跨膜AMPA受体调节蛋白亚基γ2的结构-动脉网

Structure of transmembrane AMPA receptor regulatory protein subunit γ2

Nature 等信源发布 2025-01-15 14:03



可切换为仅中文







Abstract

摘要

Transmembrane AMPA receptor regulatory proteins (TARPs) are claudin-like proteins that tightly regulate AMPA receptors (AMPARs) and are fundamental for excitatory neurotransmission. With cryo-electron microscopy (cryo-EM) we reconstruct the 36 kDa TARP subunit γ2 to 2.3 Å, which points to structural diversity among TARPs.

跨膜AMPA受体调节蛋白（TARP）是紧密调节AMPA受体（AMPARs）的claudin样蛋白，是兴奋性神经传递的基础。通过低温电子显微镜（cryo-EM），我们将36 kDa TARP亚基γ2重建为2.3Å，这表明TARP之间的结构多样性。

Our data reveals critical motifs that distinguish TARPs from claudins and define how sequence variations within TARPs differentiate subfamilies and their regulation of AMPARs..

我们的数据揭示了区分TARP和claudins的关键基序，并定义了TARP内的序列变异如何区分亚家族及其对AMPAR的调控。。

Introduction

简介

Information transfer in the brain occurs at specialized cellular junctions known as synapses, which act as neuronal communication hubs

大脑中的信息传递发生在称为突触的特殊细胞连接处，突触充当神经元通信枢纽

. Most synapses are glutamatergic, where a pre-synaptic neuron releases glutamate (Glu), and a post-synaptic neuron receives Glu. AMPARs in the post-synaptic membrane bind Glu and initiate depolarization of the post-synaptic neuron through their Glu-gated cation channels

大多数突触是谷氨酸能的，其中突触前神经元释放谷氨酸（Glu），突触后神经元接受Glu。突触后膜中的AMPAR结合Glu并通过其Glu门控阳离子通道启动突触后神经元的去极化

. TARPs are auxiliary subunits that regulate the trafficking, gating kinetics, and pharmacology of AMPARs

.TARP是调节AMPAR的运输，门控动力学和药理学的辅助亚基

TARP regulatory subunits tightly regulate AMPAR function in the post-synaptic membrane, a critical aspect of the brain’s ability to fine-tune information processing

TARP调节亚基严格调节突触后膜中的AMPAR功能，这是大脑微调信息处理能力的关键方面

. There are six TARP subtypes (TARPγ2, γ3, γ4, γ5, γ7, γ8), split into type-I (TARPγ2, γ3, γ4, γ8) and type-II (TARPγ5, γ7) families. Generally, TARPs increase the conductance of AMPARs and enhance susceptibility to pharmacological channel block

。有六种TARP亚型（TARPγ2，γ3，γ4，γ5，γ7，γ8），分为I型（TARPγ2，γ3，γ4，γ8）和II型（TARPγ5，γ7）家族。通常，TARP会增加AMPAR的电导，并增强对药理通道阻滞的敏感性

, but type-I TARPs slow desensitization and deactivation kinetics, while type-II TARPs appear to have a negative effect on gating when compared to type-I TARPs

，但是I型TARP会减慢脱敏和失活动力学，而与I型TARP相比，II型TARP似乎对门控有负面影响

. Furthermore, structural differences between TARPs in the same class underlie sensitivity to certain classes of drugs targeted to AMPAR-TARP complexes

此外，同一类别的TARP之间的结构差异是对某些针对AMPAR-TARP复合物的药物的敏感性的基础

. Since the first TARP was identified a quarter century ago (TARPγ2, also known as stargazin)

, TARPs have been recognized as a crucial component of synaptic function

，TARP已被认为是突触功能的关键组成部分

. While many foundational functional and cryo-EM studies have driven the field forward, the precise structural details of how TARPs regulate AMPARs remain ambiguous.

尽管许多基础功能和低温电磁研究推动了该领域的发展，但TARP如何调节AMPAR的确切结构细节仍然模棱两可。

Cryo-EM studies of TARP subunits have advanced our understanding of TARP structure in the context of AMPAR complexes, but the intermediate resolution has historically precluded de novo building of TARP structures

TARP亚基的低温电磁研究提高了我们对AMPAR复合物背景下TARP结构的理解，但中间分辨率在历史上排除了从头构建TARP结构的可能性

. X-ray crystallography structures of TARP homologs, such as claudins, have been indispensable for modeling TARPs

。TARP同源物（例如claudins）的X射线晶体学结构对于建模TARP是必不可少的

. Claudins are cellular junction proteins that form paracellular barriers between epithelial and endothelial cells and are functionally distinct from TARPs

.Claudins是细胞连接蛋白，在上皮细胞和内皮细胞之间形成细胞旁屏障，在功能上与TARP不同

. The reliance on claudin structures for TARP modeling has hampered the identification of distinct structural features that (1) differentiate TARPs from claudins and (2) explain the regulatory potential of TARPs for AMPARs.

。TARP建模对claudin结构的依赖阻碍了对不同结构特征的识别，这些特征（1）将TARP与claudins区分开来，以及（2）解释了TARP对AMPAR的调节潜力。

Here, we use cryo-EM to determine the structure of the prototypical TARP, TARPγ2. We identify motifs in TARPγ2 that distinguish TARP classes from one another and further differentiate TARPs from claudins. These structural features likely underlie modulatory effects exhibited by TARPs on AMPAR gating..

在这里，我们使用cryo-EM来确定原型TARP TARPγ2的结构。我们确定了TARPγ2中的基序，这些基序将TARP类别彼此区分开来，并进一步将TARP与claudins区分开来。这些结构特征可能是TARP对AMPAR门控表现出的调节作用的基础。。

Results

结果

Structure of TARPγ2

TARPγ2的结构

With cryo-EM, we reconstructed TARPγ2 to an overall resolution of 2.3 Å (2.0 Å–2.5 Å locally; Supplemental Fig.

使用cryo-EM，我们将TARPγ2重建为2.3Å（局部2.0Å–2.5Å）的总分辨率；补充图。

). Our data enables us to build most of the transmembrane domain (TMD) and extracellular domain (ECD) de novo (Fig.

)。我们的数据使我们能够从头构建大部分跨膜结构域（TMD）和细胞外结构域（ECD）（图）。

1a级

and Table

和表

). The high resolution of our reconstruction enables identification of multiple distinct structural features in the TARPγ2 extracellular domain (ECD), which sits atop its tetraspanin transmembrane (TM) helical bundle comprised of transmembrane (TM) helices TM1-4 (Fig.

)。我们重建的高分辨率使得能够识别TARPγ2胞外结构域（ECD）中的多个不同结构特征，该结构域位于由跨膜（TM）螺旋TM1-4组成的四跨膜（TM）螺旋束的顶部（图1）。

1a级

). The ECD is comprised of a five-stranded β-sheet and a single extracellular helix (ECH) that immediately precedes TM2. A previously identified disulfide bridge (DSB) between β3 (C67) and β4 (C77) strands in the ECD stabilizes the TARPγ2 ECD (Fig.

)。ECD由五链β-折叠和紧接TM2之前的单个细胞外螺旋（ECH）组成。先前确定的ECD中β3（C67）和β4（C77）链之间的二硫键（DSB）稳定了TARPγ2 ECD（图）。

1b级

) and is conserved across all TARPs and the TARP-like claudins.

)并且在所有TARP和TARP样claudins中都是保守的。

Fig. 1: Structure of TARPγ2.

