Responses to the excitatory amino acids kainate, quisqualate, N-methyl-D-aspartate (NMDA), L-glutamate and L-aspartate were recorded in mouse hippocampal neurones in cell culture, using the whole-cell configuration of the patch clamp technique. Agonists were applied rapidly from an array of flow pipes each of 250 μm diameter, positioned within 100 μm of the nerve cell body. Responses to NMDA, L-aspartate and to low concentrations of L-glutamate, recorded with glycine in the extracellular fluid, were strongly antagonized by 50 μM-zinc. Responses to kainate, quisqualate, and in glycine-free solution, responses to L-glutamate, were potentiated by 50 μM-zinc, but partially antagonized by 1 mM-zinc. On average, with 50 μM-zinc, responses to NMDA were reduced to 0.19 times control, while responses to kainate and quisqualate were increased to 1.09 and 1.14 times control. With 1 mM-zinc responses to kainate and quisqualate were reduced to 0.54 and 0.42 times control. Cadmium had a similar, though less potent action, and at 50 μM antagonized responses to NMDA but potentiated responses to kainate and quisqualate. On average, with 50 μM-cadmium, responses to NMDA were reduced to 0.39 times control, while responses to kainate and quisqualate were increased to 1.08 and 1.15 times control. With 1 mM-cadmium responses to NMDA were reduced to 0.04 times control while responses to kainate and quisqualate were reduced to 0.79 and 0.60 times control. Mercury was neurotoxic and increased the leakage current; however, no reduction of the response to NMDA was produced by 5 μM-mercury. The equilibrium dissociation constant (K(d)) for zinc antagonism of responses to NMDA, estimated from fit of a single binding site adsorption isotherm, was 13 μM; cadmium was about 4 times less potent than zinc. These effects of zinc and cadmium were nearly voltage independent. In contrast the antagonism of responses to NMDA by 150 μM-magnesium was highly voltage dependent, such that the K(d) for magnesium increased e-fold per 17.6 mV depolarization. Tye potency of zinc as an NMDA antagonist did not vary with the concentration of NMDA, and was not greatly influenced by a 1000-fold variation in the concentration of the NMDA-modulator glycine. This suggests that zinc acts as a non-competitive antagonist, and does not directly interfere with the binding of NMDA to the agonist recognition site nor with the binding of glycine to an allosteric site on the NMDA receptor complex. The potency of zinc decreased by a factor of three as the calcium concentration was raised over the range of 0.3-30 mM. In magnesium-free solutions, with 2 mM-calcium, the NMDA current-voltage relationship showed only weak rectification over the range -120 to -40 mV and remained approximately linear in the presence of 25 μM-zinc. With 15 μM-magnesium added to the extracellular solution, the NMDA current-voltage relationship had a region of negative slope conductance from -50 to -120 mV; with 25 μM-zinc, and 15 μM-magnesium, the current-voltage relationship was more linear. This most probably reflects non-additive antagonism of voltage-dependent block by Mg2+ and an essentially voltage-independent block by zinc. Our results are consistent with at least two binding sites for divalent cations on the NMDA receptor complex: one deep within the membrane electric field, most probably within the pore of the channel, which binds magnesium and related cations, and a second site near the extracellular face of the receptor which binds zinc and cadmium.
|Original language||English (US)|
|Number of pages||22|
|Journal||Journal of Physiology|
|Publication status||Published - 1989|
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