Micromolar concentrations of extracellular Zn 2ϩ 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

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... s but that the voltage-independent inhibition occurs at much lower Zn 2ϩ concentrations in NR1a-NR2A receptors (IC 50 in the nanomolar range) than in NR1a-NR2B receptors (IC 50 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 Zn 2ϩ from its binding site. By analyzing the effects of Zn 2ϩ on varied com-binations 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 Zn 2ϩ inhibition. We have observed further that under control conditions, i.e., in zero nominal Zn 2ϩ 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 Zn 2ϩ ) 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. Zn 2ϩ ions are known to be abundant in some nerve terminals and can be released in the synaptic cleft at concentrations of nearly 1 M (for review, see Smart et al., 1994) . In this context, the observation that Z n 2ϩ at micromolar concentrations inhibits NMDA responses (Peters et al., 1987; Westbrook and Mayer, 1987) immediately was given a major physiological significance. This significance was reinforced by the observation of Zn 2ϩ inhibitory effects on the NMDA components of synaptic currents (Forsythe et al., 1988; and by data indicating that Z n 2ϩ could play an important role in excitoxicity (Koh and Choi, 1988; Koh et al., 1996) . The analysis of the mechanisms of the Z n 2ϩ inhibition Christine and Choi, 1990; Legendre and Westbrook, 1990 ) revealed that Z n 2ϩ produces both a voltageindependent inhibition and a voltage-dependent block, the latter resembling that produced by Mg 2ϩ . Inhibitory effects of Zn 2ϩ on recombinant NMDA receptors were observed first on receptors expressed from whole brain RNA (Rassendren et al., 1990) and then, after the cloning of the main NMDA receptor subunits, on heteromeric receptors associating N R1 and N R2 subunits Meguro et al., 1992) and on most homomeric NR1 receptors (Hollmann et al., 1993; Z heng et al., ). Mori et al. (1992 and Sakurada et al. (1993) then analyzed the effects of mutations of a ring of asparagines found at the Q/R/N site, a critical position of the M2 segment involved in the control of the Mg 2ϩ block of NMDA channels (see McBain and Mayer, 1994) . They observed that a major reduction or even a complete suppression of the Mg 2ϩ block was associated with a mild reduction of the Zn 2ϩ block (see also Kawajiri and Dingledine, 1993) , probably because the mutations reduced or abolished the Zn 2ϩ voltage-dependent block but left intact the Zn 2ϩ voltageindependent inhibition. The present study aimed at a better separation of the voltagedependent and the voltage-independent processes in recombinant receptors. In attempting to measure the inhibitory effect of Zn 2ϩ on NR1a-NR2A receptors expressed in Xenopus oocytes and human embryonic kidney (HEK) cells, we observed inhibitions of very variable size at concentrations of a few tens of nanomolars. This was found to be attributable to the variable degree of contamination of the solutions by traces of heavy metal ions (possibly Zn 2ϩ ). By using chelators of these metals, we were able to obtain a reliable estimate of the control response and to demonstrate that the IC 50 of the voltage-independent Zn 2ϩ inhibition is highly subunit-specific, ranging from ϳ10 nM in the case of NR1a-NR2A receptors to 10 M in NR1a-NR2C receptors. In contrast, the voltage-dependent Zn 2ϩ inhibition has an IC 50 in the micromolar range in NR1a-NR2A receptors and can be suppressed selectively by a point mutation in the pore region [NR2A(N595K)]. MATERIALS AND METHODS Primary neuronal cultures Cortical and diencephalic neurons taken from 15-to 16-d-old mouse embryos were cultured for 2-5 weeks, as previously described by Ascher et al. (1988).