图1:TARPγ2的结构。

一

Cryo-EM map of TARPγ2, colored rainbow from N-terminus, NT (blue) to C-terminus, CT (red).

TARPγ2的低温电磁图，从N端，NT（蓝色）到C端，CT（红色）的彩色彩虹。

b类

Extracellular portion of the TARPγ2 model showing the β3-β4 DSB, loop anchor DSB, and TARP cleat.

TARPγ2模型的细胞外部分显示β3-β4 DSB，环锚DSB和TARP夹板。

c级

Cartoon schematic of TARPγ2 structure highlighting key structural features that rigidify the entire ECD atop the tetraspanin TMD, colored as in (

TARPγ2结构的卡通示意图，突出了关键的结构特征，这些特征使四跨膜TMD顶部的整个ECD变硬，颜色如(

一

Full size image

全尺寸图像

Table 1 Cryo-EM data collection, refinement, and validation statistics

表1 Cryo-EM数据收集，改进和验证统计

Full size table

全尺寸表

We identify two moieties in our reconstruction of TARPγ2 that distinguish TARPs from claudins. First, a π-π-π stack, which we term the TARP cleat motif, secures the TARPγ2 ECD atop the TARPγ2 TMD (Fig.

我们在TARPγ2的重建中确定了两个部分，它们将TARP与claudins区分开。首先，我们称之为TARP夹板基序的π-π-π堆栈将TARPγ2 ECD固定在TARPγ2 TMD上（图）。

1b级

). The cleat motif is formed by H60 (from β2), Y32 (TM1-β1 loop), and W178 (TM4). We also observe a second DSB in the ECD. This DSB, the loop anchor DSB, anchors the β1-β2 loop onto the β-sheet (Fig.

)。夹板基序由H60（来自β2），Y32（TM1-β1环）和W178（TM4）形成。我们还观察到ECD中的第二个DSB。这个DSB，环锚DSB，将β1-β2环锚定在β-折叠上（图）。

1b级

). The loop anchor DSB is made between C40 in the β1-β2 loop and C68 on β3. Altogether, these motifs rigidify the structure of TARPγ2 by providing additional structural interactions within the ECD and between the ECD and TMD (Fig.

)。环锚DSB位于β1-β2环中的C40和β3上的C68之间。总之，这些基序通过在ECD内以及ECD和TMD之间提供额外的结构相互作用，使TARPγ2的结构僵化（图）。

1c级

Conservation of TARP features

保护TARP功能

The TARP cleat motif is conserved in all TARPs and the TARP-like subunit germline-specific gene 1-like (GSG1L) (Fig.

TARP夹板基序在所有TARP和TARP样亚基种系特异性基因1样（GSG1L）中都是保守的（图）。

2a级

) but is absent from all claudins (Supplemental Fig.

)但所有claudins都不存在（补充图）。

). We also evaluated the conservation of the cleat motif through AlphaFold2

)。我们还通过AlphaFold2评估了夹板基序的保守性

structure prediction. This suggests that the TARP cleat motif is present in all mammalian TARPs (Supplemental Fig.

结构预测。这表明TARP夹板基序存在于所有哺乳动物TARP中（补充图）。

3a级

). Interestingly, while the TARP cleat motif is conserved in all TARPs, the loop anchor DSB is not (Fig.

)。有趣的是，虽然TARP夹板基序在所有TARP中都是保守的，但环锚DSB不是（图）。

2a级

). Structure prediction in AlphaFold2 (Supplemental Fig.

)。AlphaFold2中的结构预测（补充图）。

3b级

) also points to the loop anchor DSB being conserved in type-I TARPs but not in type-II TARPs. Thus, while our structure pointed us to look at the conservation of the cleat motif and loop anchor DSB, this was already predicted by AlphaFold2 (Supplemental Fig.

)还指出环锚DSB在I型TARP中保守，但在II型TARP中不保守。因此，虽然我们的结构让我们看到了夹板基序和环锚DSB的保守性，但AlphaFold2已经预测了这一点（补充图）。

Fig. 2: Conservation of structural features among TARP family members.

图2:TARP家族成员结构特征的保守性。

一

Multiple sequence alignment demonstrating the relative conservation of the TARP Cleat Motif, β3-β4 DSB, and Loop Anchor DSB between TARP family members. Intensity of the shade of purple represents the percent identity. Type-II TARPs are labeled in green. Loop Anchor DSB is unique to type-I and excluded from type-II TARPs.

多序列比对证明了TARP家族成员之间TARP夹板基序，β3-β4 DSB和环锚DSB的相对保守性。紫色阴影的强度代表同一性的百分比。II型防水布标记为绿色。环锚DSB是I型独有的，不包括在II型防水布中。

b类

Alignment of TARPγ2 structure in cyan with other TARP family members (TARPγ3, pink, PDB: 8C2H; TARPγ5, green, PDB: 7RZ5; TARPγ8, purple, PDB: 8AYN; GSG1L, red, PDB: 7RZ9).

青色TARPγ2结构与其他TARP家族成员的比对（TARPγ3，粉红色，PDB:8C2H；TARPγ5，绿色，PDB:7RZ5；TARPγ8，紫色，PDB:8AYN；GSG1L，红色，PDB:7RZ9）。

c级

Zoomed in view of TARP extracellular domains illustrating differing orientations in the β1-β2 loops.

放大TARP胞外域的视图，说明β1-β2环中的不同方向。

View of the TARP cleat motif illustrating conservation among all TARP family members.

TARP夹板图案的视图说明了所有TARP家族成员之间的保护。

Model of predicted β1-β2 loop orientations between type-I and type-II TARPs illustrating distinct potential contacts between TARP subtypes and AMPARs.

I型和II型TARP之间预测的β1-β2环取向的模型，说明了TARP亚型和AMPAR之间不同的潜在接触。

Full size image

全尺寸图像

Surprisingly, the TARP cleat motif and loop anchor DSB are within previous TARP structures but not identified. Previously determined structures of TARPs are overall like our structure of TARPγ2 (Fig.

令人惊讶的是，TARP夹板基序和环锚DSB在以前的TARP结构中，但未被识别。先前确定的TARP结构总体上类似于我们的TARPγ2结构（图）。

2b级

), and the loop anchor DSB is within structures of TARPγ3

)，并且环锚DSB位于TARPγ3的结构内

and TARPγ8

和 TARPγ8

, and even previously published structures of TARPγ2

，甚至是以前发表的TARP 2结构

. However, it is absent, as expected, in the structure of the type-II TARP, TARPγ5

然而，正如预期的那样，II型TARP的结构中不存在TARPγ5

(Fig.

（图。

2摄氏度

) and the TARP-like subunit GSG1L

)和TARP样亚基GSG1L

(Fig.

（图。

2摄氏度

). In contrast, the TARP cleat motif is conserved in all TARPγ3, γ5, and γ8 subunit structures as well as GSG1L

)。相比之下，TARP夹板基序在所有TARP 3、5和8亚基结构以及GSG1L中都是保守的

(Fig.

（图。

二维

). We hypothesize that these structural details and their conservation were previously missed because of a lack of structural resolution.

)。我们假设这些结构细节及其保守性以前由于缺乏结构分辨率而被遗漏。

The dichotomy in β1-β2 loop organization between type-I and type-II TARPs has significant functional implications. For example, type-II TARPs lack the loop anchor DSB and have been observed to directly interact with AMPAR subunits that are in the A and C positions when they occupy the “X” auxiliary subunit site.

