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Proline Residues Contribute to Efficient GABAp Receptor Function
ACS Chemical Neuroscience ( IF 4.1 ) Pub Date : 2020-11-17 , DOI: 10.1021/acschemneuro.0c00483
Benjamin W Baker 1 , Dennis A Dougherty 2 , Sarah C R Lummis 1
Affiliation  

GABAp receptors are homomeric pentameric ligand-gated ion channels (pLGICs) and are useful for probing the molecular details of the mechanism of action in this important protein family. Here, we explore the role of proline (Pro) residues by creating mutant receptors, expressing them in HEK293 cells, and using fluorescent membrane potential sensitive dye to monitor receptor activity. The data revealed that 3 of the Pro-to-alanine substitutions resulted in nonfunctional receptors (one in the Cys-loop, one in loop A and one in the β2-β3 loop), 7 resulted in increased EC50 values, and the remaining 13 resulted in receptors with properties similar to WT receptors. Further exploration of the nonfunctional mutants using expression in Xenopus laevis oocytes and whole-cell voltage-clamp electrophysiology, incorporating both canonical and noncanonical amino acids, revealed that the Pro in the Cys-loop had a preference for analogues with a high intrinsic cis bias, the Pro in loop A required a ring, and the β2-β3 loop Pro contributes to expression. The data from the Cys-loop Pro are consistent with those from other pLGICs, while those of the loop A Pro and some of the other Pros surprisingly differ. Thus, overall, the data clarify the roles of many of the Pros in the GABAp receptor and also suggest that caution must be applied in using data from one receptor to understand molecular details of all pLGICs.

中文翻译:

脯氨酸残基有助于高效 GABAp 受体功能

GABA p受体是同聚五聚体配体门控离子通道 (pLGIC),可用于探索这一重要蛋白质家族中作用机制的分子细节。在这里,我们通过创建突变受体、在 HEK293 细胞中表达它们以及使用荧光膜电位敏感染料监测受体活性来探索脯氨酸 (Pro) 残基的作用。数据显示,3 个 Pro-to-丙氨酸取代导致无功能受体(一个在 Cys 环中,一个在环 A 中,一个在 β2-β3 环中),7 个导致 EC 50值增加,其余的13 产生的受体具有与 WT 受体相似的特性。利用非洲爪蟾的表达进一步探索非功能性突变体结合经典和非经典氨基酸的卵母细胞和全细胞电压钳电生理学显示,Cys 环中的 Pro 偏爱具有高固有顺式偏倚的类似物,环 A 中的 Pro 需要一个环,而β2-β3 loop Pro 有助于表达。Cys-loop Pro 的数据与其他 pLGIC 的数据一致,而 loop A Pro 和其他一些 Pro 的数据却出人意料地不同。因此,总体而言,数据阐明了许多 Pros 在 GABA p受体中的作用,并且还表明在使用来自一种受体的数据来了解所有 pLGIC 的分子细节时必须谨慎。
更新日期:2020-12-16
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