High-Affinity Zinc Inhibition of NMDA NR1–NR2A Receptors

TLDR

Extracellular Zn²⁺ antagonizes NMDA receptors through both voltage‑independent and voltage‑dependent inhibition. The study aims to determine the relative contributions and subunit specificity of these two inhibition modes on recombinant NMDA receptors expressed in HEK 293 cells and Xenopus oocytes. Recombinant NMDA receptors with varied NR1 (NR1a or NR1b) and NR2 (NR2A, NR2B, NR2C) subunits were expressed in HEK 293 cells and Xenopus oocytes, and Zn²⁺ effects were measured to assess subunit contributions to voltage‑independent inhibition. Voltage‑dependent inhibition is similar for NR1a‑NR2A and NR1a‑NR2B receptors, whereas voltage‑independent inhibition is far more potent in NR1a‑NR2A (nanomolar IC₅₀) than in NR1a‑NR2B (micromolar IC₅₀), a high affinity due to slow Zn²⁺ dissociation; both NR1 and NR2 subunits contribute, and chelation of trace heavy metals potentiates NR1a‑NR2A responses, indicating contamination in standard solutions.

Abstract

Micromolar concentrations of extracellular Zn2+ are known to antagonize native NMDA receptors via a dual mechanism involving both a voltage-independent and a voltage-dependent inhibition. We have tried to evaluate the relative importance of these two effects and their subunit specificity on recombinant NMDA receptors expressed in HEK 293 cells and Xenopus oocytes. The comparison of NR1a-NR2A and NR1a-NR2B receptors shows that the voltage-dependent inhibition is similar in both types of receptors but that the voltage-independent inhibition occurs at much lower Zn2+ concentrations in NR1a-NR2A receptors (IC50 in the nanomolar range) than in NR1a-NR2B receptors (IC50 in the micromolar range). The high affinity of the effect observed with NR1a-NR2A receptors was found to be attributable mostly to the slow dissociation of Zn2+ from its binding site. By analyzing the effects of Zn2+ on varied combinations of NR1 (NR1a or NR1b) and NR2 (NR2A, NR2B, NR2C), we show that both the NR1 and the NR2 subunits contribute to the voltage-independent Zn2+ inhibition. We have observed further that under control conditions, i.e., in zero nominal Zn2+ solutions, the addition of low concentrations of heavy metal chelators markedly potentiates the responses of NR1a-NR2A receptors, but not of NR1a-NR2B receptors. This result suggests that traces of a heavy metal (probably Zn2+) contaminate standard solutions and tonically inhibit NR1a-NR2A receptors. Chelation of a contaminant metal also could account for the rapid NR2A subunit-specific potentiations produced by reducing compounds like DTT or glutathione.