I型和II型TARP之间β1-β2环组织的二分法具有重要的功能意义。例如，II型TARP缺乏环锚DSB，并且已经观察到当它们占据“X”辅助亚基位点时，它们直接与位于A和C位置的AMPAR亚基相互作用。

(Fig.

（图。

2e级

). However, we expect that this is not possible for type-I TARPs in the “X” site given the presence of the loop anchor DSB, which locks in the β1-β2 loop in an orientation away from the A and C AMPAR subunit positions. However, if a type-I TARP occupies the “Y” TARP position (Fig.

)。然而，我们预计，鉴于存在环锚DSB，这对于“X”位点的I型TARP是不可能的，该环锚DSB以远离A和C AMPAR亚基位置的方向锁定β1-β2环。然而，如果I型TARP占据“Y”TARP位置（图）。

2e级

), modulation of the AMPAR at subunit positions B or D by the β1-β2 loop is likely possible despite the loop anchor DSB, and is supported by observations in cryo-EM studies of type-I TARPs in complex with AMPARs

)，尽管存在环锚DSB，但仍有可能通过β1-β2环调节亚基位置B或D处的AMPAR，并且在与AMPAR复合的I型TARP的低温EM研究中得到了观察结果的支持

. Given the conformational changes associated with AMPAR gating, the presence or absence of the loop anchor DSB within type-I TARPs versus type-II TARPs could explain differences observed in electrophysiology experiments between chimeric constructs of the β1-β2 loop in type-I and type-II TARPs.

鉴于与AMPAR门控相关的构象变化，I型TARP与II型TARP中是否存在环锚DSB可以解释在电生理实验中观察到的I型和II型TARP中β1-β2环嵌合构建体之间的差异。

Role of loop anchor DSB in AMPAR regulation

环锚DSB在AMPAR调节中的作用

Based on this idea, we hypothesized that ablation of the loop anchor DSB from TARPγ2 would impair the modulatory nature of TARPγ2 on AMPARs. To test this hypothesis, we employed a concatenated cDNA construct encoding the AMPAR subunit GluA2 fused to TARPγ2 and a fluorescent GFP marker

基于这个想法，我们假设从TARPγ2消融环锚DSB会损害TARPγ2在AMPAR上的调节性质。为了验证这一假设，我们采用了一个连接的cDNA构建体，该cDNA构建体编码与TARP 2融合的AMPAR亚基GluA2和一个荧光GFP标记

(Fig.

（图。

3a级

, Methods). Transfection of this cDNA into Expi293 cells produced functional receptors that showed whole-cell AMPAR responses consistent with the characteristic modulation of AMPAR gating by TARPγ2 (Fig.

，方法）。将该cDNA转染到Expi293细胞中产生的功能性受体显示出与TARPγ2对AMPAR门控的特征性调节一致的全细胞AMPAR反应（图）。

3b级

). Interestingly, disrupting the TARPγ2 loop DSB by mutating C40 and C68 to serine (ΔDSB) resulted in reduced peak currents, likely attributed to decreased trafficking of the mutant channels to the cell surface. (Fig.

)。有趣的是，通过将C40和C68突变为丝氨酸（ΔDSB）来破坏TARPγ2环DSB会导致峰值电流降低，这可能归因于突变通道向细胞表面的运输减少。（图。

3b, c

3b，c

Fig. 3: The loop DSB is required for enhancement of AMPAR activation by TARPγ2.

图3:TARPγ2增强AMPAR活化需要环DSB。

一

Schematic representation of the GluA2 (blue)-TARPγ2 (cyan) fusion construct used for electrophysiology demonstrating the location of the ΔDSB mutation (pink).

用于电生理学的GluA2（蓝色）-TARPγ2（青色）融合构建体的示意图，证明了ΔDSB突变的位置（粉红色）。

b类

Representative traces,

代表性痕迹，

c级

current densities (two-tailed Welch’s

电流密度（双尾韦尔奇

-test,

-测试，

= 0.0026),

= 0.0026，

percentages of desensitization (two-tailed Welch’s

脱敏百分比（两尾韦尔奇

test,

，

= 0.0014) and

=0.0014）和

time constants of desensitization kinetics of AMPARs complexed with either WT or ΔDSB TARPγ2 during a 500 ms exposure to 1 mM Glutamate (two-tailed Welch’s

AMPARs与WT或DSB TARP 2复合的脱敏动力学的时间常数

-test,

-测试，

= 0.33). WT, black,

=0.33）。重量，黑色，

= 13; ΔDSB, pink,

=13；ΔDSB，粉红色，

= 9; Bars represent mean ± SEM; **

=9；条形代表平均值±SEM**

< 0.01. Source data are provided as a Source Data File.

<0.01。源数据作为源数据文件提供。

Full size image

全尺寸图像

To accurately measure desensitization, we focused on our analysis on channels with a peak current more than 200 pA. Perturbing the loop anchor DSB with ΔDSB substitutions increases the fraction of desensitized receptors compared to WT (Fig.

为了准确测量脱敏，我们重点分析了峰值电流超过200的通道 pA.与WT相比，用ΔDSB取代扰乱环锚DSB增加了脱敏受体的比例（图）。

), indicating that the loop anchor DSB is a major contributor to the ability of TARPγ2 to enhance AMPAR activation. However, desensitization kinetics were unchanged in ΔDSB receptor complexes compared to WT TARPγ2-containing receptor complexes (Fig.

)，表明环锚DSB是TARPγ2增强AMPAR活化能力的主要贡献者。然而，与含有WT TARPγ2的受体复合物相比，ΔDSB受体复合物的脱敏动力学没有变化（图）。

3e公司

), akin to what has been previously reported for deletion of the entire β1-β2 loop

)，类似于先前报道的删除整个1-2环

. We, therefore, conclude that the loop DSB is critical for the function of type-I TARPs, given that the loss of this single disulfide linkage phenocopies the loss of the entire β1-β2 loop.

因此，我们得出结论，鉴于这种单二硫键的丢失会导致整个β1-β2环的丢失，因此环DSB对于I型TARP的功能至关重要。

Given this result, we considered why the loop anchor DSB might be critical to the type-I TARP function. Previous studies examining the role of the TARP extracellular domains on AMPAR modulation show that several extracellular motifs contribute to the overall modulatory function of type-I TARPs

鉴于此结果，我们考虑了为什么循环锚DSB可能对I型TARP功能至关重要。先前研究TARP细胞外结构域在AMPAR调节中的作用的研究表明，几种细胞外基序有助于I型TARP的整体调节功能

(Fig.

（图。

). These features include (1) the β1-β2 loop, which interacts with AMPAR LBDs when TARPγ2 is at the “Y” position

)。这些特征包括（1）1-2环，当TARP 2位于“Y”位置时，它与AMPAR LBDs相互作用

; (2) the β4-TM2 linker, which interacts with the LBD when TARPγ2 is at the “X” position

；（2）当TARPγ2处于“X”位置时，β4-TM2接头与LBD相互作用

; and (3) the TM3-β5 linker, which interacts with the S1-M1 and S2-M4 linkers when TARPγ2 is arranged at the “Y” position

；和（3）TM3-β5接头，当TARPγ2排列在“Y”位置时，它与S1-M1和S2-M4接头相互作用

(Fig.

（图。

). While the loss of any of these three features from TARPγ2 alters the population of desensitized receptors relative to WT TARPγ2, the β4-TM2 loop more profoundly influences AMPAR desensitization and modulates desensitization kinetics

)。虽然TARPγ2失去这三个特征中的任何一个都会改变相对于WT TARPγ2的脱敏受体的数量，但β4-TM2环更深刻地影响AMPAR脱敏并调节脱敏动力学

. However, the exact roles of the TARP motifs are challenging to completely delineate because of the distinct “X” and “Y” positioning of the TARPs. Mutagenesis could affect TARPs at one site and not the other, making the functional roles not completely clear.

