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Identification of an inhibitory Zn 2+ binding site on the human glycine receptor α1 subunit
Author(s) -
Harvey Robert J.,
Thomas Philip,
James Colin H.,
Wilderspin Andrew,
Smart Trevor G.
Publication year - 1999
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1999.00053.x
Subject(s) - glycine , glycine receptor , protein subunit , inhibitory postsynaptic potential , binding site , chemistry , identification (biology) , gamma aminobutyric acid receptor subunit alpha 1 , receptor , biochemistry , biophysics , biology , amino acid , g alpha subunit , neuroscience , gene , botany
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 Zn 2+ ions on the human α1 subunit. 2 Glycine‐activated currents were potentiated by low concentrations of Zn 2+ (<10 μ m ) and inhibited by higher concentrations (>100 μ m ) on wild‐type α1 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 Zn 2+ was abolished leaving an overt potentiating effect at 10 μ m Zn 2+ that was exacerbated at 100 μ m Zn 2+ . 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 m m ) abolished Zn 2+ ‐induced inhibition and also the potentiation of glycine‐activated currents by Zn 2+ . 5 The reduction in glycine‐induced whole‐cell currents in the presence of high (100 μ m ) concentrations of Zn 2+ did not increase the rate of glycine receptor desensitisation. 6 Systematic mutation of extracellular histidine residues in the GlyR α1 subunit revealed that mutations H107A or H109A completely abolished inhibition of glycine‐gated currents by Zn 2+ . However, mutation of other external histidines, H210, H215 and H419, failed to prevent inhibition by Zn 2+ of glycine‐gated currents. Thus, H107 and H109 in the extracellular domain of the human GlyR α1 subunit are major determinants of the inhibitory Zn 2+ binding site. 7 An examination of Zn 2+ co‐ordination in metalloenzymes revealed that the histidine‐ hydrophobic residue‐histidine motif found to be responsible for binding Zn 2+ in the human GlyR α1 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 α1 subunit suggests that H107 and H109 participate in the formation of the inhibitory Zn 2+ binding site at the apex of a β sheet in the N‐terminal extracellular domain.