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

1. Whole-cell glycine-activated currents were recorded from human embryonic kidney (HEK) cells expressing wild-type and mutant recombinant homomeric g lycine receptors (GlyRs) to locate the inhibitory binding site for Zn2+ ion s on the human alpha 1 subunit. 2. Glycine-activated currents were potentiated by low concentrations of Zn2 + (<10 mu M) and inhibited by higher concentrations (>100 mu M) on wild-typ e alpha 1 subunit GlyRs. 3. 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 leavi ng an overt potentiating effect at 10 mu M Zn2+ that was exacerbated at 100 mu M Zn2+. 4. The identification of residues involved in the formation of the inhibito ry 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+. 5. The reduction in glycine-induced whole-cell currents in the presence of high (100 mM) concentrations of Zn2+ did not increase the rate of glycine r eceptor desensitisation. 6. Systematic mutation of extracellular histidine residues in the GlyR alph a 1 subunit revealed that mutations H107A or H109A completely abolished inh ibition of glycine-gated currents by Zn2+. However, mutation of other exter nal histidines, H210, H215 and H419, failed to prevent inhibition by Zn2+ o f glycine-gated currents. Thus, H107 and H109 in the extracellular domain o f the human GlyR alpha 1 subunit are major determinants of the inhibitory Z n2+ binding site. 7. 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 alpha 1 subunit is also shared by some of th ese enzymes. Further comparison of the structure and location of this motif with a generic model of the GlyR alpha 1 subunit suggests that H107 and H1 09 participate in the formation of the inhibitory Zn2+ binding site at the apex of a beta sheet in the N-terminal extracellular domain.