Disturbances of Ligand Potency and Enhanced Degradation of the Human Glycine Receptor at Affected Positions G160 and T162 Originally Identified in Patients Suffering from Hyperekplexia
Disturbances of Ligand Potency and Enhanced Degradation of the Human Glycine Receptor at Affected Positions G160 and T162 Originally Identified in Patients Suffering from Hyperekplexia
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DOI：
10.3389/fnmol.2015.00079
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发表时间：
2015-12-22
影响因子：
4.8
通讯作者：
Villmann, Carmen
中科院分区：
文献类型：
--
作者：
Atak, Sinem;Langlhofer, Georg;Villmann, Carmen
Cys 环受体的配体结合由五聚体受体复合物中两个相邻亚基的正侧和负侧的 N 端细胞外环结构决定。甘氨酸受体 (GlyR) 环 B 中的芳香族残基通过阳离子 7 相互作用与引入的配体发生直接相互作用。最近，我们发现从患有神经过敏症的人类患者中鉴定出的环 B 中的突变残基会干扰配体结合。在这里，我们用半胱环受体家族相关成员中发现的氨基酸交换了受影响的人类残基，以确定侧链体积对离子通道特性的影响。转染细胞系后，对 GlyR 变体进行体外表征，以分析蛋白质表达、运输、降解和离子通道功能。 GlyR α 1 G160 突变显着降低了甘氨酸的效力，这表明对邻近芳香残基的位置影响，从而导致配体结合口袋内的甘氨酸结合。 T162 α 1 突变导致的甘氨酸抑制受到干扰，是配体结合位点内受影响的生物发生和结构变化的累加效应。从内质网到内质网-高尔基体中间室、分泌性高尔基体途径以及最终细胞表面的蛋白质运输大大减少，但仍然足以传递至少在高甘氨酸浓度下发挥功能的离子通道。大多数 T162 突变蛋白在 ER 中积累，并被递送至 ER 相关的蛋白酶体降解。因此，G160 是甘氨酸结合过程中的重要决定因素。相反，1162主要影响受体生物合成，而功能性改变是其次要影响。
Ligand-binding of Cys-loop receptors is determined by N-terminal extracellular loop structures from the plus as well as from the minus side of two adjacent subunits in the pentameric receptor complex. An aromatic residue in loop B of the glycine receptor (GIyR) undergoes direct interaction with the incoming ligand via a cation-7 interaction. Recently, we showed that mutated residues in loop B identified from human patients suffering from hyperekplexia disturb ligand-binding. Here, we exchanged the affected human residues by amino acids found in related members of the Cys-loop receptor family to determine the effects of side chain volume for ion channel properties. GIyR variants were characterized in vitro following transfection into cell lines in order to analyze protein expression, trafficking, degradation and ion channel function. GIyR alpha 1 G160 mutations significantly decrease glycine potency arguing for a positional effect on neighboring aromatic residues and consequently glycine-binding within the ligand-binding pocket. Disturbed glycinergic inhibition due to T162 alpha 1 mutations is an additive effect of affected biogenesis and structural changes within the ligand-binding site. Protein trafficking from the ER toward the ER-Golgi intermediate compartment, the secretory Golgi pathways and finally the cell surface is largely diminished, but still sufficient to deliver ion channels that are functional at least at high glycine concentrations. The majority of T162 mutant protein accumulates in the ER and is delivered to ER-associated proteasomal degradation. Hence, G160 is an important determinant during glycine binding. In contrast, 1162 affects primarily receptor biogenesis whereas exchanges in functionality are secondary effects thereof.