Nevin Simon T, Cromer Brett A, Haddrill Justine L, Morton Craig J, Parker Michael W, Lynch Joseph W
Department of Physiology and Pharmacology, School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia.
J Biol Chem. 2003 Aug 1;278(31):28985-92. doi: 10.1074/jbc.M300097200. Epub 2003 May 9.
Histidines 107 and 109 in the glycine receptor (GlyR) alpha1 subunit have previously been identified as determinants of the inhibitory zinc-binding site. Based on modeling of the GlyR alpha1 subunit extracellular domain by homology to the acetylcholine-binding protein crystal structure, we hypothesized that inhibitory zinc is bound within the vestibule lumen at subunit interfaces, where it is ligated by His107 from one subunit and His109 from an adjacent subunit. This was tested by co-expressing alpha1 subunits containing the H107A mutation with alpha1 subunits containing the H109A mutation. Although sensitivity to zinc inhibition is markedly reduced when either mutation is individually incorporated into all five subunits, the GlyRs formed by the co-expression of H107A mutant subunits with H109A mutant subunits exhibited an inhibitory zinc sensitivity similar to that of the wild type alpha1 homomeric GlyR. This constitutes strong evidence that inhibitory zinc is coordinated at the interface between adjacent alpha1 subunits. No evidence was found for beta subunit involvement in the coordination of inhibitory zinc, indicating that a maximum of two zinc-binding sites per alpha1beta receptor is sufficient for maximal zinc inhibition. Our data also show that two zinc-binding sites are sufficient for significant inhibition of alpha1 homomers. The binding of zinc at the interface between adjacent alpha1 subunits could restrict intersubunit movements, providing a feasible mechanism for the inhibition of channel activation by zinc.
甘氨酸受体（GlyR）α1亚基中的组氨酸107和109先前已被确定为抑制性锌结合位点的决定因素。基于与乙酰胆碱结合蛋白晶体结构同源性对GlyR α1亚基细胞外结构域进行建模，我们推测抑制性锌结合在亚基界面处的前庭腔内，在此处它由一个亚基的His107和相邻亚基的His109连接。通过共表达含有H107A突变的α1亚基与含有H109A突变的α1亚基对此进行了测试。尽管当任一突变单独引入到所有五个亚基中时对锌抑制的敏感性显著降低，但由H107A突变亚基与H109A突变亚基共表达形成的GlyRs表现出与野生型α1同聚体GlyR相似的抑制性锌敏感性。这构成了强有力的证据，表明抑制性锌在相邻α1亚基之间的界面处配位。未发现β亚基参与抑制性锌配位的证据，表明每个α1β受体最多两个锌结合位点足以实现最大程度的锌抑制。我们的数据还表明，两个锌结合位点足以显著抑制α1同聚体。锌在相邻α1亚基之间的界面处结合可能会限制亚基间运动，为锌抑制通道激活提供了一种可行的机制。
