GABAA 受体错义变异体的致病性预测

IF 2.3 4区 化学 Q3 CHEMISTRY, MULTIDISCIPLINARY
Ya-Juan Wang, Giang H. Vu, Ting-Wei Mu
{"title":"GABAA 受体错义变异体的致病性预测","authors":"Ya-Juan Wang, Giang H. Vu, Ting-Wei Mu","doi":"10.1002/ijch.202300161","DOIUrl":null,"url":null,"abstract":"<h2> Introduction</h2>\n<p>Epilepsy is one of the most common neurological diseases in the world with a broad phenotypic spectrum.<span><sup>1</sup></span> Recent advances in genome sequencing identified an increasing number of genes that are associated with epilepsy.<span><sup>2</sup></span> According to protein functions, epilepsy-associated genes can be grouped to ion channels, enzymes and enzyme modulators, transports and receptors, and others.<span><sup>3</sup></span> Genetic epilepsy is often linked to developmental delay, movement disorder, and other comorbidities.<span><sup>4</sup></span> Due to the important role of neurotransmitter-gated ion channels in controlling the excitation-inhibition balance in the central nervous system, genes encoding these ion channels, including excitatory N-methyl-D-aspartate (NMDA) receptors and inhibitory γ-aminobutyric acid type A (GABA<sub>A</sub>) receptors, are recognized as prominent epilepsy-causing genes.<span><sup>5</sup></span> Here, we focus on GABA<sub>A</sub> receptors, the primary inhibitory neurotransmitter-gated ion channels in the human brain.<span><sup>6</sup></span> They mediate the fast inhibitory GABA-induced chloride currents and hyperpolarize the postsynaptic membranes to reduce neuronal firing.</p>\n<p>Proteostasis maintenance of GABA<sub>A</sub> receptors is essential for their function in the central nervous system.<span><sup>7</sup></span> GABA<sub>A</sub> receptors are assembled as pentamers from a specific combination of 19 subunits, including α1-α6 (GABRA1-A6), β1-β3 (GABRB1-B3), γ1-γ3 (GABRG1-G3), δ (GABRD), ϵ (GABRE), θ (GABRQ), π (GABRP), and ρ1-ρ3 (GABRR1-R3). The distribution of GABA<sub>A</sub> receptors is throughout the brain regions, and the most abundant subtype is composed of two α1 subunits, two β2 subunits, and one γ2 subunit.<span><sup>8</sup></span> To function, GABA<sub>A</sub> receptor subunits need to fold in the endoplasmic reticulum (ER) with the assistance of molecular chaperones and subsequently assemble with other subunits to form heteropentamers. The properly assembled receptors exit the ER and traffic to the plasma membrane to act as chloride channels. Unassembled and misfolded subunits are retained in the ER, which could be routed to the degradation pathway by the ER-associated degradation.<span><sup>9</sup></span> Recent quantitative proteomics analysis identified the proteostasis network that regulates the folding, assembly, trafficking, and degradation of GABA<sub>A</sub> receptors.<span><sup>10</sup></span></p>\n<p>Recent cryo-electron microscopy (cryo-EM) studies solved the high-resolution structures of pentameric GABA<sub>A</sub> receptors, including α1β2γ2 receptors<span><sup>11</sup></span> and α1β3γ2 receptors.