The glutamate and chloride permeation pathways are colocalized in individual neuronal glutamate transporter subunits
The glutamate and chloride permeation pathways are colocalized in individual neuronal glutamate transporter subunits
复制标题
DOI：
10.1523/jneurosci.4851-06.2007
复制
发表时间：
2007-03-14
影响因子：
5.3
通讯作者：
Kavanaugh, Michael P.
中科院分区：
文献类型：
--
作者：
Leary, Gregory P.;Stone, Emily F.;Kavanaugh, Michael P.
谷氨酸转运蛋白具有同源三聚体亚基结构，其具有部分延伸到脂质双层平面中的大的中央充水腔（Yernool等人，2004年）。除了摄取谷氨酸外，转运蛋白还介导在底物存在下增加的氯离子电导。氯离子通道是位于三聚体的中心孔还是位于单个亚基内一直存在争议。我们发现野生型神经元谷氨酸转运蛋白EAAT 3亚基与在R447处突变的亚基的共表达，R447是控制底物选择性的残基（Bendahan等人，2000），导致与两个不同的非相互作用转运蛋白群体一致的转运活性，与先前的工作一致，表明每个亚基独立地作用于转运底物（Awes等人，2004; Grewer等人，2005; Koch and Larsson，2005）。在野生型同源三聚体转运蛋白中，分离并测量了阴离子电导的谷氨酸浓度依赖性和谷氨酸通量的动力学，并将阴离子通道激活拟合为对应于（1）通过结合一个或多个亚基门控的中心孔和（2）每个亚基中的通道孔的分析表达式。这些数据表明，谷氨酸结合位点，运输途径和氯离子通道驻留在一个三聚体的各个亚基和功能独立。
Glutamate transporters have a homotrimeric subunit structure with a large central water-filled cavity that extends partially into the plane of the lipid bilayer (Yernool et al., 2004). In addition to uptake of glutamate, the transporters also mediate a chloride conductance that is increased in the presence of substrate. Whether the chloride channel is located in the central pore of the trimer or within the individual subunits has been controversial. We find that coexpression of wild-type neuronal glutamate transporter EAAT3 subunits with subunits mutated at R447, a residue governing substrate selectivity (Bendahan et al., 2000), results in transport activity consistent with two distinct noninteracting populations of transporters, in agreement with previous work suggesting that each subunit operates independently to transport substrate (Awes et al., 2004; Grewer et al., 2005; Koch and Larsson, 2005). In wild-type homotrimeric transporters, the glutamate concentration dependence of the anion conductance and the kinetics of glutamate flux were isolated and measured, and the anion channel activation was fitted to analytical expressions corresponding to (1) a central pore gated by binding to one or more subunits and (2) a channel pore in each subunit. The data indicate that glutamate-binding sites, transport pathways, and chloride channels reside in individual subunits in a trimer and function independently.