比特派钱包app下载中文版|ip3

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比特派钱包app下载中文版|ip3

作者：比特派钱包app下载中文版

2024-03-07 19:21:25

IP3_百度百科

百度百科网页新闻贴吧知道网盘图片视频地图文库资讯采购百科百度首页登录注册进入词条全站搜索帮助首页秒懂百科特色百科知识专题加入百科百科团队权威合作下载百科APP个人中心IP3播报讨论上传视频三磷酸肌醇收藏查看我的收藏0有用+10IP₃（inositol triphosphate，三磷酸肌醇），参与G蛋白耦联受体介导的信号转导的第二信使。在磷脂酰肌醇途径中，胞外信号分子与其相应的G蛋白偶联受体结合后，激活膜上的Gq蛋白（一种作用于磷脂酰肌醇系统的G蛋白），然后由Gp蛋白激活磷酸酯酶Cβ （phospholipase Cβ，PLC），将膜上的4，5-二磷酸脂酰肌醇（phosphatidylinositol biphosphate， PIP2）分解为两个细胞内的第二信使： DAG和IP₃，最后通过激活蛋白激酶C（protein kinase C，PKC），引起级联反应，进行细胞的应答。该通路也称IP₃、DAG、Ca2+信号通路。中文名三磷酸肌醇外文名inositol 1,4,5-trisphosphate, IP3水溶性可溶性质可以从质膜扩散到胞质溶胶分子量420.10分子式C6H15O15P3目录1简介2化学性质3信号通路4功能5相关疾病▪亨廷顿病▪阿尔茨海默病简介播报编辑肌醇三磷酸（IP3）是一种肌醇磷酸信号分子。它是通过磷脂酶C（PLC）水解位于细胞膜中的磷脂酰肌醇-4,5-二磷酸（PIP2）而产生的。与二酰基甘油（DAG）一样，IP3是生物细胞信号转导中使用的第二信使分子。但与DAG留在膜内不同，IP3是可溶的，并在细胞内扩散，与其受体结合。IP3的受体是位于内质网中的钙通道。当IP3与其受体结合后，钙离子释放到细胞质中，从而激活各种钙调节的细胞内信号。化学性质播报编辑IP3的分子结构IP3上的磷酸基根据溶液的pH存在三种不同的形式。磷原子可以通过单键与三个氧原子结合，并使用双键/二配位键与第四个氧原子结合。因此，溶液的pH通过改变磷酸基的形式，决定了其与其他分子结合的能力。磷酸基与肌醇环的结合通过磷酸酯键合实现（参见磷酸和磷酸盐）。这种键合涉及通过脱水反应将肌醇环中的一个氢氧基与一个游离的磷酸基结合。考虑到平均生理pH约为7.4，生物体内与肌醇环结合的磷酸基的主要形式是PO42-，因此IP3通常带有净负电荷，这对于使其与受体结合至关重要，因为它正是通过磷酸基与受体上带有正电荷的残基结合。IP3在其三个氢氧基形式上有三个氢键供体，另外肌醇环上第6个碳原子的氢氧基也参与了IP3的结合。 [2]信号通路播报编辑细胞内Ca2+浓度的增加通常是IP3激活的结果。当配体结合到与G蛋白耦合的G蛋白偶联受体（GPCR）时，Gq蛋白的α亚单位可以结合并激活PLC的同工酶PLC-β，导致PIP2被分解成IP3和DAG。如果受体酪氨酸激酶（RTK）参与激活该通路，同工酶PLC-γ上的酪氨酸残基能够在RTK激活时被磷酸化，这将激活PLC-γ并使其将PIP2分解为DAG和IP3。这发生在能够对生长因子（如胰岛素）产生响应的细胞中，因为生长因子是激活RTK的配体。由于其可溶性，IP3在PLC的作用下产生后，能够通过细胞质扩散到内质网（ER）或肌细胞中的肌浆网（SR）。一旦到达ER，IP3能够结合到三磷酸肌醇受体（Ins(1,4,5)P3R），这是一种位于ER表面的配体门控Ca2+通道。IP3作为门控通道的配体与Ins(1,4,5)P3R的结合，触发Ca2+通道的开放，从而释放Ca2+进入细胞质。在心肌细胞中，Ca2+的增加会激活SR上的Ryanodine受体控制的通道，通过一种称为钙致钙释放的过程导致Ca2+进一步增加。IP3还可能通过增加细胞膜上的Ca2+浓度而间接激活细胞膜上的Ca2+通道。功能播报编辑IP3的主要功能是动员储存器官中的Ca2+并调节细胞增殖以及其他需要游离钙的细胞反应。例如，在平滑肌细胞中，细胞质Ca2+浓度的增加导致肌细胞的收缩。 [3]在神经系统中，IP3充当第二信使，小脑包含最高浓度的IP3受体。 [4]有证据表明IP3受体在小脑Purkinje细胞的可塑性诱导中发挥重要作用。相关疾病播报编辑亨廷顿病亨廷顿病发生在细胞质蛋白亨廷顿（Htt）的N-末端区域额外添加了35个谷氨酰胺残基时。这种修改后的Htt称为Httexp，它使得类型1的IP3受体对IP3更为敏感，从而导致从内质网释放过多的Ca2+，使细胞质和线粒体中Ca2+浓度增加，这种增加被认为是中型多棘神经元降解的原因。 [5]阿尔茨海默病自1994年提出阿尔茨海默病的Ca2+假说以来，多项研究表明Ca2+信号紊乱是阿尔茨海默病的主要原因。家族性阿尔茨海默病与早老素1（PS1）、早老素2（PS2）和淀粉样前体蛋白（APP）基因的突变密切相关，这些基因的突变形式都被发现引起内质网中Ca2+信号的异常。已经证明PS1的突变在几种动物模型中增加了IP3介导的内质网Ca2+释放，并使用钙通道阻滞剂成功治疗阿尔茨海默病，另外还提出使用锂以减少IP3周转（turnover）作为可能的治疗方法。 [6]新手上路成长任务编辑入门编辑规则本人编辑我有疑问内容质疑在线客服官方贴吧意见反馈投诉建议举报不良信息未通过词条申诉投诉侵权信息封禁查询与解封©2024 Baidu 使用百度前必读 | 百科协议 | 隐私政策 | 百度百科合作平台 | 京ICP证030173号京公网安备110000020000