然而，由于TARP的“X”和“Y”位置不同，因此很难完全描述TARP基序的确切作用。诱变可能会影响一个位点而不是另一个位点的TARP，从而使功能作用不完全清楚。

Fig. 4: Model of AMPAR regulation by TARPγ2.

图4:TARPγ2调节AMPAR的模型。

Cartoon representation of TARPγ2 showing extracellular features that are critical for AMPAR modulation. The β1-β2 loop, the Loop Anchor DSB, and the TM3-Β5 loop (pink) are predicted to modulate AMPAR gating from TARPs in the “Y” position and, when mutated, increase the fraction of desensitized AMPARs compared to WT TARPγ2.

TARPγ2的卡通表示显示了对AMPAR调节至关重要的细胞外特征。预计β1-β2环，环锚定DSB和TM3-Β5环（粉红色）会在“Y”位置调节TARP的AMPAR门控，并且当突变时，与WT TARPγ2相比，会增加脱敏AMPAR的比例。

The β4-TM2 loop (purple) is predicted to modulate AMPAR gating from the “X” position and, in addition to regulating the fraction of desensitized AMPARs, also regulates .

预计β4-TM2环（紫色）会从“X”位置调节AMPAR门控，除了调节脱敏AMPAR的比例外，还可以调节。

des

des公司

Full size image

全尺寸图像

Discussion

讨论

To explain how these disparate structural features are all required for TARPγ2 to enhance activation via reducing the population of desensitized receptors, we propose a model in which these motifs orchestrate a network of extracellular interactions (Fig.

为了解释TARPγ2如何通过减少脱敏受体的数量来增强激活，这些不同的结构特征都是必需的，我们提出了一个模型，其中这些基序协调了细胞外相互作用的网络（图）。

). The loop anchor DSB enables the β1-β2 loop to interact with AMPARs while at the “Y” site. Loss of this DSB ablates the TARPγ2 effect on the activated state. On the other hand, the β4-TM2 loop, by which TARPγ2 interacts with AMPARs while at the “X” site, is also critical for enhancing AMPAR activation.

)。环锚DSB使β1-β2环能够在“Y”位点与AMPAR相互作用。该DSB的丢失消除了TARPγ2对激活状态的影响。另一方面，TARPγ2在“X”位点与AMPAR相互作用的β4-TM2环对于增强AMPAR活化也至关重要。

Thus, the presence of both motifs at disparate sites around the AMPAR is critical for the effect of TARPs on AMPAR activation by limiting the population of desensitized receptors. Meanwhile, the β4-TM2 loop affects the desensitization kinetics via mechanisms that are not completely clear. This may explain how TARP stoichiometry can tune AMPAR function through differential occupancy of the “X” and “Y” sites.

因此，通过限制脱敏受体的数量，两个基序在AMPAR周围不同位点的存在对于TARP对AMPAR活化的影响至关重要。同时，β4-TM2环通过尚不完全清楚的机制影响脱敏动力学。这可以解释TARP化学计量如何通过“X”和“Y”位点的差异占用来调节AMPAR功能。

Supporting this idea is that type-II TARPs and GSG1L modulate AMPARs via the β1-β2 loop while occupying the “X” site. The TARP cleat fixes the orientation of the TARP ECD atop the transmembrane helical bundle. A high-resolution structural analysis of how the TARP motifs change during AMPAR gating is required to fully delineate these details..

支持这一想法的是，II型TARP和GSG1L通过β1-β2环调节AMPAR，同时占据“X”位点。TARP夹板将TARP ECD的方向固定在跨膜螺旋束的顶部。需要对AMPAR门控过程中TARP基序如何变化进行高分辨率结构分析，以充分描述这些细节。。

The TARP cleat motif also plays a significant role in distinguishing TARPs from claudins. Both TARPs and claudins share the same overall structural fold (i.e., tetraspanin with a five-stranded extracellular β-sheet). However, claudins have strong oligomerization properties, where they self-oligomerize to form paracellular barriers.

TARP夹板图案在区分TARP和claudins方面也起着重要作用。TARP和claudins都具有相同的整体结构折叠（即具有五链细胞外β-折叠的四跨膜蛋白）。然而，claudins具有很强的寡聚特性，在那里它们会自我寡聚形成细胞旁屏障。

A similar phenomenon has not been reported for TARP proteins. We propose that the TARP cleat motif plays a role in preventing oligomerization in TARPs, enabling their complexation with AMPARs and other synaptic proteins..

TARP蛋白也没有类似的现象报道。我们建议TARP夹板基序在防止TARP寡聚化方面发挥作用，使其能够与AMPAR和其他突触蛋白复合。。

In sum, we report the structure of TARPγ2, and how precise moieties in the ECD account for tune AMPAR function. In addition, we precisely define how TARPs are differentiated from claudins, which may explain the critical point of divergence between the structurally related proteins that are functionally distinct.

总之，我们报告了TARPγ2的结构，以及ECD中的精确部分如何解释调谐AMPAR功能。此外，我们精确定义了TARP与claudins的区别，这可能解释了功能不同的结构相关蛋白之间分歧的临界点。

Our findings provide a framework for future studies to understand the function of TARPs and foundations to target TARPs in structure-based drug design against AMPAR-related neurological disorders..

我们的研究结果为未来的研究提供了一个框架，以了解TARP的功能以及在基于结构的药物设计中针对AMPAR相关神经系统疾病靶向TARP的基础。。

Methods

方法

Image processing

图像处理

The initial stages of cryo-EM sample preparation and data collection were carried out on the GluA2-TARPγ2 complex

在GluA2-TARPγ2复合物上进行了低温电磁样品制备和数据收集的初始阶段

. From this data, a 2.80 Å AMPAR-TARPγ2 local map (Supplemental Fig.

根据这些数据，一个2.80 AMPAR-TARPγ2局部图（补充图）。

1a级

), symmetry expansion was used to refine the structure of TARPγ2. To achieve this, we applied C4 symmetry to the AMPAR-TARP particles (Supplemental Fig.

)，使用对称展开来细化TARPγ2的结构。为了实现这一点，我们将C4对称性应用于AMPAR-TARP颗粒（Supplemental Fig.）。

1a级

). We masked one TARPγ2 in the AMPAR-TARPγ2, then inverted this mask, and subtracted the inverted mask from all particle images. We then used the subtracted particle images, coupled with the original TARPγ2 mask (non-inverted) applied to the complete AMPAR-TARPγ2 complex cryo-EM map reference to refine the final cryo-EM reconstruction of TARPγ2 (Supplemental Fig. .

)。我们在AMPAR-TARPγ2中屏蔽了一个TARPγ2，然后反转了这个掩模，并从所有粒子图像中减去了反转的掩模。然后，我们使用减去的粒子图像，再加上应用于完整AMPAR-TARPγ2复合低温电磁图参考的原始TARPγ2掩模（非倒置），以完善TARPγ2的最终低温电磁重建（补充图。

1b级

Model building, refinement, and structural analysis

模型构建、改进和结构分析

Coot

库特

was used to build a polyalanine chain into TARPγ2 map. Bulky resides from sequence information were used to anchor the building. A previously determined structure of TARPγ2 (pdb 5WEO) and a structure predicted from AlphaFold2 (AlphaFold Protein Structure Database, #AF-O88602) were used as reference.

