J Biol Chem.
January 1, 2017;
Unique Contributions of an Arginine Side Chain to Ligand Recognition in a Glutamate-gated Chloride Channel.
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.
J Biol Chem.
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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.