【射频芯片指标】——三阶截断点IP3 - 知乎

【射频芯片指标】——三阶截断点IP3 - 知乎切换模式写文章登录/注册【射频芯片指标】——三阶截断点IP3利尔达利尔达工作人员　　由于放大器存在非线性效应（BJT的e指数转移特性和FET的二次函数转移特性），当输入两个频率很接近的功率信号Pi（f1）和Pi（f2）时，在放大器输出端会产生两个三阶交调分量Po（2f2-f1）和Po（2f1-f2）,如果f1和f2频点比较接近，那么2f1-f2和2f2-f1就会比较靠近f1和f2，一般的滤波器很难滤除。由此可以想象如果f1，f2是工作信道的边界，那么经过放大器后在工作信道带外将会产生杂散泄露。　　IMD的值是衡量三阶交调失真与有用信号之间差异的量，IMD=Po（f2）-Po（2f2-f1）（单位dBc），可以看出IMD越大，发射机的线性度越好。　　在对数标系中，双音输入输出功率和三阶交调输出功率关系如下图所示：　　Po（f2）与Pi（f2）关系曲线的斜率是1，Po（2f2-f1）与Pi（f2）的关系曲线的斜率是3，两条曲线的交点定义为IP3：输入功率就叫IIP3，输出功率就叫OIP3; 由此读者可以理解IP3的含义，即当输入功率达到IIP3时，有用信号的功率达到OIP3，三阶交调的功率也是OIP3，此时IMD为0dBc，这种情况是输出信号最恶劣的情形。　　另外要提及的是发射机的无失真动态范围df，由上图相似三角形可轻易求出df：df（dB）=2/3[OIP3(dBm)-Po,mds(dBm)]，Po,mds定义为放大器最小输出功率，它的值一般规定为比放大器的输出噪声功率大3dB，由此可以算出Po,mds（dBm）=KTB（dBm）+G（dB）+F（dB）+3dB；从曲线意义上来理解df，即当输入功率为某一数值P时，三阶交调的功率正好淹没于放大器的最小输出功率里面，此时相当于三阶交调对有用信号几乎没有干扰，所以此时的有用信号输出功率与放大器最小输出功率之差定义为无失真动态范围。　　还要谈及一点的是级联系统，虽然提高了系统的增益，但是整体的噪声系数F提高了，Po,mds也提高了，级联系统的OIP3降低了，所以根据上面的公式，整个系统的df降低了。发布于 2019-08-14 09:17射频赞同 371 条评论分享喜欢收藏申请

