmed
Cymes, Probing ion-channel pores one proton at a time. 2005, Pubmed
Daeffler, Functional evaluation of key interactions evident in the structure of the eukaryotic Cys-loop receptor GluCl. 2014, Pubmed , Xenbase
Dahan, A fluorophore attached to nicotinic acetylcholine receptor beta M2 detects productive binding of agonist to the alpha delta site. 2004, Pubmed , Xenbase
Dougherty, In vivo incorporation of non-canonical amino acids by using the chemical aminoacylation strategy: a broadly applicable mechanistic tool. 2014, Pubmed , Xenbase
Edgar, MUSCLE: multiple sequence alignment with high accuracy and high throughput. 2004, Pubmed
Furukawa, Subunit arrangement and function in NMDA receptors. 2005, Pubmed
Hampson, Probing the ligand-binding domain of the mGluR4 subtype of metabotropic glutamate receptor. 1999, Pubmed
Hibbs, Principles of activation and permeation in an anion-selective Cys-loop receptor. 2011, Pubmed
Hidalgo, Revealing the architecture of a K+ channel pore through mutant cycles with a peptide inhibitor. 1995, Pubmed , Xenbase
Huang, Effect of extracellular pH on GABA-activated current in rat recombinant receptors and thin hypothalamic slices. 1999, Pubmed
Judice, Probing the mechanism of staphylococcal nuclease with unnatural amino acids: kinetic and structural studies. 1993, Pubmed
Kash, Coupling of agonist binding to channel gating in the GABA(A) receptor. 2003, Pubmed
Kawamoto, Arginine-481 mutation abolishes ligand-binding of the AMPA-selective glutamate receptor channel alpha1-subunit. 1997, Pubmed
Krishnakumar, Experimental challenges of sense codon reassignment: an innovative approach to genetic code expansion. 2014, Pubmed
Kunishima, Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. 2000, Pubmed
Laube, Molecular determinants of agonist discrimination by NMDA receptor subunits: analysis of the glutamate binding site on the NR2B subunit. 1997, Pubmed
Le, Site-specific and regiospecific installation of methylarginine analogues into recombinant histones and insights into effector protein binding. 2013, Pubmed
Lynagh, Molecular basis for convergent evolution of glutamate recognition by pentameric ligand-gated ion channels. 2015, Pubmed
McCavera, An ivermectin-sensitive glutamate-gated chloride channel from the parasitic nematode Haemonchus contortus. 2009, Pubmed , Xenbase
Mehler, The role of hydrophobic microenvironments in modulating pKa shifts in proteins. 2002, Pubmed
Moroz, The ctenophore genome and the evolutionary origins of neural systems. 2014, Pubmed
Nowak, In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system. 1998, Pubmed , Xenbase
Pless, Unnatural amino acids as probes of ligand-receptor interactions and their conformational consequences. 2013, Pubmed
Rajagopal, Teaching old receptors new tricks: biasing seven-transmembrane receptors. 2010, Pubmed
Smart, Synaptic neurotransmitter-gated receptors. 2012, Pubmed
Wilkinson, Site-directed mutagenesis as a probe of enzyme structure and catalysis: tyrosyl-tRNA synthetase cysteine-35 to glycine-35 mutation. 1983, Pubmed
Wolstenholme, Glutamate-gated chloride channels. 2012, Pubmed
Yu, Agonist and antagonist binding in human glycine receptors. 2014, Pubmed