<span><sup>12</sup></span> The pentameric receptors are arranged as β-α1-β-α1-γ2 counterclockwise when viewed from the synaptic cleft (Figure 1A). Each pentamer has two binding sites for the neurotransmitter, GABA, at the interfaces between β subunits and α1 subunits. Residues from β subunits constitute the principal binding site, denoted as “positive” (+) side, whereas residues from α1 subunits constitute the complementary binding site, denoted as “negative” (−) side. Each subunit shares a common structural scaffold, including a large extracellular N-terminal domain (NTD), four transmembrane helices (TM1-TM4), and loops connecting transmembrane helices (a short intracellular TM1–2 loop, a short extracellular TM2–3 loop, and a long intracellular TM3–4 loop), and a short extracellular C-terminus (Figure 1B, 1C). The secondary structures of the NTD contain two α-helices, ten β-sheets (β1-β10), and connecting loops (Figure 1C, 1D). GABA<sub>A</sub> receptors belong to the Cys-loop receptor superfamily.<span><sup>7</sup></span> The signature Cys-loop in GABA<sub>A</sub> receptor subunits is designated as loop 7. Biochemical studies revealed that several segments in GABA<sub>A</sub> receptor subunits play an important role in binding the ligand: the binding loops in the principal side are called loop A–C, whereas the binding loops in the complementary side are called loop D–F (Figure 1C, 1D).\n</p>\n<figure><picture>\n<source media=\"(min-width: 1650px)\" srcset=\"/cms/asset/bea630e5-c7bf-4e25-942a-1534d6a58d6b/ijch202300161-fig-0001-m.jpg\"/><img alt=\"Details are in the caption following the image\" data-lg-src=\"/cms/asset/bea630e5-c7bf-4e25-942a-1534d6a58d6b/ijch202300161-fig-0001-m.jpg\" loading=\"lazy\" src=\"/cms/asset/aee6a2ea-37a8-4a49-9335-ac2ce9a50873/ijch202300161-fig-0001-m.png\" title=\"Details are in the caption following the image\"/></picture><figcaption>\n<div><strong>Figure 1</strong><div>Open in figure viewer<i aria-hidden=\"true\"></i><span>PowerPoint</span></div>\n</div>\n<div>\n<p>Structures and sequence alignment of GABA<sub>A</sub> receptors. (A) Cartoon representation of pentameric α1βγ2 receptors, built from 6X3S.pdb. The principal side of one subunit is denoted as “+”, whereas the complementary side of one subunit is denoted as “−”. (B) The schematic of the primary protein sequence of a GABA<sub>A</sub> receptor subunit. NTD, N-terminal domain; M1-M4, transmembrane helices 1 to 4. (C) The secondary structures of a GABA<sub>A</sub> receptor subunit. The two cysteines in the signature Cys-loop are colored in yellow. (D) The sequence alignment of major human GABA<sub>A</sub> receptor subunits, including α1, β2, β3, and γ2. The residue positions that harbor clinical missense variants are highlighted. According to ClinVar annotation, pathogenic variants are colored in red, uncertain in yellow, and benign in green.</p></div>\n</figcaption>\n</figure>\n<p>To date, over 1000 clinical variants in genes encoding GABA<sub>A</sub> receptor subunits have been recorded in ClinVar (www.clinvar.com), including missense, nonsense, and frameshift variants. However, the clinical significance of these variants is not adequately addressed since most of them lack functional characterization and many of them are classified as uncertain or conflicting interpretations. For the limited number of characterized GABA<sub>A</sub> receptor variants, accumulating evidence indicated that proteostasis deficiency that resulted from misfolding and excessive degradation of the variants is a major disease-causing mechanism.<span><sup>13</sup></span> Adapting the ER proteostasis network pharmacologically corrected the misfolding and restored the surface trafficking and thus ion channel function for a variety of pathogenic GABA<sub>A</sub> receptor variants.<span><sup>14</sup></span> Another important disease-causing mechanism is that the missense variants result in the channel gating defects and altered electrophysiological properties, such as current kinetics, current amplitude, and ligand potency.