用于将聚丙氨酸链构建到TARPγ2图谱中。来自序列信息的庞大住宅被用来固定建筑物。先前确定的TARPγ2（pdb 5WEO）结构和从AlphaFold2（AlphaFold蛋白质结构数据库，#AF-O88602）预测的结构用作参考。

Isolde.

伊索尔德

and Phenix

和菲尼克斯

were used to refine the model. Quality of the model was assessed with MolProbity

被用来改进模型。用MolProbity评估模型的质量

. Visualizations and domain measurements were performed in ChimeraX

可视化和域测量是在ChimeraX中进行的

. The software was compiled and accessed via the SBGrid Consortium

.该软件是通过SBGrid Consortium编译和访问的

Sequence analysis

All sequence alignments were done with ClustalW

所有序列比对均使用ClustalW完成

and analyzed in Jalview

并在Jalview进行了分析

Structure prediction

结构预测

TARP structure predictions of TARPγ2, γ3, γ4, γ5, γ7, γ8 of human, rat, mouse species were used from AlphaFold2. For each TARP subunit structure prediction, the respective amino acids corresponding to the cleat motif and disulfide bridge were determined. Cleat motif measurements were taken by calculating the distance between the Cα’s of histidine to tyrosine and Cα’s of tyrosine to tryptophan.

使用AlphaFold2对人，大鼠，小鼠物种的TARPγ2，γ3，γ4，γ5，γ7，γ8进行TARP结构预测。对于每个TARP亚基结构预测，确定了对应于cleat基序和二硫键的各个氨基酸。通过计算组氨酸的Cα与酪氨酸和酪氨酸的Cα与色氨酸之间的距离来进行Cleat基序测量。

Calculations were performed using the Biopython.PDB package..

使用Biopython进行计算。PDB包。。

AlphaFold2 accession numbers of models: AF-Q9Y698, AF-A0JNG9, AF-O88602, AF-Q71RJ2, AF-Q9JJV5, AF-Q0VD05, AF-O60359, AF-Q8VHX0, AF-A0A3Q1LKG2, AF-Q9JJV4, AF-Q8VHW9, AF-Q9UBN1, AF-E1BEI3, AF-Q8VHW4, AF-Q8VHW8, AF-Q9UF02, AF-E1BIG3, AF-P62956, AF-P62957, AF-P62955, AF-Q8WXS5, AF-F1MV40, AF-Q8VHW2, AF-Q8VHW5..

AlphaFold2型号：AF-Q9Y698、AF-A0JNG9、AF-O88602、AF-Q71RJ2、AF-Q9JJV5、AF-Q0VD05、AF-O60359、AF-Q8VHX0、AF-A0A3Q1LKG2、AF-Q9JJV4、AF-Q8VHW9、AF-Q9UBN1、AF-E1BEI3、AF-Q8VHW4、AF-Q8VHW8、AF-Q9UF02、AF-E1BIG3、AF-P62956、AF-P62957、AF-P62955、AF-Q8WXS5、AF-F1MV40、AF-Q8VHW2、AF-Q8VHW5。

Electrophysiological recordings

电生理记录

40 ml of Expi293 Gnti

40毫升Expi293 Gnti

-（笑声）

(Gibco, A39240) cells at a concentration of 1.75*10

（Gibco，A39240）细胞浓度为1.75*10

cells/ml were transfected by mixing 8 μg GluA2-Tarpγ2 (WT) or GluA2-ΔDSB plasmid with 40 μl of polyethylenimine (PEI; Polysciences, 24765) diluted in cell culture media. Cells were incubated with DNA:PEI complexes at 37 °C for 24 h, followed by a further 48 h incubation at 30 °C to facilitate the mutant channel trafficking to the cell surface.

通过将8μgGluA2-Tarpγ2（WT）或GluA2-ΔDSB质粒与在细胞培养基中稀释的40μl聚乙烯亚胺（PEI；Polysciences，24765）混合来转染细胞/ml。将细胞与DNA:PEI复合物在37℃下孵育24小时，然后在30℃下再孵育48小时以促进突变通道运输至细胞表面。

Cells were then added to 12-mm coverslips coated with 0.1 mg/ml poly-.

然后将细胞加入涂有0.1mg/ml聚-的12毫米盖玻片中。

-lysine for recordings. Whole-cell patch-clamp recordings were performed using pulled borosilicate glass (Sutter Instrument). Pipettes with 2–5 MΩ resistance were filled with internal solution (mM): 110 CsF, 30 CsCl, 4 NaCl, 0.5 CaCl

-赖氨酸用于录音。使用拉动的硼硅酸盐玻璃（Sutter Instrument）进行全细胞膜片钳记录。电阻为2-5MΩ的移液管充满内部溶液（mM）：110脑脊液，

, 10 HEPES and 5 EGTA (adjusted to pH 7.4 with CsOH). The extracellular solution (ECS) consisted of (mM): 150 NaCl, 3 KCl, 2 CaCl

，10个HEPES和5个EGTA（用CsOH调节至pH 7.4）。细胞外溶液（ECS）由（mM）组成：150 NaCl，3 KCl，2 CaCl

and 10 HEPES adjusted to pH 7.4 with NaOH. Cells were lifted and exposed with ECS that contained 1 mM glutamate for 500 ms intervals using the SF-77C perfusion fast-step system (Warner Instruments), which was set up as previously described

用NaOH将10个HEPES调节至pH 7.4。使用SF-77C灌注快速步进系统（Warner Instruments），将细胞提起并暴露于含有1mM谷氨酸的EC中，间隔500ms

. ECS without glutamate was applied for 2 s between each 500 ms interval. Recordings were performed using Axopatch 700B amplifier and Digidata 1550B (Molecular Devices) at −60 mV hold potential and acquired at 1 kHz using pCLAMP10.7 software (Molecular Devices). Data analyses were focused on the peak currents above 200 pA to ensure an accurate measurement of desensitization and its kinetics..

不含谷氨酸的EC在每500 ms间隔之间应用2 s。使用Axopatch 700B放大器和Digidata 1550B（Molecular Devices）在-60 mV保持电位下进行记录，并使用pCLAMP10.7软件（Molecular Devices）在1 kHz下采集。数据分析集中在200 pA以上的峰值电流上，以确保准确测量脱敏及其动力学。。

The percentage of GluA2-Tarpγ2 desensitization was calculated as depicted below:

GluA2 Tarpγ2脱敏的百分比计算如下：

$$\left(100-\left(\frac{{Steady\; State}}{{Peak\; Current}}\right)\right) * 100\%$$

$$\左（100-\左（\分数{{稳态}}{{峰值}；电流}}\右）\右）*100 \%$$

(1)

Desensitization kinetics (

脱敏动力学(

desensitization

脱敏

) were obtained from fitting traces to a standard first-order Chebyshev exponential with a 4-pt smoothing filter (Clampfit 10.7).

)通过使用4-pt平滑滤波器（Clampfit 10.7）将迹线拟合到标准的一阶切比雪夫指数来获得。

Reporting summary

报告摘要

Further information on research design is available in the

有关研究设计的更多信息，请参阅

Nature Portfolio Reporting Summary

自然投资组合报告摘要

linked to this article.

链接到本文。

Data availability

数据可用性

The cryo-EM reconstruction is deposited into the Electron Microscopy Data Bank (EMDB) at accession number

低温电磁重建以登录号保存在电子显微镜数据库（EMDB）中

EMD-43242

. The structural model generated from cryo-EM is deposited in the Protein Data Bank (pdb) at accession number

由cryo-EM生成的结构模型以登录号保存在蛋白质数据库（pdb）中

8VHV

. Electrophysiology data are provided as a Source Data File.

