Identification of an inhibitory Zn2+ binding site on the human glycine receptor alpha1 subunit.
1. 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 alpha1 subunit. 2. Glycine-activated currents were potentiated by low concentrations of Zn2+ (<10 microM) and inhibited by higher concentrations (>100 microM) on wild-type alpha1 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 leaving an overt potentiating effect at 10 microM Zn2+ that was exacerbated at 100 microM Zn2+. 4. 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+. 5. The reduction in glycine-induced whole-cell currents in the presence of high (100 microM) concentrations of Zn2+ did not increase the rate of glycine receptor desensitisation. 6. Systematic mutation of extracellular histidine residues in the GlyR alpha1 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 alpha1 subunit are major determinants of the inhibitory Zn2+ 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 alpha1 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 alpha1 subunit suggests that H107 and H109 participate in the formation of the inhibitory Zn2+ binding site at the apex of a beta sheet in the N-terminal extracellular domain.
Harvey
R J
RJ
Department of Pharmacology and Department of Pharmaceutical Chemistry, The School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, UK.
Thomas
P
P
James
C H
CH
Wilderspin
A
A
Smart
T G
TG
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004594:Electrophysiology; D005109:Extracellular Matrix / Q000378:metabolism; D005822:Genetic Vectors; D005998:Glycine / Q000502:physiology; D006639:Histidine / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics / Q000502:physiology; D018408:Patch-Clamp Techniques; D018009:Receptors, Glycine / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D018009:Receptors, Glycine; D006639:Histidine; D015032:Zinc; D004047:Diethyl Pyrocarbonate; D005998:Glycine