Allosteric Inhibition of a Vesicular Glutamate Transporter by an Isoform-Specific Antibody.
Allosteric Inhibition of a Vesicular Glutamate Transporter by an Isoform-Specific Antibody.
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DOI：
10.1021/acs.biochem.1c00375
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发表时间：
2021-08-17
期刊：
影响因子：
2.9
通讯作者：
Edwards RH
中科院分区：
文献类型：
--
作者：
Eriksen J;Li F;Stroud RM;Edwards RH
谷氨酸在兴奋性神经传递中的作用依赖于其通过囊泡谷氨酸转运体（VGLUT）转运到突触囊泡中。三种VGLUT亚型在神经系统中表现出互补的分布，并且每种亚型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。为了开发新的VGLUT分子探针，我们提出了六个小鼠单克隆抗体VGLUT 2。所有六个结合到VGLUT 2的结构化区域，五个结合到腔面，一个结合到胞质。其中两个是特异性的VGLUT 2，而其他四个结合VGLUT 1和2;没有检测VGLUT 3。抗体8 E11识别跨越C结构域中三个胞外环的表位，这解释了VGLUT 1和2的识别，但不识别VGLUT 3。8 E11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，因此它可能通过限制构象变化来变构地抑制功能。同种型特异性还表明，变构抑制提供了一种区分密切相关的转运蛋白的机制。
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.
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影响因子：
4.8
作者：
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通讯作者：
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