Allosteric Inhibition of a Vesicular Glutamate Transporter by an Isoform-Specific Antibody,Biochemistry

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Allosteric Inhibition of a Vesicular Glutamate Transporter by an Isoform-Specific Antibody
Biochemistry ( IF 2.9 ) Pub Date : 2021-07-28 , DOI: 10.1021/acs.biochem.1c00375
Jacob Eriksen ₁ , Fei Li _{1,

2} , Robert M Stroud ₂ , Robert H Edwards ₁

Affiliation

The role of glutamate in excitatory neurotransmission depends on its transport into synaptic vesicles by the vesicular glutamate transporters (VGLUTs). The three VGLUT isoforms exhibit a complementary distribution in the nervous system, and the knockout of each produces severe, pleiotropic neurological effects. However, the available pharmacology lacks sensitivity and specificity, limiting the analysis of both transport mechanism and physiological role. To develop new molecular probes for the VGLUTs, we raised six mouse monoclonal antibodies to VGLUT2. All six bind to a structured region of VGLUT2, five to the luminal face, and one to the cytosolic. Two are specific to VGLUT2, whereas the other four bind to both VGLUT1 and 2; none detect VGLUT3. Antibody 8E11 recognizes an epitope spanning the three extracellular loops in the C-domain that explains the recognition of both VGLUT1 and 2 but not VGLUT3. 8E11 also inhibits both glutamate transport and the VGLUT-associated chloride conductance. Since the antibody binds outside the substrate recognition site, it acts allosterically to inhibit function, presumably by restricting conformational changes. The isoform specificity also shows that allosteric inhibition provides a mechanism to distinguish between closely related transporters.

中文翻译：

异构体特异性抗体对囊泡谷氨酸转运蛋白的变构抑制

谷氨酸在兴奋性神经传递中的作用取决于其通过囊泡谷氨酸转运蛋白 (VGLUT) 转运到突触小泡中。三种 VGLUT 亚型在神经系统中表现出互补分布，并且每一种的敲除都会产生严重的多效性神经效应。然而，现有的药理学缺乏敏感性和特异性，限制了对转运机制和生理作用的分析。为了开发 VGLUT 的新分子探针，我们培养了六种针对 VGLUT2 的小鼠单克隆抗体。所有六个都与 VGLUT2 的结构化区域结合，五个与腔面结合，一个与细胞溶质结合。其中两个是 VGLUT2 特有的，而另外四个则同时与 VGLUT1 和 2 结合；没有检测到 VGLUT3。抗体 8E11 识别跨越 C 域中三个细胞外环的表位，这解释了识别 VGLUT1 和 2 但不识别 VGLUT3。8E11 还抑制谷氨酸转运和 VGLUT 相关的氯离子电导。由于抗体在底物识别位点外结合，它变构地抑制功能，可能是通过限制构象变化。同种型特异性还表明，变构抑制提供了一种机制来区分密切相关的转运蛋白。

更新日期：2021-08-17

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