Characterization of the Effects of Charged Residues in the Intracellular Loop on Ion Permeation in α1 Glycine Receptor Channels
Characterization of the Effects of Charged Residues in the Intracellular Loop on Ion Permeation in α1 Glycine Receptor Channels
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DOI：
10.1074/jbc.m806618200
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发表时间：
2009-01-23
影响因子：
4.8
通讯作者：
Moorhouse, Andrew J.
中科院分区：
文献类型：
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作者：
Carland, Jane E.;Cooper, Michelle A.;Moorhouse, Andrew J.
Cys环受体通道介导神经系统中的快速突触传递。以M2为界的跨膜孔是其离子渗透性能的重要决定因素。细胞内域的门户也是阳离子Cys环受体中渗透途径的一部分，螺旋MA拉伸中的带电残基部分衬在这些开口中，深刻影响通道电导。目前尚不清楚类似的门户网站是否有助于阴离子半胱氨酸环受体的渗透途径。因此，我们研究了带电残基内的建议MA拉伸的同聚甘氨酸α 1受体的功能特性的影响。MA延伸中多达8个碱性残基同时突变为带负电荷的谷氨酸，并且野生型和突变体亚基在HEK-293细胞中表达。所有八个残基的突变产生了无功能的受体。在负膜电位（从92.2 +/- 2.8到60.0 +/- 2.2皮西门子）下，观察到谷氨酸盐存在于377、378、385和386位置（4 E亚基）时电导的最大降低。包括额外的谷氨酸残基在这个亚基没有进一步降低电导。中和这些残基（4A亚基）导致电导适度降低（80.5 +/- 2.3皮西门子）。正电位下的电导率没有明显影响。阴离子对阳离子的选择性和浓度-响应关系不受4A或4 E突变的影响。我们的研究结果确定了甘氨酸受体中影响电导的基本残基，这表明门户网站的扩展渗透途径的一部分，但M2划定的通道孔是甘氨酸受体中渗透性能的主要决定因素。
The Cys loop receptor channels mediate fast synaptic transmission in the nervous system. The M2-demarcated transmembrane pore is an important determinant of their ion permeation properties. Portals within the intracellular domain are also part of the permeation pathway in cationic Cys loop receptors, with charged residues in a helical MA stretch partially lining these openings profoundly affecting channel conductance. It is unknown whether analogous portals contribute to the permeation pathway in anionic Cys loop receptors. We therefore investigated the influence of charged residues within the proposed MA stretch on functional properties of the homomeric glycine alpha 1 receptor. Up to eight basic residues in the MA stretch were concurrently mutated to a negatively charged glutamate, and wild-type and mutant subunits were expressed in HEK-293 cells. Mutation of all eight residues produced a non-functional receptor. The greatest reduction in conductance at negative membrane potentials (from 92.2 +/- 2.8 to 60.0 +/- 2.2 picosiemens) was observed with glutamate present at the 377, 378, 385, and 386 positions (the 4E subunit). Inclusion of additional glutamate residues within this subunit did not decrease conductance further. Neutralizing these residues (the 4A subunit) caused a modest decrease in conductance (80.5 +/- 2.3 picosiemens). Outward conductance at positive potentials was not markedly affected. Anion to cation selectivity and concentration-response relationships were unaffected by the 4A or 4E mutations. Our results identify basic residues affecting conductance in the glycine receptor, suggesting that portals are part of the extended permeation pathway but that the M2-demarcated channel pore is the dominant determinant of permeation properties in glycine receptors.