.电生理数据作为源数据文件提供。

Source data

源数据

are provided with this paper.

随本文提供。

References

参考文献

Diering, G. H. & Huganir, R. L. The AMPA receptor code of synaptic plasticity.

Diering，G.H。和Huganir，R.L。突触可塑性的AMPA受体代码。

Neuron

神经元

100

, 314–329 (2018).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Hansen, K. B. et al. Structure, function, and pharmacology of glutamate receptor ion channels.

Hansen，K.B.等人。谷氨酸受体离子通道的结构、功能和药理学。

Pharm. Rev.

制药公司 Rev.

, 298–487 (2021).

Article

文章

PubMed

PubMed Central

PubMed 中央

MATH

数学

Google Scholar

谷歌学者

Twomey, E. C., Yelshanskaya, M. V. & Sobolevsky, A. I. Structural and functional insights into transmembrane AMPA receptor regulatory protein complexes.

Twomey，E.C.，Yelshanskaya，M.V。和Sobolevsky，A.I。跨膜AMPA受体调节蛋白复合物的结构和功能见解。

J. Gen. Physiol.

J、一般生理学。

151

, 1347–1356 (2019).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Brown, P. M. G. E., McGuire, H. & Bowie, D. Stargazin and cornichon-3 relieve polyamine block of AMPA receptors by enhancing blocker permeation.

Brown，P.M.G.E.，McGuire，H.＆Bowie，D.Stargazin和cornichon-3通过增强阻滞剂渗透来缓解AMPA受体的多胺阻滞。

J. Gen. Physiol.

J、一般生理学。

150

, 67–82 (2017).

Article

文章

PubMed

Google Scholar

谷歌学者

Carrillo, E. et al. Memantine inhibits calcium-permeable AMPA receptors. Preprint at

Carrillo，E。等人。美金刚抑制钙渗透性AMPA受体。预印于

https://doi.org/10.1101/2024.07.02.601784

(2024).

Zhang, D. et al. Modulatory mechanisms of TARP γ8-selective AMPA receptor therapeutics.

Zhang，D。等。TARPγ8选择性AMPA受体治疗的调节机制。

Nat. Commun.

Nat.普通。

, 1659 (2023).

Article

文章

ADS

CAS

中科院

PubMed

PubMed Central

PubMed 中央

MATH

数学

Google Scholar

谷歌学者

Letts, V. A. et al. The mouse stargazer gene encodes a neuronal Ca2+-channel gamma subunit.

Letts，V.A。等人。小鼠观星者基因编码神经元Ca2+通道γ亚基。

Nat. Genet.

晚上，基因

, 340–347 (1998).

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Tomita, S., Shenoy, A., Fukata, Y., Nicoll, R. A. & Bredt, D. S. Stargazin interacts functionally with the AMPA receptor glutamate-binding module.

Tomita，S.，Shenoy，A.，Fukata，Y.，Nicoll，R.A。＆Bredt，D.S。Stargazin与AMPA受体谷氨酸结合模块在功能上相互作用。

Neuropharmacology

神经药理学

, 87–91 (2007).

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Twomey, E. C., Yelshanskaya, M. V., Grassucci, R. A., Frank, J. & Sobolevsky, A. I. Elucidation of AMPA receptor-stargazin complexes by cryo-electron microscopy.

Twomey，E.C.，Yelshanskaya，M.V.，Grassucci，R.A.，Frank，J。＆Sobolevsky，A.I。通过冷冻电子显微镜阐明AMPA受体-星形胶质复合物。

Science

科学

353

, 83–86 (2016).

Article

文章

ADS

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Twomey, E. C., Yelshanskaya, M. V., Grassucci, R. A., Frank, J. & Sobolevsky, A. I. Channel opening and gating mechanism in AMPA-subtype glutamate receptors.

Twomey，E.C.，Yelshanskaya，M.V.，Grassucci，R.A.，Frank，J。＆Sobolevsky，A.I。AMPA亚型谷氨酸受体的通道开放和门控机制。

Nature

自然

549

, 60–65 (2017).

Article

文章

ADS

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Twomey, E. C., Yelshanskaya, M. V., Grassucci, R. A., Frank, J. & Sobolevsky, A. I. Structural bases of desensitization in AMPA receptor-auxiliary subunit complexes.

Twomey，E.C.，Yelshanskaya，M.V.，Grassucci，R.A.，Frank，J。＆Sobolevsky，A.I。AMPA受体辅助亚基复合物中脱敏的结构基础。

Neuron

神经元

, 569–580.e5 (2017).

。

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Twomey, E. C., Yelshanskaya, M. V., Vassilevski, A. A. & Sobolevsky, A. I. Mechanisms of channel block in calcium-permeable AMPA receptors.

Twomey，E.C.，Yelshanskaya，M.V.，Vassilevski，A.A。和Sobolevsky，A.I。钙渗透性AMPA受体通道阻滞的机制。

Neuron

神经元

, 956–968.e4 (2018).

，956-968.e4（2018）。

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Zhao, Y., Chen, S., Swensen, A. C., Qian, W.-J. & Gouaux, E. Architecture and subunit arrangement of native AMPA receptors elucidated by cryo-EM.

Zhao，Y.，Chen，S.，Swensen，A.C.，Qian，W.-J.＆Gouaux，E。通过cryo-EM阐明的天然AMPA受体的结构和亚基排列。

Science

科学

364

, 355–362 (2019).

Article

文章

ADS

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Yu, J. et al. Hippocampal AMPA receptor assemblies and mechanism of allosteric inhibition.

。

Nature

自然

594

, 448–453 (2021).

Article

文章

ADS

CAS

中科院

PubMed

PubMed Central

PubMed 中央

MATH

数学

Google Scholar

谷歌学者

Herguedas, B. et al. Mechanisms underlying TARP modulation of the GluA1/2-γ8 AMPA receptor.

Herguedas，B。等人。TARP调节GluA1/2-γ8 AMPA受体的机制。

Nat. Commun.

Nat.普通。

, 734 (2022).

Article

文章

ADS

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Chen, S. et al. Activation and desensitization mechanism of AMPA receptor-TARP complex by Cryo-EM.

。

Cell

细胞

170

, 1234–1246.e14 (2017).

，1234-1246.e14（2017）。

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

MATH

数学

Google Scholar

谷歌学者

Zhao, Y., Chen, S., Yoshioka, C., Baconguis, I. & Gouaux, E. Architecture of fully occupied GluA2 AMPA receptor-TARP complex elucidated by cryo-EM.

Zhao，Y.，Chen，S.，Yoshioka，C.，Baconguis，I。＆Gouaux，E。通过cryo-EM阐明的完全占据的GluA2 AMPA受体TARP复合物的结构。

Nature

自然

536

, 108–111 (2016).

Article

文章

ADS

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Suzuki, H. et al. Crystal structure of a claudin provides insight into the architecture of tight junctions.

Suzuki，H。等人。claudin的晶体结构提供了对紧密连接结构的深入了解。

Science

科学

344

, 304–307 (2014).

Zihni, C., Mills, C., Matter, K. & Balda, M. S. Tight junctions: from simple barriers to multifunctional molecular gates.

Zihni，C.，Mills，C.，Matter，K。＆Balda，M.S。紧密连接：从简单的屏障到多功能分子门。

Nat. Rev. Mol. Cell Biol.