生理学中的IP3是什么？ - 知乎

生理学中的IP3是什么？ - 知乎首页知乎知学堂发现等你来答切换模式登录/注册生理学人体人体解剖学人体构造人体生理学生理学中的IP3是什么？关注者2被浏览15,561关注问题写回答邀请回答好问题添加评论分享1 个回答默认排序惧色医学狗关注IP3（inositol triphosphate，三磷酸肌醇），参与G蛋白耦联受体接到的信号转导的第二信使。在磷脂酰肌醇途径中，胞外信号分子与其相应的G蛋白偶联受体结合后，激活膜上的Gp蛋白（一种作用于磷脂酰肌醇系统的G蛋白），然后由Gp蛋白激活磷酸酯酶Cβ （phospholipase Cβ，PLC），将膜上的4，5-二磷酸脂酰肌醇（phosphatidylinositol biphosphate， PIP2）分解为两个细胞内的第二信使： DAG和IP3，最后通过激活蛋白激酶C（protein kinase C，PKC），引起级联反应，进行细胞的应答。该通路也称IP3、DAG、Ca2+信号通路。发布于 2020-04-19 14:01赞同 43 条评论分享收藏喜欢收起

IP3_百度百科

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IP3

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IP₃（inositol triphosphate，三磷酸肌醇），參與G蛋白耦聯受體介導的信號轉導的第二信使。在磷脂酰肌醇途徑中，胞外信號分子與其相應的G蛋白偶聯受體結合後，激活膜上的Gq蛋白（一種作用於磷脂酰肌醇系統的G蛋白），然後由Gp蛋白激活磷酸酯酶Cβ （phospholipase Cβ，PLC），將膜上的4，5-二磷酸脂酰肌醇（phosphatidylinositol biphosphate， PIP2）分解為兩個細胞內的第二信使： DAG和IP₃，最後通過激活蛋白激酶C（protein kinase C，PKC），引起級聯反應，進行細胞的應答。該通路也稱IP₃、DAG、Ca2+信號通路。

中文名

三磷酸肌醇

外文名

inositol 1,4,5-trisphosphate, IP3

水溶性

可溶

性質

可以從質膜擴散到胞質溶膠

分子量

420.10

分子式

C6H15O15P3

簡介

化學性質

信號通路

功能

相關疾病

▪

亨廷頓病

▪

阿爾茨海默病

IP3簡介

肌醇三磷酸（IP3）是一種肌醇磷酸信號分子。它是通過磷脂酶C（PLC）水解位於細胞膜中的磷脂酰肌醇-4,5-二磷酸（PIP2）而產生的。與二酰基甘油（DAG）一樣，IP3是生物細胞信號轉導中使用的第二信使分子。但與DAG留在膜內不同，IP3是可溶的，並在細胞內擴散，與其受體結合。IP3的受體是位於內質網中的鈣通道。當IP3與其受體結合後，鈣離子釋放到細胞質中，從而激活各種鈣調節的細胞內信號。

IP3化學性質

IP3的分子結構

IP3上的磷酸基根據溶液的pH存在三種不同的形式。磷原子可以通過單鍵與三個氧原子結合，並使用雙鍵/二配位鍵與第四個氧原子結合。因此，溶液的pH通過改變磷酸基的形式，決定了其與其他分子結合的能力。磷酸基與肌醇環的結合通過磷酸酯鍵合實現（參見磷酸和磷酸鹽）。這種鍵合涉及通過脱水反應將肌醇環中的一個氫氧基與一個遊離的磷酸基結合。考慮到平均生理pH約為7.4，生物體內與肌醇環結合的磷酸基的主要形式是PO42-，因此IP3通常帶有淨負電荷，這對於使其與受體結合至關重要，因為它正是通過磷酸基與受體上帶有正電荷的殘基結合。IP3在其三個氫氧基形式上有三個氫鍵供體，另外肌醇環上第6個碳原子的氫氧基也參與了IP3的結合。

[2]

