AbstractBestrophin-1 (Best1) is an anion channel genetically linked to vision-threatening retinal degenerative channelopathies. Here, we identify interactions between Best1 and both isoforms of glutamic acid decarboxylases (GAD65 and GAD67), elucidate the distinctive influences of GAD65 and GAD67 on Best1’s permeability to various anions/neurotransmitters, discover the functionality of Best1 as a γ-Aminobutyric acid (GABA) type A receptor, and solve the structure of GABA-bound Best1.

摘要Bestrophin-1（Best1）是一种阴离子通道，与威胁视力的视网膜退行性通道病遗传相关。在这里，我们确定了Best1与谷氨酸脱羧酶（GAD65和GAD67）的两种同工型之间的相互作用，阐明了GAD65和GAD67对Best1对各种阴离子/神经递质的渗透性的独特影响，发现了Best1作为γ-氨基丁酸（GABA）a型受体的功能，并解决了GABA结合的Best1的结构。

GAD65 and GAD67 both promote Best1-mediated Cl− currents, but only GAD65 drastically enhances the permeability of Best1 to glutamate and GABA, for which GAD67 has no effect. GABA binds to Best1 on an extracellular site and stimulates Best1-mediated Cl− currents at the nano-molar concentration level.

GAD65和GAD67都能促进Best1介导的Cl-电流，但只有GAD65能显着增强Best1对谷氨酸和GABA的渗透性，而GAD67对此没有影响。GABA在细胞外位点与Best1结合，并在纳摩尔浓度水平上刺激Best1介导的Cl-电流。

The physiological role of GAD65 as a cell type-specific binding partner and facilitator of Best1 is demonstrated in retinal pigment epithelial cells. Together, our results reveal critical regulators of Best1 and inform a network of membrane transport metabolons formed between bestrophin channels and glutamate metabolic enzymes..

GAD65作为细胞类型特异性结合伴侣和Best1促进剂的生理作用在视网膜色素上皮细胞中得到证实。总之，我们的研究结果揭示了Best1的关键调节因子，并为bestrophin通道和谷氨酸代谢酶之间形成的膜转运代谢网络提供了信息。。

IntroductionThe human BEST1 gene encodes a Ca2+-activated anion channel (bestrophin-1, Best1) predominantly expressed in retinal pigment epithelium (RPE)1, and its genetic mutations, of which over 350 have been identified, cause a spectrum of at least five retinal degenerative diseases collectively known as bestrophinopathies2,3.

引言人类BEST1基因编码主要在视网膜色素上皮（RPE）1中表达的Ca2+激活的阴离子通道（bestrophin-1，BEST1），其基因突变已被鉴定出350多种，导致至少五种视网膜退行性疾病，统称为bestrophinopathies2,3。

The patients are susceptible to progressive vision loss which may eventually lead to blindness, and no treatments are currently available2,3,4. Therefore, understanding the physiological properties and cellular regulators of Best1 is critical for the development of treatment strategy for bestrophinopathies.A clinical feature manifested by Best1 mutation carriers is an abnormal electrooculogram (EOG) light peak (LP), which represents the depolarization of the basolateral membrane of RPE due to the activation of a Cl− conductance triggered by the increase of intracellular Ca2+ concentration ([Ca2+]i)5,6,7.

患者易患进行性视力丧失，最终可能导致失明，目前尚无治疗方法2,3,4。因此，了解Best1的生理特性和细胞调节剂对于开发bestrophinopathies的治疗策略至关重要。Best1突变携带者表现出的一个临床特征是异常眼电图（EOG）光峰（LP），它代表了RPE基底外侧膜的去极化，这是由于细胞内Ca2+浓度增加触发的Cl-电导的激活（[Ca2+]i）5,6,7。

We previously demonstrated that Best1 is the anion channel responsible for conducting this Ca2+-dependent Cl− currents in human RPE8,9,10,11. However, several lines of evidence strongly suggest the existence of uncharacterized RPE-specific facilitator(s) of Best1 in vivo.Firstly, although the currents in RPE cells conducted by endogenous or exogenously supplemented Best1 are similar to each other, they are significantly bigger compared to those conducted by transiently expressed Best1 in HEK293 cells8,9,12,13.

