Paired-Cysteine Scanning Reveals Conformationally Sensitive Proximity between the TM4b-4c Loop and TM8 of the Glutamate Transporter EAAT1
Paired-Cysteine Scanning Reveals Conformationally Sensitive Proximity between the TM4b-4c Loop and TM8 of the Glutamate Transporter EAAT1
复制标题
配对半胱氨酸扫描揭示了谷氨酸转运蛋白 EAAT1 的 TM4b-4c 环和 TM8 之间的构象敏感接近度
DOI：
10.1021/acschemneuro.9b00048
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发表时间：
2019-05-01
影响因子：
5
通讯作者：
Zhang, Xiuping
中科院分区：
文献类型：
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作者：
Qu, Shaogang;Zhang, Wenlong;Zhang, Xiuping
兴奋性氨基酸转运蛋白（EAATs）从突触间隙吸收神经递质谷氨酸并维持谷氨酸浓度低于神经毒性水平。近年来，热稳定型EAAT1的晶体结构已被报道；然而，关于跨膜结构域（TM） 4b-4c环的功能机制知之甚少，该环在哺乳动物eaat中含有超过50个氨基酸，而这些氨基酸在原核同源物中是不存在的。为了探索TM4在转运周期中的空间位置和功能，我们在无半胱氨酸版本的EAAT1 CL-EAAT1中引入TM4b-4c环和TM8之间的配对半胱氨酸替换。我们观察到Cu(II)（1,10-菲罗啉）(3)（CuPh）对双取代的V238C/I469C和A243C/I469C变异有明显的抑制作用，但对相应的单取代的CL-EAAT1或其他20多种双半胱氨酸变异没有明显的抑制作用。二硫苏糖醇处理部分恢复了CuPh处理过的V238C/I469C和A243C/1469C双取代变异体的摄取活性，证实CuPh对这些变异体的影响是由于分子内二硫键的形成。谷氨酸、KCl和D， l -三- β -苯氧基-天冬氨酸削弱了V238C/I469C变体的CuPh抑制，但只有KCl削弱了V243C/1469C变体的CuPh抑制，这表明TM4b-4c环和TM8在EAAT1的内向构象中相互分离。我们的研究结果表明，TM4b-4c环和TM8在运输周期中位置接近，而在面向内的构象中间距较小。
Excitatory amino acid transporters (EAATs) take up the neurotransmitter glutamate from the synaptic cleft and maintain glutamate concentrations below neurotoxic levels. Recently, the crystal structures of thermostable EAAT1 variants have been reported; however, little is understood regarding the functional mechanism of the transmembrane domain (TM) 4b-4c loop, which contains more than 50 amino acids in mammalian EAATs that are absent in prokaryotic homologues. To explore the spatial position and function of TM4 during the transport cycle, we introduced pairwise cysteine substitutions between the TM4b-4c loop and TM8 in a cysteine-less version of EAAT1, CL-EAAT1. We observed pronounced inhibition of transport by Cu(II)(1,10-phenanthroline)(3) (CuPh) for doubly substituted V238C/I469C and A243C/I469C variants, but not for corresponding singly substituted CL-EAAT1 or for more than 20 other double-cysteine variants. Dithiothreitol treatment partially restored the uptake activity of the CuPh-treated V238C/I469C and A243C/1469C doubly substituted variants, confirming that the effects of CuPh on these variants were due to the formation of intramolecular disulfide bonds. Glutamate, KCl, and D,L-threo-beta-benzyloxy-aspartate weakened CuPh inhibition of the V238C/I469C variant, but only KCl weakened CuPh inhibition of the V243C/1469C variant, suggesting that the TM4b-4c loop and TM8 are separated from each other in the inward-facing conformations of EAAT1. Our results suggest that the TM4b-4c loop and TM8 are positioned in close proximity during the transport cycle and are less closely spaced in the inward-facing conformation.