IP3信號通路

細胞內Ca2+濃度的增加通常是IP3激活的結果。當配體結合到與G蛋白耦合的G蛋白偶聯受體（GPCR）時，Gq蛋白的α亞單位可以結合並激活PLC的同工酶PLC-β，導致PIP2被分解成IP3和DAG。如果受體酪氨酸激酶（RTK）參與激活該通路，同工酶PLC-γ上的酪氨酸殘基能夠在RTK激活時被磷酸化，這將激活PLC-γ並使其將PIP2分解為DAG和IP3。這發生在能夠對生長因子（如胰島素）產生響應的細胞中，因為生長因子是激活RTK的配體。由於其可溶性，IP3在PLC的作用下產生後，能夠通過細胞質擴散到內質網（ER）或肌細胞中的肌漿網（SR）。一旦到達ER，IP3能夠結合到三磷酸肌醇受體（Ins(1,4,5)P3R），這是一種位於ER表面的配體門控Ca2+通道。IP3作為門控通道的配體與Ins(1,4,5)P3R的結合，觸發Ca2+通道的開放，從而釋放Ca2+進入細胞質。在心肌細胞中，Ca2+的增加會激活SR上的Ryanodine受體控制的通道，通過一種稱為鈣致鈣釋放的過程導致Ca2+進一步增加。IP3還可能通過增加細胞膜上的Ca2+濃度而間接激活細胞膜上的Ca2+通道。

IP3功能

IP3的主要功能是動員儲存器官中的Ca2+並調節細胞增殖以及其他需要遊離鈣的細胞反應。例如，在平滑肌細胞中，細胞質Ca2+濃度的增加導致肌細胞的收縮。

[3]

在神經系統中，IP3充當第二信使，小腦包含最高濃度的IP3受體。

[4]

有證據表明IP3受體在小腦Purkinje細胞的可塑性誘導中發揮重要作用。

IP3相關疾病

IP3亨廷頓病

亨廷頓病發生在細胞質蛋白亨廷頓（Htt）的N-末端區域額外添加了35個谷氨醯胺殘基時。這種修改後的Htt稱為Httexp，它使得類型1的IP3受體對IP3更為敏感，從而導致從內質網釋放過多的Ca2+，使細胞質和線粒體中Ca2+濃度增加，這種增加被認為是中型多棘神經元降解的原因。

[5]

IP3阿爾茨海默病

自1994年提出阿爾茨海默病的Ca2+假説以來，多項研究表明Ca2+信號紊亂是阿爾茨海默病的主要原因。家族性阿爾茨海默病與早老素1（PS1）、早老素2（PS2）和澱粉樣前體蛋白（APP）基因的突變密切相關，這些基因的突變形式都被發現引起內質網中Ca2+信號的異常。已經證明PS1的突變在幾種動物模型中增加了IP3介導的內質網Ca2+釋放，並使用鈣通道阻滯劑成功治療阿爾茨海默病，另外還提出使用鋰以減少IP3週轉（turnover）作為可能的治療方法。

[6]

參考資料

Inositol trisphosphate and calcium signalling mechanisms

．ScienceDirect[引用日期2023-12-12]

Structural insights into the regulatory mechanism of IP3 receptor

．ScienceDirect[引用日期2023-12-12]

Signal transduction and regulation in smooth muscle

．Nature[引用日期2023-12-12]

Inositol 1,4,5-trisphosphate receptor binding: autoradiographic localization in rat brain

．PubMed[引用日期2023-12-12]

Deranged neuronal calcium signaling and Huntington disease

．ScienceDirect[引用日期2023-12-12]

Michael J. Berridge．The Inositol Trisphosphate/Calcium Signaling Pathway in Health and Disease：American Physiological Society，2016

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IP3的概述圖（1張）

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失真1--线性度概念(1dB压缩点,IP3,OP3) - 知乎