我们先前证明Best1是负离子通道，负责在人RPE8、9、10、11中传导这种Ca2+依赖性Cl-电流。然而，有几条证据强烈表明体内存在Best1的未表征RPE特异性促进因子。首先，尽管内源性或外源性补充的Best1在RPE细胞中传导的电流彼此相似，但与HEK293细胞中瞬时表达的Best1传导的电流相比，它们显着更大8,9,12,13。

Secondly, Best1 has been shown by cell-based approaches to mediate the transport of essential metabolites/neurotransmitters which are much bulkier in size compared to Cl−, such as glutamate in RPE14, as well as GABA and glutamate in astrocytes15,16,17,18,19,20. However, purified chicken Best1 is impermeable to glutamate21, while Ca2+-boun.

其次，基于细胞的方法已经证明Best1可以介导必需代谢物/神经递质的转运，这些代谢物/神经递质的体积比Cl-大得多，例如RPE14中的谷氨酸，以及星形胶质细胞中的GABA和谷氨酸15,16,17,18,19,20。然而，纯化的鸡Best1对谷氨酸不渗透21，而Ca2+-boun。

Data availability

数据可用性

The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-45915 (BEST1 + GABA open), EMD-45916 (BEST1 + GABA intermediate state 1), EMD-45917 (BEST1 + GABA intermediate state 2), EMD-45918 (BEST1 + GABA closed). The atomic coordinates have been deposited in the Protein Data Bank (PDB) under accession codes 9CTQ (BEST1 + GABA open), 9CTR (BEST1 + GABA intermediate state 1), 9CTS (BEST1 + GABA intermediate state 2), 9CTT (BEST1 + GABA closed).

低温电磁密度图已保存在电子显微镜数据库（EMDB）中，登录号为EMD-45915（BEST1+ GABA open），EMD-45916（BEST1+ GABA中间状态1），EMD-45917（BEST1+ GABA中间状态2），EMD-45918（BEST1+ GABA closed）。原子坐标已保存在蛋白质数据库（PDB）中，登录号为9CTQ（BEST1+ GABA open），9CTR（BEST1+ GABA中间状态1），9CTS（BEST1+ GABA中间状态2），9CTT（BEST1+ GABA closed）。

Previously published models 8D1O and 8D1I are used. The source data underlying Figs. 1–7 and Supplementary Fig. 2 are provided in a Source Data file. Source data are provided in this paper..

使用以前发布的型号8D1O和8D1I。图1-7和补充图2的基础源数据在源数据文件中提供。本文提供了源数据。。

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Kittredge, A., Ward, N., Hopiavuori, A., Zhang, Y. & Yang, T. Expression and purification of mammalian bestrophin ion channels. J Vis Exp. https://doi.org/10.3791/57832 (2018).Download referencesAcknowledgementsWe thank Renato Bruni at Center on Membrane Protein Production and Analysis (COMPPÅ) for providing the bacterial expression vector, and the Unrestricted Grant from Research to Prevent Blindness (RPB) to the Department of Ophthalmology at Columbia University.

Kittredge，A.，Ward，N.，Hopiavuori，A.，Zhang，Y。＆Yang，T。哺乳动物bestrophin离子通道的表达和纯化。J Vis Exp。https://doi.org/10.3791/57832（2018年）。下载参考文献致谢我们感谢膜蛋白生产和分析中心（COMPPÅ）的Renato Bruni提供了细菌表达载体，以及哥伦比亚大学眼科预防失明研究（RPB）的无限制拨款。

Cryo-EM data were collected within the Columbia Cryo-EM Core. T.Y. was supported by NIH grants R35GM149252, R01GM127652 and R24EY028758, the Irma T. Hirschl/Monique Weill-Caulier Research Award (CU20-4313) and RPB Career Advancement Award (CU22-1892).Author informationAuthor notesThese authors contributed equally: Jiali Wang, Aaron P.

低温电磁数据是在哥伦比亚低温电磁核心内收集的。T、 Y.得到了NIH拨款R35GM149252，R01GM127652和R24EY028758，Irma T.Hirschl/Monique Weill Caulier研究奖（CU20-4313）和RPB职业发展奖（CU22-1892）的支持。作者信息作者注意到这些作者做出了同样的贡献：王佳丽，Aaron P。

Owji.Authors and AffiliationsDepartment of Ophthalmology, Columbia University, New York, NY, USAJiali Wang, Aaron P. Owji, Alec Kittredge, Zada Clark, Yu Zhang & Tingting YangAuthorsJiali WangView author publicationsYou can also search for this author in.

