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

被引:85
作者
Harvey, RJ
Thomas, P
James, CH
Wilderspin, A
Smart, TG
机构
[1] Univ London, Sch Pharm, Dept Pharmacol, London WC1N 1AX, England
[2] Univ London, Sch Pharm, Dept Pharmaceut Chem, London WC1N 1AX, England
来源
JOURNAL OF PHYSIOLOGY-LONDON | 1999年 / 520卷 / 01期
关键词
D O I
10.1111/j.1469-7793.1999.00053.x
中图分类号
Q189 [神经科学];
学科分类号
071006 ;
摘要
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 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-type 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 leaving 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 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 mM) concentrations of Zn2+ did not increase the rate of glycine receptor desensitisation. 6. Systematic mutation of extracellular histidine residues in the GlyR alpha 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 alpha 1 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 alpha 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 alpha 1 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.
引用
收藏
页码:53 / 64
页数:12
相关论文
共 29 条
[1]   RELEASE OF ENDOGENOUS ZN-2+ FROM BRAIN-TISSUE DURING ACTIVITY [J].
ASSAF, SY ;
CHUNG, SH .
NATURE, 1984, 308 (5961) :734-736
[2]  
Becker C-M, 1995, Neuroscientist, V1, P130
[3]  
BLOOMENTHAL AB, 1994, MOL PHARMACOL, V46, P1156
[4]   Identification and mechanism of action of two histidine residues underlying high-affinity Zn2+ inhibition of the NMDA receptor [J].
Choi, YB ;
Lipton, SA .
NEURON, 1999, 23 (01) :171-180
[5]  
Fisher JL, 1998, J NEUROSCI, V18, P2944
[6]  
FREDERICKSON CJ, 1989, INT REV NEUROBIOL, V31, P145
[7]   Predicted structure of the extracellular region of ligand-gated ion-channel receptors shows SH2-like and SH3-like domains forming the ligand-binding site [J].
Gready, JE ;
Ranganathan, S ;
Schofield, PR ;
Matsuo, Y ;
Nishikawa, K .
PROTEIN SCIENCE, 1997, 6 (05) :983-998
[8]   ALPHA-SUBUNIT VARIANTS OF THE HUMAN GLYCINE RECEPTOR - PRIMARY STRUCTURES, FUNCTIONAL EXPRESSION AND CHROMOSOMAL LOCALIZATION OF THE CORRESPONDING GENES [J].
GRENNINGLOH, G ;
SCHMIEDEN, V ;
SCHOFIELD, PR ;
SEEBURG, PH ;
SIDDIQUE, T ;
MOHANDAS, TK ;
BECKER, CM ;
BETZ, H .
EMBO JOURNAL, 1990, 9 (03) :771-776
[9]   ZN2+ - AN ENDOGENOUS MODULATOR OF LIGAND-GATED AND VOLTAGE-GATED ION CHANNELS [J].
HARRISON, NL ;
GIBBONS, SJ .
NEUROPHARMACOLOGY, 1994, 33 (08) :935-952
[10]   IMPROVED GREEN FLUORESCENCE [J].
HEIM, R ;
CUBITT, AB ;
TSIEN, RY .
NATURE, 1995, 373 (6516) :663-664