Nat。Rev。Mol。Cell Biol。

, 564–580 (2016).

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold.

Jumper，J.等人。使用AlphaFold进行高度准确的蛋白质结构预测。

Nature

自然

596

, 583–589 (2021).

Article

文章

ADS

CAS

中科院

PubMed

PubMed Central

PubMed 中央

MATH

数学

Google Scholar

谷歌学者

Zhang, D. et al. Structural mobility tunes signalling of the GluA1 AMPA glutamate receptor.

Zhang，D。等人。结构迁移率调节GluA1 AMPA谷氨酸受体的信号传导。

Nature

自然

1–6

https://doi.org/10.1038/s41586-023-06528-0

(2023).

Zhang, D., Watson, J. F., Matthews, P. M., Cais, O. & Greger, I. H. Gating and modulation of a hetero-octameric AMPA glutamate receptor.

Zhang，D.，Watson，J.F.，Matthews，P.M.，Cais，O。＆Greger，I.H。异源八聚体AMPA谷氨酸受体的门控和调节。

Nature

自然

594

, 454–458 (2021).

Article

文章

ADS

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Klykov, O., Gangwar, S. P., Yelshanskaya, M. V., Yen, L. & Sobolevsky, A. I. Structure and desensitization of AMPA receptor complexes with type II TARP γ5 and GSG1L.

Klykov，O.，Gangwar，S.P.，Yelshanskaya，M.V.，Yen，L。＆Sobolevsky，A.I。AMPA受体复合物与II型TARPγ5和GSG1L的结构和脱敏作用。

Mol. Cell

摩尔电池

, 4771–4783.e7 (2021).

，4771–4783.e7（2021）。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Gangwar, S. P. et al. Modulation of GluA2-γ5 synaptic complex desensitization, polyamine block and antiepileptic perampanel inhibition by auxiliary subunit cornichon-2.

Gangwar，S.P.等人。辅助亚基cornichon-2对GluA2-γ5突触复合物脱敏、多胺阻滞和抗癫痫药perampanel抑制的调节。

Nat. Struct. Mol. Biol.

自然结构。分子生物学。

, 1481–1494 (2023).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Hale, W. D. et al. Allosteric competition and inhibition in AMPA receptors.

Hale，W.D.等人。AMPA受体的变构竞争和抑制。

Nat. Struct. Mol. Biol.

自然结构。分子生物学。

1–11

https://doi.org/10.1038/s41594-024-01328-0

(2024).

Riva, I., Eibl, C., Volkmer, R., Carbone, A. L. & Plested, A. J. Control of AMPA receptor activity by the extracellular loops of auxiliary proteins.

Riva，I.，Eibl，C.，Volkmer，R.，Carbone，A.L。＆Plested，A.J。通过辅助蛋白的细胞外环控制AMPA受体活性。

eLife

生活

, e28680 (2017).

，e28680（2017）。

Article

文章

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Dawe, G. B. et al. Distinct structural pathways coordinate the activation of AMPA receptor-auxiliary subunit complexes.

Dawe，G.B。等人。不同的结构途径协调AMPA受体辅助亚基复合物的激活。

Neuron

神经元

, 1264–1276 (2016).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

MATH

数学

Google Scholar

谷歌学者

Cais, O. et al. Mapping the interaction sites between AMPA receptors and TARPs reveals a role for the receptor N-terminal domain in channel gating.

绘制AMPA受体和TARP之间相互作用位点的图谱揭示了受体N末端结构域在通道门控中的作用。

Cell Rep.

细胞代表。

, 728–740 (2014).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

MATH

数学

Google Scholar

谷歌学者

Tomita, S. et al. Stargazin modulates AMPA receptor gating and trafficking by distinct domains.

Tomita，S。等人Stargazin通过不同的结构域调节AMPA受体门控和运输。

Nature

自然

435

, 1052–1058 (2005).

Article

文章

ADS

CAS

中科院

PubMed

MATH

数学

Google Scholar

谷歌学者

Hawken, N. M., Zaika, E. I. & Nakagawa, T. Engineering defined membrane-embedded elements of AMPA receptor induces opposing gating modulation by cornichon 3 and stargazin.

Hawken，N.M.，Zaika，E.I。＆Nakagawa，T。工程定义的AMPA受体膜嵌入元件诱导cornichon 3和stargazin的相反门控调节。

J. Physiol.

J、生理学。

595

, 6517–6539 (2017).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics.

Emsley，P。＆Cowtan，K。Coot：分子图形的模型构建工具。

Acta Cryst. D.

Cryst会议记录。D。

, 2126–2132 (2004).

Article

文章

MATH

数学

Google Scholar

谷歌学者

Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps.

Croll，T.I.ISOLDE：一个物理真实的环境，用于将模型构建成低分辨率电子密度图。

Acta Crystallogr. D. Struct. Biol.

。D、结构。生物。

, 519–530 (2018).

Article

文章

ADS

CAS

中科院

PubMed

PubMed Central

PubMed 中央

MATH

数学

Google Scholar

谷歌学者

Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix.

Liebschner，D.等人，《利用X射线、中子和电子测定大分子结构：Phenix的最新进展》。

Acta Cryst. D.

Cryst会议记录。D。

, 861–877 (2019).

Article

文章

CAS

中科院

MATH

数学

Google Scholar

谷歌学者

Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation.

Williams，C.J.等人，《MolProbity：更多更好的参考数据，用于改进全原子结构验证》。

Protein Sci.

蛋白质科学。

, 293–315 (2018).

Article

文章

CAS

中科院

PubMed

MATH

数学

Google Scholar

谷歌学者

Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers.

Pettersen，E.F.等人，《UCSF ChimeraX：研究人员、教育者和开发人员的结构可视化》。

Protein Sci.

蛋白质科学。

, 70–82 (2021).

Article

文章

CAS

中科院

PubMed

MATH

数学

Google Scholar

谷歌学者

Morin, A. et al. Collaboration gets the most out of software.

Morin，A.等人，协作从软件中获得最大收益。

eLife

生活

, e01456 (2013).

，e01456（2013）。

Article

文章

ADS

PubMed

PubMed Central

PubMed 中央

MATH

数学

Google Scholar

谷歌学者

Larkin, M. A. et al. Clustal W and Clustal X version 2.0.

Larkin，M.A.等人，Clustal W和Clustal X 2.0版。

Bioinformatics

生物信息学

, 2947–2948 (2007).

Article

文章

CAS

中科院

PubMed

MATH

数学

Google Scholar

谷歌学者

Waterhouse, A. M., Procter, J. B., Martin, D. M. A., Clamp, M., Barton, G. J. Jalview Version 2 - A multiple sequence alignment editor and analysis workbench.

Waterhouse，A.M.，Procter，J.B.，Martin，D.M.A.，Clamp，M.，Barton，G.J.Jalview版本2-多序列比对编辑器和分析工作台。

Bioinformatics.

生物信息学。

, 1189–1191 (2009).

Li, R.-C., Ben-Chaim, Y., Yau, K.-W. & Lin, C.-C. Cyclic-nucleotide–gated cation current and Ca2+-activated Cl current elicited by odorant in vertebrate olfactory receptor neurons.

。

Proc. Natl Acad. Sci. USA

普罗克。阿卡德之夜滑雪。美国

113

, 11078–11087 (2016).