</p>\n<p>Here, we applied two state-of-the-art modeling tools, namely AlphaMissense<span><sup>15</sup></span> and Rhapsody,<span><sup>16</sup></span> to comprehensively predict the pathogenicity of saturating missense variants in the major subunits of GABA<sub>A</sub> receptors (α1, β2, β3, and γ2). AlphaMissense represents a major technological advance in the field of the prediction of missense variants by integrating structural context and evolutionary conservation.<span><sup>15</sup></span> Other machine learning-based prediction approaches have limitations on the training databases, which are prone to human bias,<span><sup>17</sup></span> lack of precise structural information,<span><sup>18</sup></span> or inadequate genetic evolutionary constraint.<span><sup>19</sup></span> AlphaMissense overcomes these limitations by combining the following strengths. First, AlphaMissense utilizes the training datasets with weak labels from population frequency data; second, AlphaMissense fine-tunes highly accurate protein structures afforded by AlphaFold;<span><sup>20</sup></span> third, AlphaMissense is capable of learning evolutionary constraints according to amino acid sequences. Consequently, AlphaMissense outperforms other prediction models across multiple clinical benchmarks.<span><sup>15</sup></span> Recently, AlphaMissense has been applied to predict pathogenicity of cystic fibrosis transmembrane conductance regulator (CFTR) variants and the results correlated well with certain clinical benchmarks.<span><sup>21</sup></span> Here, we also compared the AlphaMissense and Rhapsody predictions with the ClinVar clinical benchmarks, aiming to provide insights for clinical interpretation and guidance for future experimental investigation of missense variants in GABA<sub>A</sub> receptors.</p>","PeriodicalId":14686,"journal":{"name":"Israel Journal of Chemistry","volume":"33 1","pages":""},"PeriodicalIF":2.3000,"publicationDate":"2024-01-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Pathogenicity Prediction of GABAA Receptor Missense Variants\",\"authors\":\"Ya-Juan Wang, Giang H. Vu, Ting-Wei Mu\",\"doi\":\"10.1002/ijch.202300161\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<h2> Introduction</h2>\\n<p>Epilepsy is one of the most common neurological diseases in the world with a broad phenotypic spectrum.<span><sup>1</sup></span> Recent advances in genome sequencing identified an increasing number of genes that are associated with epilepsy.<span><sup>2</sup></span> According to protein functions, epilepsy-associated genes can be grouped to ion channels, enzymes and enzyme modulators, transports and receptors, and others.<span><sup>3</sup></span> Genetic epilepsy is often linked to developmental delay, movement disorder, and other comorbidities.<span><sup>4</sup></span> Due to the important role of neurotransmitter-gated ion channels in controlling the excitation-inhibition balance in the central nervous system, genes encoding these ion channels, including excitatory N-methyl-D-aspartate (NMDA) receptors and inhibitory γ-aminobutyric acid type A (GABA<sub>A</sub>) receptors, are recognized as prominent epilepsy-causing genes.<span><sup>5</sup></span> Here, we focus on GABA<sub>A</sub> receptors, the primary inhibitory neurotransmitter-gated ion channels in the human brain.<span><sup>6</sup></span> They mediate the fast inhibitory GABA-induced chloride currents and hyperpolarize the postsynaptic membranes to reduce neuronal firing.