Neto proteins regulate gating of the kainate-type glutamate receptor GluK2 through two binding sites
Neto proteins regulate gating of the kainate-type glutamate receptor GluK2 through two binding sites
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Neto 蛋白通过两个结合位点调节红藻氨酸型谷氨酸受体 GluK2 的门控
DOI：
10.1074/jbc.ra119.008631
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发表时间：
2019-11-22
影响因子：
4.8
通讯作者：
Shi, Yun Stone
中科院分区：
文献类型：
--
作者：
Li, Yan-Jun;Duan, Gui-Fang;Shi, Yun Stone
神经纤毛蛋白和tolloid-like（Neto）蛋白Neto 1和Neto 2是海人酸型谷氨酸受体（KAR）的辅助亚基，其调节KAR运输和门控。然而，Netos如何结合和调节KAR的生物物理功能仍不清楚。在这里，我们发现谷氨酸受体离子型红藻氨酸2（GluK 2）的N-末端结构域（NTD）结合Neto蛋白的第一个补体C1 r/C1 s-Uegf-BMP（CUB）结构域（即NTD-CUB 1相互作用），并且GluK 2的核心（GluK 2 ΔNTD）通过CUB 1 s以外的结构域（核心-Neto相互作用）结合Netos。在HEK 293 T细胞中使用电生理学分析，我们研究了这些相互作用对GluK 2门控的影响，包括失活、脱敏和从脱敏中恢复。我们发现，NTD删除不影响GluK 2快速门控动力学，脱敏，和失活。我们还观察到Neto 1和Neto 2差异调节GluK 2快速门控动力学，这在很大程度上依赖于NTD-CUB 1相互作用。NTD去除促进GluK 2从脱敏中恢复，表明NTD稳定GluK 2脱敏状态。与Neto 1或Neto 2的共表达也加速了GluK 2从脱敏中的恢复，这完全依赖于NTD-CUB 1的相互作用。此外，我们表明，NTD-CUB 1相互作用涉及带正电荷的残基之间的电吸引力在GluK2_NTD和带负电荷的CUB 1域。这些电荷的中和消除了NTD-CUB 1相互作用对GluK 2门控的调节作用。我们的结论是，KARs结合Netos通过至少两个网站和NTD-CUB 1相互作用的关键调节Neto介导的GluK 2门控。
The neuropilin and tolloid-like (Neto) proteins Neto1 and Neto2 are auxiliary subunits of kainate-type glutamate receptors (KARs) that regulate KAR trafficking and gating. However, how Netos bind and regulate the biophysical functions of KARs remains unclear. Here, we found that the N-terminal domain (NTD) of glutamate receptor ionotropic kainate 2 (GluK2) binds the first complement C1r/C1s-Uegf-BMP (CUB) domain of Neto proteins (i.e. NTD-CUB1 interaction) and that the core of GluK2 (GluK2ΔNTD) binds Netos through domains other than CUB1s (core-Neto interaction). Using electrophysiological analysis in HEK293T cells, we examined the effects of these interactions on GluK2 gating, including deactivation, desensitization, and recovery from desensitization. We found that NTD deletion does not affect GluK2 fast gating kinetics, the desensitization, and the deactivation. We also observed that Neto1 and Neto2 differentially regulate GluK2 fast gating kinetics, which largely rely on the NTD-CUB1 interactions. NTD removal facilitated GluK2 recovery from desensitization, indicating that the NTD stabilizes the GluK2 desensitization state. Co-expression with Neto1 or Neto2 also accelerated GluK2 recovery from desensitization, which fully relied on the NTD-CUB1 interactions. Moreover, we demonstrate that the NTD-CUB1 interaction involves electric attraction between positively charged residues in the GluK2_NTD and negatively charged ones in the CUB1 domains. Neutralization of these charges eliminated the regulatory effects of the NTD-CUB1 interaction on GluK2 gating. We conclude that KARs bind Netos through at least two sites and that the NTD-CUB1 interaction critically regulates Neto-mediated GluK2 gating.