Identification of amino acid residues in GluR1 responsible for ligand binding and desensitization

Abstract

Although GluR1(o) and GluR3(o) are homologous at the amino acid level, GluR3(o) desensitizes approximately threefold faster than GluR1(o). By creating chimeras of GluR1(o) and GluR3(o) and point amino acid exchanges in their S2 regions, two residues were identified to be critical for GluR1(o) desensitization: Y716 and the R/G RNA-edited site, R757. With creation of the double-point mutant (Y716F, R757G)GluR1(o), complete exchange of the desensitization rate of GluR1(o) to that of GluR3(o) was obtained. In addition, both the potency and affinity of the subtype-selective agonist bromohomoibotenic acid were exchanged by the Y716F mutation. A model is proposed of the AMPA receptor binding site whereby a hydrogen-bonding matrix of water molecules plays an important role in determining both ligand affinity and receptor desensitization properties. Residues Y716 in GluR1 and F728 in GluR3 differentially interact with this matrix to affect the binding affinity of some ligands, providing the possibility of developing subtype-selective compounds.

Fig. 1.

Chimeric constructs of GluR1_o and GluR3_o. The GluR1_o sequence is represented bylight gray areas, and the GluR3_o sequence is represented by black areas.Numbering above the wild-type sequences refers to amino acid position number, starting from the initiation methionine. The corresponding restriction enzyme sites in the cDNA used to create these constructs are also indicated. The amino acid length of each protein is given in square brackets. Note that chimeras 1 and 2 were previously named NG1-CG3 and NG3-CG1, respectively (Banke et al., 1997), but have been renamed here for simplicity. TMD I through TMD IV are indicated as boxes; N, N terminus; C, C terminus. This figure is not drawn to scale.

Fig. 2.

l-glutamate and (R,S)-BrHIBO desensitization at GluR1_o and GluR3_o. A, Comparison of representative currents evoked by 10 mml-glutamate on outside-out patches expressing GluR1_o (left, scale bar = 40 pA) or GluR3_o (right, scale bar = 60 pA), respectively. l-glutamate was applied by fast application on outside-out patches as shown above the traces (V_h = −60 mV). Each trace was fitted to a monoexponential equation (line over traces), and τ was determined; τ (GluR1_o), 4.2 msec; τ (GluR3_o), 1.5 msec. B, Current evoked by 1 mm (R,S)-BrHIBO on outside-out patches expressing GluR1_o (left, scale bar = 250 pA) or GluR3_o (right, scale bar = 50 pA); τ (GluR1_o), 4.0 msec; τ (GluR3_o), 1.8 msec. C, Mean τ ± SEM for GluR1_o (two left bars) and GluR3_o (two right bars). Filled bars, 10 mml-glutamate (left, n = 13; right,n = 16); open bars, 1 mm(R,S)-BrHIBO (left, right,n = 5).

Fig. 3.

Desensitization at chimeric AMPARs. Comparison of 10 mml-glutamate-evoked currents (mean ± SEM) from, starting at top left, GluR1_o, GluR3_o, GluR4c_o, and chimeric receptors (Ch) 1–7 expressed in X. laevis oocytes. The chimeric constructs are shown on the left, as described in Figure1. l-glutamate was applied by fast application on outside-out patches (V_h = −60 mV). Data were fit to a monoexponential equation (see Materials and Methods), and τ was determined.

Fig. 4.

Desensitization at GluR1_o S2 region point mutants. A, An alignment of GluR1_o and GluR3_o amino acid sequences in the region between TMD III and TMD IV (S2 region). N, N terminus; C, terminus; TMDs are represented by boxes. This section of the figure is not drawn to scale. B, A histogram of the mean desensitization rate constant (τ) for GluR1_o point mutants, with amino acids numbered as in A. The 10–90% rise time for the double mutants (pooled) was 275 ± 21 μsec compared with 343 ± 39 μsec for GluR1_o (10 fastest patches), which was not statistically significantly different. Significant differences versus GluR1_o are indicated as follows: ^##p < 0.001,^#p < 0.01.

Fig. 5.

Deactivation and recovery from desensitization.A, Recovery from desensitization in an outside-out patch expressing wild-type GluR1_o receptors.l-glutamate (10 mm) was applied for 1 msec at the following time intervals (in msec): 0, 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, and 800. τ_rec was 152 msec for this experiment. B, Histogram showing recovery from desensitization for GluR1_o and the multiple-point mutants.C, Deactivation and desensitization in an outside-out patch expressing GluR1_o. Deactivation and desensitization were obtained by application of 10 mml-glutamate for 1 and 100 msec, respectively, as shown above the traces. Traces were fitted to a monoexponential equation with the following results: τ_deact, 0.98 msec; τ, 3.12 msec. D, Histogram showing deactivation rate constants for GluR1_o, GluR3_o , and GluR1_o mutants. ^*p < 0.05, significantly different from GluR1_o.

Fig. 6.

Pharmacology of mutant and wild-type AMPARs. Potency (EC₅₀) and affinity (K_i) of (R,S)-BrHIBO at wild-type and mutant AMPARs are shown. A, Receptors were expressed in X. laevis oocytes, and steady-state currents were measured after application of increasing concentrations of (R,S)-BrHIBO. EC₅₀ was determined by fitting data to a logistic equation as described in Materials and Methods. Curves are mean ± SEM from 5 to 16 oocytes.B, Receptors were expressed in Sf9 cell membranes and drug affinity was measured by (R,S)-[³H]AMPA competition binding assays. K_i was determined by nonlinear, iterative fitting of the data as described in Materials and Methods. Shown are the mean ± SD of triplicate determinations from single experiments (replicated 3–6 times). ○, GluR1_o(EC₅₀ = 7.2 ± 2.8 μm;n = 5; K_i = 183 nm); ●, GluR3_o (EC₅₀ = 198 ± 31 μm; n = 5;K_i = 9.53 μm); ▵, (Y716F)GluR1_o (EC₅₀ = 200 ± 27 μm; n = 16;K_i = 3.69 μm); ▴, (F728Y)GluR3_o (EC₅₀ = 2.6 ± 0.4 μm; n = 5;K_i = 423 nm).

Fig. 7.

Binding site models. Models ofl-glutamate (A, D), (S)-BrHIBO (B, E), and (S)-AMPA (C, F) binding to GluR1_o (A–C) and GluR3_o(D–F). The seven amino acid residues that have been shown to interact directly with ligands in the GluR2-S1S2 constructs correspond to the homologous residues Y464, P492, T494, R499, S668, T669, and E719 in GluR1_o (numbering from the initiation methionine) or with Y474, P502, T504, R509, S680, T681, and E731 in GluR3_o. Residues are numbered (in Aand D), with the homologous GluR2 residue (Armstrong et al., 1998) indicated in parentheses. Some residues have been removed for clarity. Hydrogen bonds are denoted by dashed lines. W1, W2, and W3 are binding site water molecules. O1 represents the hydroxyl group of AMPA or BrHIBO and O2 is the isoxazole oxygen.

Fig. 8.

Ab initio modeling of ligand binding. Simplified models of AMPA (I) and MeHIBO (II) binding to GluR1 (TYR) and GluR3 (PHE) under constrained optimization according toab initio molecular orbital theory (see Materials and Methods). Quantum chemical energy calculations yield total energies (Hartree) at B3LYP/6–31+G(d): I-TYR, −859.7460; II-TYR, −859.7462; I-PHE, −784.5093; II-PHE, −784.5069. Relative energy differences: TYR ΔE, −0.18 kcal/mol; PHE ΔE, +1.47 kcal/mol.