Allosteric Modulation of an Excitatory Amino Acid Transporter: The Subtype-Selective Inhibitor UCPH-101 Exerts Sustained Inhibition of EAAT1 through an Intramonomeric Site in the Trimerization Domain
Allosteric Modulation of an Excitatory Amino Acid Transporter: The Subtype-Selective Inhibitor UCPH-101 Exerts Sustained Inhibition of EAAT1 through an Intramonomeric Site in the Trimerization Domain
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DOI：
10.1523/jneurosci.3396-12.2013
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发表时间：
2013-01-16
影响因子：
5.3
通讯作者：
Jensen, Anders A.
中科院分区：
文献类型：
--
作者：
Abrahamsen, Bjarke;Schneider, Nicole;Jensen, Anders A.
在本研究中，阐明了人类兴奋性氨基酸转运蛋白亚型1（EAAT 1）及其啮齿动物直系同源物GLAST的第一类选择性抑制剂活性的作用机制和分子基础。先前报道的UCPH-101和UCPH-102对EAAT 1的特异性超过EAAT 2和EAAT 3，证明其也延伸至EAAT 4和EAAT 5亚型。有趣的是，短暂暴露于UCPH-101诱导EAAT 1的持久失活状态，而密切相关的类似物所产生的抑制作用本质上更可逆。与此一致，UCPH-101解除转运蛋白阻断的动力学性质比UCPH-102的动力学性质慢得多。UCPH-101表现出对EAAT 1的非竞争性抑制，其在GLAST中的结合位点已在一项精心设计的诱变研究中被描绘出来。GLAST的TM 3、TM 4c和TM 7a中几个残基的取代对UCPH-101的抑制效力和/或功效具有不利影响，而不显著影响（S）-谷氨酸或竞争性EAAT抑制剂TBOA的药理学性质。因此，提出UCPH-101靶向GLAST单体的“三聚化结构域”中的主要疏水性裂缝，并且该抑制剂被证明抑制通过其唯一结合的单体的摄取，并且不影响通过GLAST三聚体中的其他单体的底物易位。UCPH-101抑制的变构模式强调了EAAT三聚化结构域的功能重要性，并证明了通过配体结合到远离其“转运结构域”的区域来调节转运蛋白功能的可行性。"
In the present study, the mechanism of action and molecular basis for the activity of the first class of selective inhibitors of the human excitatory amino acid transporter subtype 1 (EAAT1) and its rodent ortholog GLAST are elucidated. The previously reported specificity of UCPH-101 and UCPH-102 for EAAT1 over EAAT2 and EAAT3 is demonstrated to extend to the EAAT4 and EAAT5 subtypes as well. Interestingly, brief exposure to UCPH-101 induces a long-lasting inactive state of EAAT1, whereas the inhibition exerted by closely related analogs is substantially more reversible in nature. In agreement with this, the kinetic properties of UCPH-101 unblocking of the transporter are considerably slower than those of UCPH-102. UCPH-101 exhibits noncompetitive inhibition of EAAT1, and its binding site in GLAST has been delineated in an elaborate mutagenesis study. Substitutions of several residues in TM3, TM4c, and TM7a of GLAST have detrimental effects on the inhibitory potency and/or efficacy of UCPH-101 while not affecting the pharmacological properties of (S)-glutamate or the competitive EAAT inhibitor TBOA significantly. Hence, UCPH-101 is proposed to target a predominantly hydrophobic crevice in the "trimerization domain" of the GLAST monomer, and the inhibitor is demonstrated to inhibit the uptake through the monomer that it binds to exclusively and not to affect substrate translocation through the other monomers in the GLAST trimer. The allostericmodeof UCPH-101 inhibition underlines the functional importance of the trimerization domain of the EAAT and demonstrates the feasibility of modulating transporter function through ligand binding to regions distant from its "transport domain."