Cysteine Scanning Mutagenesis of TM4b-4c Loop of Glutamate Transporter EAAT1 Reveals Three Conformationally Sensitive Residues
Cysteine Scanning Mutagenesis of TM4b-4c Loop of Glutamate Transporter EAAT1 Reveals Three Conformationally Sensitive Residues
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谷氨酸转运蛋白 EAAT1 TM4b-4c 环的半胱氨酸扫描诱变揭示了三个构象敏感残基
DOI：
10.1124/mol.117.111245
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发表时间：
2018-07
影响因子：
3.6
通讯作者：
Shaogang Qu
中科院分区：
文献类型：
--
作者：
Wenlong Zhang;Xiuping Zhang;Shaogang Qu
谷氨酸能突触递质通过兴奋性氨基酸转运蛋白（EAAT）从突触间隙清除，EAAT负责回收谷氨酸并将其转运到神经元和神经胶质细胞中。为了探索EAAT 1（褐家鼠）的TM 4 b-4c环的结构作用，将57个氨基酸残基中的每一个突变为半胱氨酸。13个单突变体具有非常低的转运活性。水的可及性的引入的半胱氨酸从其余的突变体，然后探索膜渗透和膜不渗透巯基试剂在不同的条件下。F190 C、V238 C和A243 C受MTSET影响，而Q189 C、F190 C、V238 C、A243 C和L244 C对MTSEA敏感。Q189 C和L244 C转运活性在钾的存在下降低，这预计有利于转运蛋白的内向构象。因此，L244 C受到谷氨酸的保护。MTSEA对A243 C的修饰可通过钾和谷氨酸或dl-苏型-β-苄氧基天冬氨酸来增强。根据这些结果，我们认为残基F190 C，V238 C和A243 C可能位于细胞外表面附近，并且TM 4 b-4c环在细胞表面上形成多个凹入的膜环。或者，F190 C、V238 C和A243 C可能在暴露于MTSET的转运途径中起作用。此外，Q189 C，A243 C和L244 C是构象敏感的，并可能在运输循环中发挥作用。
Glutamatergic synaptic transmitters are cleared from the synaptic cleft through excitatory amino acid transporters (EAATs) that are responsible for recycling glutamate and transporting it into neurons and glial cells. To probe the structural role of the TM4b-4c loop of EAAT1 (Rattus norvegicus), each of the 57 amino acid residues was mutated to cysteine. Thirteen of the single mutants have very low transport activity. Aqueous accessibility of the introduced cysteines from the remaining mutants was then explored by membrane-permeant and membrane-impermeant sulfhydryl reagents in different conditions. F190C, V238C, and A243C were affected by MTSET, whereas Q189C, F190C, V238C, A243C, and L244C were sensitive to MTSEA. Q189C and L244C transport activity was diminished in the presence of potassium, which is expected to favor the inward-facing conformation of the transporter. Inversely, L244C was protected by glutamate. The modification of A243C by MTSEA was enhanced by either potassium and glutamate or dl-threo-β-benzyloxyaspartate. From these results, we suggest that residues F190C, V238C, and A243C may be located near the extracellular surface, and the TM4b-4c loop forms multiple reentrant membrane loops on the cell surface. Alternatively, F190C, V238C, and A243C may function in the transport pathway, which is exposed to MTSET. In addition, Q189C, A243C, and L244C are conformationally sensitive and may play a role in the transport cycle.
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影响因子：
64.8
作者：
通讯作者：
--
影响因子：
64.8
作者：
KANAI, Y;HEDIGER, MA
通讯作者：
HEDIGER, MA
影响因子：
5.3
作者：
Levy, LM;Warr, O;Attwell, D
通讯作者：
Attwell, D
影响因子：
5.3
作者：
Koch, Hans Peter;Brown, Ronald Lane;Larsson, Hans Peter
通讯作者：
Larsson, Hans Peter
影响因子：
5.3
作者：
Leary, Gregory P.;Stone, Emily F.;Kavanaugh, Michael P.
通讯作者：
Kavanaugh, Michael P.