Article

文章

ADS

CAS

中科院

PubMed

PubMed Central

PubMed 中央

MATH

数学

Google Scholar

谷歌学者

Download references

下载参考资料

Acknowledgements

致谢

We thank members of the Twomey and Huganir labs for insightful discussions, and Junhua Yang and Niki Gooya for technical assistance with electrophysiological recordings. All cryo-EM data was collected at the Beckman Center for Cryo-EM at Johns Hopkins with assistance from D. Sousa and D. Ding. E.C.T.

我们感谢Twomey和Huganir实验室的成员进行了深入的讨论，并感谢Junhua Yang和Niki Gooya在电生理记录方面提供的技术援助。在D.Sousa和D.Ding的帮助下，所有低温电磁数据均在约翰·霍普金斯大学贝克曼低温电磁中心收集。E、 C.T。

is supported by National Institutes of Health (NIH) grant R35GM154904, the Searle Scholars Program (Kinship Foundation #22098168) and the Diana Helis Henry Medical Research Foundation (#142548). R.L.H. is supported by NIH grants R01 NS036715 and R01 MH112152. Z.Q. is supported by NIH grants R35 GM124824, R01 NS118014, and RF1 NS134549.

由美国国立卫生研究院（NIH）资助R35GM154904，塞尔学者计划（亲属基金会#22098168）和戴安娜·海利斯·亨利医学研究基金会（#142548）支持。R、 L.H.得到了NIH拨款R01 NS036715和R01 MH112152的支持。Z、 Q.得到了NIH拨款R35 GM124824，R01 NS118014和RF1 NS134549的支持。

W.D.H. is supported by NIH grant K99 MH132811..

W、 D.H.得到了NIH拨款K99 MH132811的支持。。

Author information

作者信息

Author notes

作者注释

These authors contributed equally: W. Dylan Hale, Alejandra Montaño Romero, Nicholas Koylass.

这些作者做出了同样的贡献：W.Dylan Hale，Alejandra Montaño Romero，Nicholas Koylass。

Authors and Affiliations

作者和隶属关系

Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA

所罗门·H·斯奈德，约翰·霍普金斯大学医学院神经科学系，美国马里兰州巴尔的摩

W. Dylan Hale, Alejandra Montaño Romero, Collin R. Warrick, Zhaozhu Qiu, Richard L. Huganir & Edward C. Twomey

W、迪伦·黑尔、亚历杭德拉·蒙塔尼奥·罗梅罗、科林·R·沃里克、邱兆珠、理查德·L·胡加尼尔和爱德华·C·托米

Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA

约翰·霍普金斯大学医学院生物物理与生物物理化学系，美国马里兰州巴尔的摩

W. Dylan Hale, Alejandra Montaño Romero & Edward C. Twomey

W、迪伦·黑尔、亚历杭德拉·蒙塔尼奥·罗梅罗和爱德华·C·托米

Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

约翰·霍普金斯大学医学院生理学系，美国马里兰州巴尔的摩

Nicholas Koylass & Zhaozhu Qiu

尼古拉斯·柯拉斯和邱兆珠

Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA

约翰·霍普金斯大学医学院药理学和分子科学系，美国马里兰州巴尔的摩

Collin R. Warrick

科林·R·沃里克

Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA

约翰·霍普金斯大学医学院神经外科，美国马里兰州巴尔的摩

Zhaozhu Qiu

赵竹秋

Kavli Neuroscience Discovery Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA

约翰·霍普金斯大学医学院卡夫利神经科学发现研究所，美国马里兰州巴尔的摩

Richard L. Huganir

理查德·L·休根

The Beckman Center for Cryo-EM at Johns Hopkins, Johns Hopkins University School of Medicine, Baltimore, MD, USA

美国马里兰州巴尔的摩市约翰·霍普金斯大学医学院约翰·霍普金斯大学贝克曼冷冻电镜中心

Edward C. Twomey

爱德华·C·托米

Diana Helis Henry Medical Research Foundation, New Orleans, LA, USA

戴安娜·海利斯·亨利医学研究基金会，美国洛杉矶新奥尔良

Edward C. Twomey

爱德华·C·托米

Authors

作者

W. Dylan Hale

W、迪伦·海尔

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

Google Scholar

谷歌学者

Alejandra Montaño Romero

亚历杭德拉·蒙塔尼奥·罗梅罗

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

Google Scholar

谷歌学者

Nicholas Koylass

尼古拉斯科伊拉斯

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

Google Scholar

谷歌学者

Collin R. Warrick

科林·R·沃里克

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

Google Scholar

谷歌学者

Zhaozhu Qiu

赵竹秋

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

Google Scholar

谷歌学者

Richard L. Huganir

理查德·L·休根

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

Google Scholar

谷歌学者

Edward C. Twomey

爱德华·C·托米

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

Google Scholar

谷歌学者

Contributions

捐款

E.C.T. and R.L.H. supervised all aspects and planning of this research. E.C.T., A.M.R., and W.D.H. designed the project. E.C.T. and W.D.H. wrote the manuscript with input from all authors. W.D.H. prepared samples for cryo-EM, collected cryo-EM data, processed cryo-EM data, analyzed data, and built models with E.C.T.

E、 C.T.和R.L.H.监督了这项研究的各个方面和计划。E、 C.T.、A.M.R.和W.D.H.设计了该项目。E、 C.T.和W.D.H.在所有作者的意见下撰写了手稿。W、。

A.M.R. assisted with analysis, structure prediction, model building, and in uncovering the conserved TARP motifs. C.R.W. performed biochemistry and imaging experiments with W.D.H. critical for the manuscript review. N.K. performed the electrophysiological experiments and analyzed the data. Z.Q. supervised the electrophysiological study and wrote the results with N.K..

A、 M.R.协助分析，结构预测，模型构建以及揭示保守的TARP基序。C、 R.W.与W.D.H.进行了生物化学和成像实验，这对手稿审查至关重要。N、 K.进行了电生理实验并分析了数据。Z、 Q.监督电生理研究，并与N.K.一起写下结果。。

Corresponding authors

通讯作者

Correspondence to

通信对象

Richard L. Huganir

理查德·L·休根

或

Edward C. Twomey

爱德华·C·托米

Ethics declarations

道德宣言

Competing interests

相互竞争的利益

R.L.H. is a scientific cofounder and Scientific Advisory Board member of Neumora Therapeutics. The remaining authors declare no competing interests.

R、 L.H.是Neumora Therapeutics的科学联合创始人和科学顾问委员会成员。其余作者声明没有利益冲突。

Peer review

同行评审

Peer review information

同行评审信息

Nature Communications

自然通讯

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

。

Additional information

其他信息

Publisher’s note

出版商注释

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Springer Nature在已发布的地图和机构隶属关系中的管辖权主张方面保持中立。

Supplementary information

补充信息

Supplementary Information

补充信息

Reporting Summary

报告摘要

Transparent Peer Review file

透明的同行评审文件

Source data

源数据

Source Data

源数据

Rights and permissions

权限和权限

Open Access

开放存取

This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material.

本文是根据知识共享署名非商业性NoDerivatives 4.0国际许可证授权的，该许可证允许以任何媒介或格式进行任何非商业性使用，共享，分发和复制，只要您对原始作者和来源给予适当的信任，提供知识共享许可证的链接，并指出您是否修改了许可材料。

You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this licence, visit .

要查看此许可证的副本，请访问。

http://creativecommons.org/licenses/by-nc-nd/4.0/

Reprints and permissions

重印和许可

About this article

关于本文

Cite this article

引用本文

Hale, W.D., Romero, A.M., Koylass, N.

黑尔（W.D.），罗梅罗（Romero），上午（Koylass）。

et al.