奥吉。作者和附属机构哥伦比亚大学眼科，纽约，纽约，USAJiali Wang，Aaron P.Owji，Alec Kittredge，Zada Clark，Yu Zhang＆Tingting Yang作者JIALI WangView作者出版物您也可以在中搜索该作者。

PubMed Google ScholarAaron P. OwjiView author publicationsYou can also search for this author in

PubMed Google ScholarAaron P.OwjiView作者出版物您也可以在

PubMed Google ScholarAlec KittredgeView author publicationsYou can also search for this author in

PubMed Google ScholarAlec KittredgeView作者出版物您也可以在

PubMed Google ScholarZada ClarkView author publicationsYou can also search for this author in

PubMed Google ScholarZada ClarkView作者出版物您也可以在

PubMed Google ScholarYu ZhangView author publicationsYou can also search for this author in

PubMed Google ScholarYu ZhangView作者出版物您也可以在

PubMed Google ScholarTingting YangView author publicationsYou can also search for this author in

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PubMed Google ScholarContributionsJ.W. designed research, performed patch clamp recordings, analyzed data and made figures; A.P.O. designed research, performed protein purification and cryo-EM experiments, analyzed data, made figures and helped write the paper; A.K. designed research, generated constructs, purified proteins, performed immunoblotting and made figures; Z.C.

PubMed谷歌学术贡献。W、 设计研究，进行膜片钳记录，分析数据并制作数字；A、 P.O.设计研究，进行蛋白质纯化和冷冻电镜实验，分析数据，制作数字并帮助撰写论文；A、 K.设计研究，生成构建体，纯化蛋白质，进行免疫印迹并制作数字；Z、 C。

maintained HEK293 and RPE culture; Y.Z. and T.Y. designed research, analyzed data, made figures and wrote the paper.Corresponding authorsCorrespondence to.

维持HEK293和RPE培养；Y、 Z.和T.Y.设计研究，分析数据，绘制图表并撰写论文。通讯作者通讯。

Yu Zhang or Tingting Yang.Ethics declarations

张宇或杨婷婷。道德宣言

Competing interests

相互竞争的利益

Provisional patents on using GABA analogs (no. 63/591,019) and GAD proteins (63/692,462) as Best1 activators have been filled by Columbia University, listing A.P.O., Y.Z., and T.Y. as inventors for the former and Y.Z. and T.Y. as inventors for the latter. The remaining authors declare no competing interests..

哥伦比亚大学（Columbia University）已经申请了使用GABA类似物（编号63/591019）和GAD蛋白（63/692462）作为Best1激活剂的临时专利，其中A.P.O.、Y.Z.和T.Y.是前者和Y的发明者。Z、 和T.Y.作为后者的发明者。其余作者声明没有利益冲突。。

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Nature Communications thanks Tomohiro Nishizawa and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

《自然通讯》感谢西泽友弘和另一位匿名审稿人对这项工作的同行评审所做的贡献。可以获得同行评审文件。

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Reprints and permissionsAbout this articleCite this articleWang, J., Owji, A.P., Kittredge, A. et al. GAD65 tunes the functions of Best1 as a GABA receptor and a neurotransmitter conducting channel.

转载和许可本文引用本文Wang，J.，Owji，A.P.，Kittredge，A。等人。GAD65调节Best1作为GABA受体和神经递质传导通道的功能。

Nat Commun 15, 8051 (2024). https://doi.org/10.1038/s41467-024-52039-5Download citationReceived: 30 October 2023Accepted: 23 August 2024Published: 14 September 2024DOI: https://doi.org/10.1038/s41467-024-52039-5Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

《国家公社》158051（2024）。https://doi.org/10.1038/s41467-024-52039-5Download引文接收日期：2023年10月30日接收日期：2024年8月23日发布日期：2024年9月14日OI：https://doi.org/10.1038/s41467-024-52039-5Share本文与您共享以下链接的任何人都可以阅读此内容：获取可共享链接对不起，本文目前没有可共享的链接。复制到剪贴板。

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Chloride channelsCryoelectron microscopyIon transport

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