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Identification of an inhibitory Zn²⁺ binding site on the human glycine receptor 1 subunit

Robert J. Harvey, Philip Thomas, Colin H. James *, Andrew Wilderspin * and Trevor G. Smart

Whole-cell glycine-activated currents were recorded from human embryonic kidney (HEK) cells expressing wild-type and mutant recombinant homomeric glycine receptors (GlyRs) to locate the inhibitory binding site for Zn²⁺ ions on the human 1 subunit.

Glycine-activated currents were potentiated by low concentrations of Zn²⁺ (<10 µM) and inhibited by higher concentrations (>100 µM) on wild-type 1 subunit GlyRs.

Lowering the external pH from 7·4 to 5·4 inhibited the glycine responses in a competitive manner. The inhibition caused by Zn²⁺ was abolished leaving an overt potentiating effect at 10 µM Zn²⁺ that was exacerbated at 100 µM Zn²⁺.

The identification of residues involved in the formation of the inhibitory binding site was also assessed using diethylpyrocarbonate (DEPC), which modifies histidines. DEPC (1 mM) abolished Zn²⁺-induced inhibition and also the potentiation of glycine-activated currents by Zn²⁺.

The reduction in glycine-induced whole-cell currents in the presence of high (100 µM) concentrations of Zn²⁺ did not increase the rate of glycine receptor desensitisation.

Systematic mutation of extracellular histidine residues in the GlyR 1 subunit revealed that mutations H107A or H109A completely abolished inhibition of glycine-gated currents by Zn²⁺. However, mutation of other external histidines, H210, H215 and H419, failed to prevent inhibition by Zn²⁺ of glycine-gated currents. Thus, H107 and H109 in the extracellular domain of the human GlyR 1 subunit are major determinants of the inhibitory Zn²⁺ binding site.

An examination of Zn²⁺ co-ordination in metalloenzymes revealed that the histidine- hydrophobic residue-histidine motif found to be responsible for binding Zn²⁺ in the human GlyR 1 subunit is also shared by some of these enzymes. Further comparison of the structure and location of this motif with a generic model of the GlyR 1 subunit suggests that H107 and H109 participate in the formation of the inhibitory Zn²⁺ binding site at the apex of a sheet in the N-terminal extracellular domain.

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