</p>\\n<p>Proteostasis maintenance of GABA<sub>A</sub> receptors is essential for their function in the central nervous system.<span><sup>7</sup></span> GABA<sub>A</sub> receptors are assembled as pentamers from a specific combination of 19 subunits, including α1-α6 (GABRA1-A6), β1-β3 (GABRB1-B3), γ1-γ3 (GABRG1-G3), δ (GABRD), ϵ (GABRE), θ (GABRQ), π (GABRP), and ρ1-ρ3 (GABRR1-R3). The distribution of GABA<sub>A</sub> receptors is throughout the brain regions, and the most abundant subtype is composed of two α1 subunits, two β2 subunits, and one γ2 subunit.<span><sup>8</sup></span> To function, GABA<sub>A</sub> receptor subunits need to fold in the endoplasmic reticulum (ER) with the assistance of molecular chaperones and subsequently assemble with other subunits to form heteropentamers. The properly assembled receptors exit the ER and traffic to the plasma membrane to act as chloride channels. Unassembled and misfolded subunits are retained in the ER, which could be routed to the degradation pathway by the ER-associated degradation.<span><sup>9</sup></span> Recent quantitative proteomics analysis identified the proteostasis network that regulates the folding, assembly, trafficking, and degradation of GABA<sub>A</sub> receptors.<span><sup>10</sup></span></p>\\n<p>Recent cryo-electron microscopy (cryo-EM) studies solved the high-resolution structures of pentameric GABA<sub>A</sub> receptors, including α1β2γ2 receptors<span><sup>11</sup></span> and α1β3γ2 receptors.<span><sup>12</sup></span> The pentameric receptors are arranged as β-α1-β-α1-γ2 counterclockwise when viewed from the synaptic cleft (Figure 1A). Each pentamer has two binding sites for the neurotransmitter, GABA, at the interfaces between β subunits and α1 subunits. Residues from β subunits constitute the principal binding site, denoted as “positive” (+) side, whereas residues from α1 subunits constitute the complementary binding site, denoted as “negative” (−) side. Each subunit shares a common structural scaffold, including a large extracellular N-terminal domain (NTD), four transmembrane helices (TM1-TM4), and loops connecting transmembrane helices (a short intracellular TM1–2 loop, a short extracellular TM2–3 loop, and a long intracellular TM3–4 loop), and a short extracellular C-terminus (Figure 1B, 1C). The secondary structures of the NTD contain two α-helices, ten β-sheets (β1-β10), and connecting loops (Figure 1C, 1D). GABA<sub>A</sub> receptors belong to the Cys-loop receptor superfamily.<span><sup>7</sup></span> The signature Cys-loop in GABA<sub>A</sub> receptor subunits is designated as loop 7. Biochemical studies revealed that several segments in GABA<sub>A</sub> receptor subunits play an important role in binding the ligand: the binding loops in the principal side are called loop A–C, whereas the binding loops in the complementary side are called loop D–F (Figure 1C, 1D).\\n</p>\\n<figure><picture>\\n<source media=\\\"(min-width: 1650px)\\\" srcset=\\\"/cms/asset/bea630e5-c7bf-4e25-942a-1534d6a58d6b/ijch202300161-fig-0001-m.jpg\\\"/><img alt=\\\"Details are in the caption following the image\\\" data-lg-src=\\\"/cms/asset/bea630e5-c7bf-4e25-942a-1534d6a58d6b/ijch202300161-fig-0001-m.jpg\\\" loading=\\\"lazy\\\" src=\\\"/cms/asset/aee6a2ea-37a8-4a49-9335-ac2ce9a50873/ijch202300161-fig-0001-m.png\\\" title=\\\"Details are in the caption following the image\\\"/></picture><figcaption>\\n<div><strong>Figure 1</strong><div>Open in figure viewer<i