Molecular determinants of coordinated proton and zinc inhibition of N-methyl-D-aspartate NR1/NR2A receptors

Abstract

Modulation of the N-methyl-d-aspartate (NMDA)-selective glutamate receptors by extracellular protons and Zn(2+) may play important roles during ischemia in the brain and during seizures. Recombinant NR1/NR2A receptors exhibit a much higher apparent affinity for voltage-independent Zn(2+) inhibition than receptors with other subunit combinations. Here, we show that the mechanism of this apparent high-affinity, voltage-independent Zn(2+) inhibition for NR2A-containing receptors results from the enhancement of proton inhibition. We also show that the N-terminal leucine/isoleucine/valine binding protein (LIVBP)-like domain of the NR2A subunit contains critical determinants of the apparent high-affinity, voltage-independent Zn(2+) inhibition. Mutations H42A, H44G, or H128A greatly increase the Zn(2+) IC(50) (by up to approximately 700-fold) with no effect on the potencies of glutamate and glycine or on voltage-dependent block by Mg(2+). Furthermore, the amino acid residue substitution H128A, which mediates the largest effect on the apparent high-affinity Zn(2+) inhibition among all histidine substitutions we tested, is also critical to the pH-dependency of Zn(2+) inhibition. Our data revealed a unique interaction between two important extracellular modulators of NMDA receptors.

Figure 1

Zn²⁺ inhibition is pH dependent for NR2A but not NR2B-containing receptors. (A) Inhibition of NR1-1a(D669N)/NR2A receptors by 1 μM added Zn²⁺ at two indicated pHs from the same oocyte. (B) Inhibition of NR1-1a(D669N)/NR2B receptor by 15 μM added Zn²⁺ at two indicated pHs from the same oocyte. All control currents for NR2A-containing receptors were recorded in the presence of 10 μM EDTA. The NR1(D669N) mutant was used to shift the pH sensitivity to a range where formation of Zn²⁺ hydroxide complex is negligible at alkaline pHs. (C) Plot of the degree of Zn²⁺ inhibition at various pH values for NR1-1a(D669N) containing NR2A (−20 to −40 mV) or NR2B (−15 to −30 mV) receptors. (D) Wild-type NR1-1a receptors containing NR2A or NR2B subunit in the presence of 3 μM and 15 μM added Zn²⁺, respectively, confirm the observation in C. ○, Outward responses recorded at +20 mV for NR1-1a/NR2B. All error bars are SEM. Numbers in parentheses are the number of oocytes.

Figure 2

The voltage-independent Zn²⁺ inhibition of NR1-1a/NR2A reflects the enhancement of proton inhibition. (A) Zn²⁺ causes an alkaline-shift of the proton inhibition curve of NR1-1a/NR2A receptors. Proton inhibition curve was measured both in the presence of 1 μM EDTA to remove the contaminant Zn²⁺ in the recording solution and in the presence of 1 μM Zn²⁺. All current responses were normalized to the current responses at the most alkaline pH 8.8. For pH 8.8 and pH 8.2, 6.64 μM and 1.48 μM Zn²⁺, respectively, were added to the recording solutions to achieve 1 μM free Zn²⁺. Error bars are SEM (whenever larger than the symbol). (B) Zn²⁺ inhibition curves at various pH values were predicted as described in Materials and Methods. The residual responses should be pH dependent when the apparent high affinity Zn²⁺ site is saturated. (C) Correlation between predicted residual currents (dotted line) and experimentally obtained residual currents (solid dot) of NR1/NR2A receptors at pHs 6.8, 7.3, and 8.0. (D–F) Zn²⁺ inhibition curves recorded at three different pH values. As predicted, the residual responses are pH dependent. The IC₅₀ values of Zn²⁺ inhibition at pHs 6.8, 7.3, and 8.0 are 74.2 ± 9.0 nM, 23.9 ± 2.9 nM, and 4.4 ± 1.0 nM, respectively. For pH 8.0, Zn²⁺ inhibition curve was obtained with buffered Zn²⁺ by 1 mM ADA. All currents were measured in the presence of saturating concentrations of glutamate (20–50 μM) and glycine (10–30 μM).

Figure 3

Histidines in the LIVBP-like domain of NR2A are critical for the apparent high-affinity Zn²⁺ inhibition. (A) Schematic of the NR2A subunit showing the positions of histidine in the LIVBP-like domain of NR2A that were investigated. The labels at the bottom of this figure indicate putative signal peptide (sp), the region with homology to the Leu/Ile/Val binding protein (LIVBP; ref. 31), the four membrane-associated domains (TM1–4; ref. 1), the agonist binding domains (S1, S2; ref. 32, 33), the region controlling the slow component of desensitization (preM1; refs. and 35). (B) Mean-fitted IC₅₀ values determined for voltage-independent Zn²⁺ inhibition of NR1-1a/NR2A receptors containing the histidine mutations. These histidines were categorized into three groups based on their effect on the IC₅₀ values of the voltage-independent Zn²⁺ inhibition. Group I histidines, when mutated, have micromolar IC₅₀ values (**). Group II histidines, when mutated, have moderate but significant effect on the IC₅₀ values compared with wild-type (P < 0.01, ANOVA and Newman–Keul's post hoc test). Group III histidines do not have significantly different IC₅₀ values compared with wild-type (P > 0.05, ANOVA and Newman–Keul's post hoc tests). (C) Composite Zn²⁺ inhibition curves are shown for NR2A mutants (H42A, H44G, and H128A) and wild-type NR1-1a/NR2A. Zn²⁺ inhibition of glutamate-evoked NR2A wild-type and mutant receptor-mediated currents were recorded at −20 to −30 mV. Zn²⁺ concentrations (0.007 to 2.6 μM) were achieved by using tricine buffer. Values are mean ± SEM. Numbers in parentheses are the number of oocytes.

Figure 4

Mutations of histidine 128 abolished pH-dependency of Zn²⁺ inhibition of NR1/NR2A receptors. (A) Zn²⁺ inhibition of glutamate-evoked NR1-1a/NR2A(H42A) receptor-mediated currents recorded in 3 μM added Zn²⁺ at −20 mV in pHs 6.8 and 7.8. (B) Zn²⁺ inhibition of glutamate-evoked NR1-1a/NR2A(H44G) receptor-mediated currents recorded in 10 μM added Zn²⁺ at −20 mV in pHs 6.8 and 7.8. (C) Zn²⁺ inhibition of glutamate-evoked NR1-1a/NR2A(H128A) receptor-mediated currents recorded in 16 μM added Zn²⁺ at −30 mV in pHs 6.8 and 7.8. All control solutions contain 10 μM EDTA to chelate any contaminant Zn²⁺. (D) The pH-dependency of voltage-independent Zn²⁺ inhibition of NR2A-containing receptors harboring H42A or H44G mutations is similar to that of wild-type NR1/NR2A receptors (dotted line, data from Fig. 1D). (E) Exchange of His128 to Ala is sufficient to abolish the pH-dependency of voltage-independent Zn²⁺ inhibition. Free Zn²⁺ concentration is compensated at pH 8.2 for the formation of zinc hydroxide complexes. Error bars are SEM. Numbers in parentheses are the number of oocytes.