Hsiao Bernard, Mihalak Karla B, Repicky Sarah E, Everhart Drew, Mederos Ana H, Malhotra Arun, Luetje Charles W
Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami, Miami, FL 33101, USA.
Mol Pharmacol. 2006 Jan;69(1):27-36. doi: 10.1124/mol.105.015164. Epub 2005 Sep 27.
We have shown previously that the function of neuronal nicotinic acetylcholine receptors can be modulated by zinc. This modulation varies from potentiation to inhibition, depending on receptor subunit composition and zinc concentration, with the alpha4beta2 and alpha4beta4 receptors displaying the most dramatic potentiation. In this study, we used site-directed mutagenesis to identify glutamate 59 and histidine 162 on the rat alpha4 subunit as potential mediators of zinc potentiation. By modeling the extracellular domain of the receptor pentamer, we locate these residues to two subunit-subunit interfaces that alternate with the two acetylcholine-binding interfaces. Substitution of a cysteine at either position allows additional reduction of zinc potentiation upon treatment with the methanethiosulfonate reagents N-biotinoylaminoethyl methanethiosulfonate (MTSEA-biotin) and [2-(trimethylammonium)ethyl] methanethiosulfonate. Mutagenesis and methanethiosulfonate treatment are most effective at position 162, and the presence of zinc hinders the reaction of MTSEA-biotin with the substituted cysteine at this position, suggesting that alpha4His162 participates in forming a coordination site for zinc. Mutagenesis and methanethiosulfonate treatment are less effective at position 59, suggesting that whereas alpha4Glu59 may be near the zinc coordination site, it may not be participating in coordination of the zinc ion. It is noteworthy that the position of alpha4Glu59 within the neuronal nAChR is identical to that of a residue that lines the benzodiazepine-binding site on GABA(A) receptors. We suggest that the zinc potentiation sites on neuronal nAChRs are structurally and functionally similar to the benzodiazepine-binding sites on GABA(A) receptors.
我们之前已经表明，神经元烟碱型乙酰胆碱受体的功能可被锌调节。这种调节从增强到抑制各不相同，这取决于受体亚基组成和锌浓度，其中α4β2和α4β4受体表现出最显著的增强作用。在本研究中，我们使用定点诱变来确定大鼠α4亚基上的谷氨酸59和组氨酸162作为锌增强作用的潜在介导因子。通过对受体五聚体的细胞外结构域进行建模，我们将这些残基定位到与两个乙酰胆碱结合界面交替的两个亚基-亚基界面上。在任一位置取代一个半胱氨酸，在用甲硫基磺酸盐试剂N-生物素酰氨基乙基甲硫基磺酸盐（MTSEA-生物素）和[2-(三甲基铵)乙基]甲硫基磺酸盐处理后，可进一步降低锌的增强作用。诱变和甲硫基磺酸盐处理在位置162处最为有效，并且锌的存在会阻碍MTSEA-生物素与该位置取代的半胱氨酸反应，这表明α4His162参与形成锌的配位位点。诱变和甲硫基磺酸盐处理在位置59处效果较差，这表明虽然α4Glu59可能靠近锌配位位点，但它可能不参与锌离子的配位。值得注意的是，α4Glu59在神经元nAChR中的位置与位于GABA(A)受体苯二氮䓬结合位点的一个残基的位置相同。我们认为，神经元nAChRs上的锌增强位点在结构和功能上与GABA(A)受体上的苯二氮䓬结合位点相似。