aria-hidden=\\\"true\\\"></i><span>PowerPoint</span></div>\\n</div>\\n<div>\\n<p>Structures and sequence alignment of GABA<sub>A</sub> receptors. (A) Cartoon representation of pentameric α1βγ2 receptors, built from 6X3S.pdb. The principal side of one subunit is denoted as “+”, whereas the complementary side of one subunit is denoted as “−”. (B) The schematic of the primary protein sequence of a GABA<sub>A</sub> receptor subunit. NTD, N-terminal domain; M1-M4, transmembrane helices 1 to 4. (C) The secondary structures of a GABA<sub>A</sub> receptor subunit. The two cysteines in the signature Cys-loop are colored in yellow. (D) The sequence alignment of major human GABA<sub>A</sub> receptor subunits, including α1, β2, β3, and γ2. The residue positions that harbor clinical missense variants are highlighted. According to ClinVar annotation, pathogenic variants are colored in red, uncertain in yellow, and benign in green.</p></div>\\n</figcaption>\\n</figure>\\n<p>To date, over 1000 clinical variants in genes encoding GABA<sub>A</sub> receptor subunits have been recorded in ClinVar (www.clinvar.com), including missense, nonsense, and frameshift variants. However, the clinical significance of these variants is not adequately addressed since most of them lack functional characterization and many of them are classified as uncertain or conflicting interpretations. For the limited number of characterized GABA<sub>A</sub> receptor variants, accumulating evidence indicated that proteostasis deficiency that resulted from misfolding and excessive degradation of the variants is a major disease-causing mechanism.<span><sup>13</sup></span> Adapting the ER proteostasis network pharmacologically corrected the misfolding and restored the surface trafficking and thus ion channel function for a variety of pathogenic GABA<sub>A</sub> receptor variants.<span><sup>14</sup></span> Another important disease-causing mechanism is that the missense variants result in the channel gating defects and altered electrophysiological properties, such as current kinetics, current amplitude, and ligand potency.</p>\\n<p>Here, we applied two state-of-the-art modeling tools, namely AlphaMissense<span><sup>15</sup></span> and Rhapsody,<span><sup>16</sup></span> to comprehensively predict the pathogenicity of saturating missense variants in the major subunits of GABA<sub>A</sub> receptors (α1, β2, β3, and γ2). AlphaMissense represents a major technological advance in the field of the prediction of missense variants by integrating structural context and evolutionary conservation.<span><sup>15</sup></span> Other machine learning-based prediction approaches have limitations on the training databases, which are prone to human bias,<span><sup>17</sup></span> lack of precise structural information,<span><sup>18</sup></span> or inadequate genetic evolutionary constraint.<span><sup>19</sup></span> AlphaMissense overcomes these limitations by combining the following strengths. First, AlphaMissense utilizes the training datasets with weak labels from population frequency data; second, AlphaMissense fine-tunes highly accurate protein structures afforded by AlphaFold;<span><sup>20</sup></span> third, AlphaMissense is capable of learning evolutionary constraints according to amino acid sequences. Consequently, AlphaMissense outperforms other prediction models across multiple clinical benchmarks.<span><sup>15</sup></span> Recently, AlphaMissense has been applied to predict pathogenicity of cystic fibrosis transmembrane conductance regulator (CFTR) variants and the results correlated well with certain clinical benchmarks.<span><sup>21</sup></span> Here, we also compared the AlphaMissense and Rhapsody predictions with the ClinVar clinical benchmarks, aiming to provide insights for clinical interpretation and guidance for future experimental investigation of missense variants in GABA<sub>A</sub> receptors.</p>\",\"PeriodicalId\":14686,\"journal\":{\"name\":\"Israel Journal of Chemistry\",\"volume\":\"33 1\",\"pages\":\"\"},\"PeriodicalIF\":2.3000,\"publicationDate\":\"2024-01-26\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Israel Journal of Chemistry\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://doi.org/10.1002/ijch.202300161\",\"RegionNum\":4,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"CHEMISTRY, MULTIDISCIPLINARY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Israel Journal of Chemistry","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1002/ijch.202300161","RegionNum":4,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"CHEMISTRY, MULTIDISCIPLINARY","Score":null,"Total":0}
引用次数: 0

摘要

导言癫痫是世界上最常见的神经系统疾病之一,具有广泛的表型谱1。4 由于神经递质门控离子通道在控制中枢神经系统兴奋-抑制平衡中的重要作用,编码这些离子通道(包括兴奋性 N-甲基-D-天冬氨酸(NMDA)受体和抑制性γ-氨基丁酸 A 型(GABAA)受体)的基因被认为是主要的癫痫致病基因。GABAA 受体是人脑中主要的抑制性神经递质门控离子通道。6 GABAA 受体介导由 GABA 诱导的快速抑制性氯离子电流,并使突触后膜超极化,从而降低神经元的发射。GABAA 受体是由α1-α6(GABRA1-A6)、β1-β3(GABRB1-B3)、γ1-γ3(GABRG1-G3)、δ(GABRD)、ϵ(GABRE)、θ(GABRQ)、π(GABRP)和ρ1-ρ3(GABRR1-R3)等 19 个亚基的特定组合而成的五聚体。GABAA 受体分布在整个脑区,最丰富的亚型由两个 α1 亚基、两个 β2 亚基和一个 γ2 亚基组成。8 GABAA 受体亚基需要在分子伴侣的帮助下在内质网(ER)中折叠,然后与其他亚基组装成异源五聚体。正确组装的受体离开 ER,进入质膜,发挥氯离子通道的作用。未组装和折叠错误的亚基被保留在 ER 中,可通过 ER 相关降解作用进入降解途径。9 最近的定量蛋白质组学分析确定了调控 GABAA 受体折叠、组装、运输和降解的蛋白质稳态网络。最近的低温电子显微镜(cryo-EM)研究解决了五聚体 GABAA 受体(包括 α1β2γ2 受体11 和 α1β3γ2 受体12 )的高分辨率结构。每个五聚体在 β 亚基和 α1 亚基之间的界面上都有两个神经递质 GABA 的结合位点。来自 β 亚基的残基构成主要结合位点,称为 "正"(+)面,而来自 α1 亚基的残基构成互补结合位点,称为 "负"(-)面。每个亚基都有一个共同的结构支架,包括一个大的胞外 N 端结构域(NTD)、四个跨膜螺旋(TM1-TM4)、连接跨膜螺旋的环路(一个短的胞内 TM1-2 环路、一个短的胞外 TM2-3 环路和一个长的胞内 TM3-4 环路)以及一个短的胞外 C 端(图 1B、1C)。NTD 的二级结构包括两个 α-螺旋、十个 β-片(β1-β10)和连接环(图 1C、1D)。GABAA 受体属于 Cys 环状受体超家族7 。生化研究发现,GABAA 受体亚基中的几个片段在与配体结合时起着重要作用:主侧的结合环称为环 A-C,而互补侧的结合环称为环 D-F(图 1C、1D)。(A)根据 6X3S.pdb 构建的五聚体 α1βγ2 受体的图示。一个亚基的主侧表示为 "+",而一个亚基的互补侧表示为"-"。(B) GABAA 受体亚基的主要蛋白质序列示意图。NTD,N-末端结构域;M1-M4,跨膜螺旋 1 至 4。 (C) GABAA 受体亚基的二级结构。标志性 Cys 环中的两个半胱氨酸用黄色标出。(D) 人类 GABAA 受体主要亚基的序列比对,包括 α1、β2、β3 和 γ2。含有临床错义变异的残基位置高亮显示。根据 ClinVar 的注释,致病变异用红色表示,不确定变异用黄色表示,良性变异用绿色表示。迄今为止,ClinVar (www.clinvar.com) 已记录了超过 1000 个编码 GABAA 受体亚基的基因中的临床变异,包括错义、无义和框移变异。然而,由于这些变异大多缺乏功能特征描述,而且许多变异被归类为不确定或相互矛盾的解释,因此这些变异的临床意义并未得到充分探讨。 对于数量有限的 GABAA 受体变体,不断积累的证据表明,变体的错误折叠和过度降解导致的蛋白稳态缺陷是主要的致病机制。另一个重要的致病机制是,错义变体导致通道门控缺陷和电生理特性改变,如电流动力学、电流振幅和配体效力。在这里,我们应用了两种最先进的建模工具,即 AlphaMissense15 和 Rhapsody16,来全面预测 GABAA 受体主要亚基(α1、β2、β3 和 γ2)中饱和错义变体的致病性。AlphaMissense 结合了结构背景和进化保护,是错义变异预测领域的一项重大技术进步。15 其他基于机器学习的预测方法在训练数据库方面存在局限性,容易出现人为偏差、17 缺乏精确的结构信息18 或遗传进化约束不足19 等问题。首先,AlphaMissense 利用了来自种群频率数据的弱标签训练数据集;其次,AlphaMissense 对 AlphaFold 提供的高精度蛋白质结构进行了微调;20 第三,AlphaMissense 能够根据氨基酸序列学习进化约束。最近,AlphaMissense 被应用于预测囊性纤维化跨膜传导调节器(CFTR)变体的致病性,其结果与某些临床基准有很好的相关性21。在此,我们还将 AlphaMissense 和 Rhapsody 的预测结果与 ClinVar 临床基准进行了比较,旨在为临床解释提供见解,并为未来 GABAA 受体错义变体的实验研究提供指导。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Pathogenicity Prediction of GABAA Receptor Missense Variants

Pathogenicity Prediction of GABAA Receptor Missense Variants

Introduction

Epilepsy is one of the most common neurological diseases in the world with a broad phenotypic spectrum.1 Recent advances in genome sequencing identified an increasing number of genes that are associated with epilepsy.2 According to protein functions, epilepsy-associated genes can be grouped to ion channels, enzymes and enzyme modulators, transports and receptors, and others.3 Genetic epilepsy is often linked to developmental delay, movement disorder, and other comorbidities.4 Due to the important role of neurotransmitter-gated ion channels in controlling the excitation-inhibition balance in the central nervous system, genes encoding these ion channels, including excitatory N-methyl-D-aspartate (NMDA) receptors and inhibitory γ-aminobutyric acid type A (GABAA) receptors, are recognized as prominent epilepsy-causing genes.5 Here, we focus on GABAA receptors, the primary inhibitory neurotransmitter-gated ion channels in the human brain.6 They mediate the fast inhibitory GABA-induced chloride currents and hyperpolarize the postsynaptic membranes to reduce neuronal firing.

Proteostasis maintenance of GABAA receptors is essential for their function in the central nervous system.7 GABAA receptors are assembled as pentamers from a specific combination of 19 subunits, including α1-α6 (GABRA1-A6), β1-β3 (GABRB1-B3), γ1-γ3 (GABRG1-G3), δ (GABRD), ϵ (GABRE), θ (GABRQ), π (GABRP), and ρ1-ρ3 (GABRR1-R3). The distribution of GABAA receptors is throughout the brain regions, and the most abundant subtype is composed of two α1 subunits, two β2 subunits, and one γ2 subunit.8 To function, GABAA receptor subunits need to fold in the endoplasmic reticulum (ER) with the assistance of molecular chaperones and subsequently assemble with other subunits to form heteropentamers. The properly assembled receptors exit the ER and traffic to the plasma membrane to act as chloride channels. Unassembled and misfolded subunits are retained in the ER, which could be routed to the degradation pathway by the ER-associated degradation.9 Recent quantitative proteomics analysis identified the proteostasis network that regulates the folding, assembly, trafficking, and degradation of GABAA receptors.10

Recent cryo-electron microscopy (cryo-EM) studies solved the high-resolution structures of pentameric GABAA receptors, including α1β2γ2 receptors11 and α1β3γ2 receptors.12 The pentameric receptors are arranged as β-α1-β-α1-γ2 counterclockwise when viewed from the synaptic cleft (Figure 1A). Each pentamer has two binding sites for the neurotransmitter, GABA, at the interfaces between β subunits and α1 subunits. Residues from β subunits constitute the principal binding site, denoted as “positive” (+) side, whereas residues from α1 subunits constitute the complementary binding site, denoted as “negative” (−) side. Each subunit shares a common structural scaffold, including a large extracellular N-terminal domain (NTD), four transmembrane helices (TM1-TM4), and loops connecting transmembrane helices (a short intracellular TM1–2 loop, a short extracellular TM2–3 loop, and a long intracellular TM3–4 loop), and a short extracellular C-terminus (Figure 1B, 1C). The secondary structures of the NTD contain two α-helices, ten β-sheets (β1-β10), and connecting loops (Figure 1C, 1D). GABAA receptors belong to the Cys-loop receptor superfamily.7 The signature Cys-loop in GABAA receptor subunits is designated as loop 7. Biochemical studies revealed that several segments in GABAA receptor subunits play an important role in binding the ligand: the binding loops in the principal side are called loop A–C, whereas the binding loops in the complementary side are called loop D–F (Figure 1C, 1D).

Details are in the caption following the image
Figure 1
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Structures and sequence alignment of GABAA receptors. (A) Cartoon representation of pentameric α1βγ2 receptors, built from 6X3S.pdb. The principal side of one subunit is denoted as “+”, whereas the complementary side of one subunit is denoted as “−”. (B) The schematic of the primary protein sequence of a GABAA receptor subunit. NTD, N-terminal domain; M1-M4, transmembrane helices 1 to 4. (C) The secondary structures of a GABAA receptor subunit. The two cysteines in the signature Cys-loop are colored in yellow. (D) The sequence alignment of major human GABAA receptor subunits, including α1, β2, β3, and γ2. The residue positions that harbor clinical missense variants are highlighted. According to ClinVar annotation, pathogenic variants are colored in red, uncertain in yellow, and benign in green.

To date, over 1000 clinical variants in genes encoding GABAA receptor subunits have been recorded in ClinVar (www.clinvar.com), including missense, nonsense, and frameshift variants. However, the clinical significance of these variants is not adequately addressed since most of them lack functional characterization and many of them are classified as uncertain or conflicting interpretations. For the limited number of characterized GABAA receptor variants, accumulating evidence indicated that proteostasis deficiency that resulted from misfolding and excessive degradation of the variants is a major disease-causing mechanism.13 Adapting the ER proteostasis network pharmacologically corrected the misfolding and restored the surface trafficking and thus ion channel function for a variety of pathogenic GABAA receptor variants.14 Another important disease-causing mechanism is that the missense variants result in the channel gating defects and altered electrophysiological properties, such as current kinetics, current amplitude, and ligand potency.

Here, we applied two state-of-the-art modeling tools, namely AlphaMissense15 and Rhapsody,16 to comprehensively predict the pathogenicity of saturating missense variants in the major subunits of GABAA receptors (α1, β2, β3, and γ2). AlphaMissense represents a major technological advance in the field of the prediction of missense variants by integrating structural context and evolutionary conservation.15 Other machine learning-based prediction approaches have limitations on the training databases, which are prone to human bias,17 lack of precise structural information,18 or inadequate genetic evolutionary constraint.19 AlphaMissense overcomes these limitations by combining the following strengths. First, AlphaMissense utilizes the training datasets with weak labels from population frequency data; second, AlphaMissense fine-tunes highly accurate protein structures afforded by AlphaFold;20 third, AlphaMissense is capable of learning evolutionary constraints according to amino acid sequences. Consequently, AlphaMissense outperforms other prediction models across multiple clinical benchmarks.15 Recently, AlphaMissense has been applied to predict pathogenicity of cystic fibrosis transmembrane conductance regulator (CFTR) variants and the results correlated well with certain clinical benchmarks.21 Here, we also compared the AlphaMissense and Rhapsody predictions with the ClinVar clinical benchmarks, aiming to provide insights for clinical interpretation and guidance for future experimental investigation of missense variants in GABAA receptors.

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来源期刊
Israel Journal of Chemistry
Israel Journal of Chemistry 化学-化学综合
CiteScore
6.20
自引率
0.00%
发文量
62
审稿时长
6-12 weeks
期刊介绍: The fledgling State of Israel began to publish its scientific activity in 1951 under the general heading of Bulletin of the Research Council of Israel, which quickly split into sections to accommodate various fields in the growing academic community. In 1963, the Bulletin ceased publication and independent journals were born, with Section A becoming the new Israel Journal of Chemistry. The Israel Journal of Chemistry is the official journal of the Israel Chemical Society. Effective from Volume 50 (2010) it is published by Wiley-VCH. The Israel Journal of Chemistry is an international and peer-reviewed publication forum for Special Issues on timely research topics in all fields of chemistry: from biochemistry through organic and inorganic chemistry to polymer, physical and theoretical chemistry, including all interdisciplinary topics. Each topical issue is edited by one or several Guest Editors and primarily contains invited Review articles. Communications and Full Papers may be published occasionally, if they fit with the quality standards of the journal. The publication language is English and the journal is published twelve times a year.
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