失真1--线性度概念(1dB压缩点,IP3,OP3) - 知乎首发于Analog/RF IC Design切换模式写文章登录/注册失真1--线性度概念(1dB压缩点,IP3,OP3)Shuiniu死磕RFIC线性度的相关基本概念在很多教材上都已经说明，最近再次温故，收益匪浅，同时对失真进行深入的研究和学习，分享下心得。本文主要介绍下线性度的基本概念，包括1dB压缩点,IP3,OP3，注重公式的推导。电路存在非线性是个普遍现象，通常用1dB压缩点和三阶交调点来描述电路的非线性。可以用泰勒级数展开来表达电路的输入输出特性：其中a1作为电路的小信号增益,a2和a3是高阶非线性系数，当输入一个正弦信号x(t)=Acos(wt) ，带入上式，忽略三次以上的非线性，得到：其中第一项是直流分量，直流分量是由偶次谐波产生的，如果是全差分电路，那么偶次谐波将被消除，直流分量也就消除了，但是当电路存在失配时仍会导致有限的偶次谐波。第二项为基波分量，是想要的分量；后二项分别是二次谐波和三次谐波，谐波分量的幅值和输入信号幅值A的关系分别是平方关系和三次方关系。当电路存在选频网络时，高次谐波分量被滤除，通过隔直电容或者全差分电路时消除直流分量后，只剩下基波分量，如果不存在着非线性，即a2=a3=0, 那么理想的线性系统的输出为：而非线性导致输出为:增益为 : 输入输出响应偏离线性关系，对于射频电路，通常a1*a3<1，因此增益受到压缩，当增益偏离线性增益1dB时，对应的输入信号幅值为输入1dB压缩点 A_{in,1dB} ，对应的输出信号幅值为输出1dB压缩点A_{out,1dB} ，输入输出幅值也可以用功率值替代，表示为 IP_{1dB} 和OP_{1dB} 。如图所示表示1dB压缩点，计算输入1dB压缩点，即增益降低1dB的输入幅度值：除了用1dB压缩点描述非线性外，另一种描述非线性的方法是互调失真，当输入二个频率的信号，不同频率分量会产生互调分量 2w1-w2 和 2w2-w1 ，这些分量可能会对输出信号产生干扰，令 x(t)=A1cos(w1t)+A2cos(w2t) ，忽略三次以上的非线性，带入上面公式得：经化简产生的基波频率分量为：二阶交调 \omega_{1}\pm\omega_{2} 分量为：三阶交调 2\omega_{1}\pm\omega_{2} 分量为：三阶交调 2\omega_{2}\pm\omega_{1} 分量为：令二个输入频率的幅度相同，即 A1=A2=A ，且假设A是个比较小的值，可简化基波分量为 a_{1}A(cos\omega_{1}t+cos\omega_{2}t) ，把二阶交调归一化到基波分量上，得到：把三阶交调归一化到基波分量（三阶交调的高频分量不考虑），得到：在差分系统中，二阶非线性被抵消了，因此，更加关注三阶交调点，当基波幅度和三阶交调幅度相等时，即令 IM_{3 } =1 ，得到输出三阶交调点：对应的输出表示为输出三阶交调 A_{OIP3} ，如图s所示表示三阶交调点，比较输入1dB压缩点和输入三阶交调点，得到：利用MOS晶体管构成的差分对时，忽略电路失调失配的影响，有下面结论：提高差分对MOS管的过驱动电压有利于提高线性度。当多个子系统级联时，可以推导出以下结论：其中 A_{IP3} 表示整体系统的输入三阶交调点， a_{n} 表示第n级子系统的增益， A_{IP3,n} 表示第n级子系统的三阶交调点。如果级联的每一级的增益都大于1，那么后级对整体系统的影响更大，直观的理解是信号每经过一级，都被放大，因此产生的非线性更加严重。好了，总结完毕，后期再会。发布于 2020-06-10 22:53非线性谐波失真模拟电路赞同 518 条评论分享喜欢收藏申请转载文章被以下专栏收录Analog/RF IC Des

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【译】三阶截点（IP3）的物理意义 | Inoki in the world

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三阶互调的计算及IP3测试原理和方法 - 21ic电子网

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三阶互调的计算及IP3测试原理和方法

时间：2017-12-14 14:24:12

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ip3

三阶互调

测试原理

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[导读]三阶交截点(IP3)是衡量通信系统线性度的一个重要指标，他反映了系统受到强信号干扰时互调失真的大小。当系统的IP3较高时，要精确测试IP3会比较困难，因为测试环境中各种因素(如测试配件的隔离度、线性度和匹配性等)都容易影响高IP3的测试。

三阶交截点(IP3)是衡量通信系统线性度的一个重要指标，他反映了系统受到强信号干扰时互调失真的大小。当系统的IP3较高时，要精确测试IP3会比较困难，因为测试环境中各种因素(如测试配件的隔离度、线性度和匹配性等)都容易影响高IP3的测试。下面将简略介绍IP3的测试原理，详细分析高IP3的测试方法。

1　IP3测试原理

在无线通信设备中，器件(如放大器、混频器、调制/解调器等)的非线性通常会使同时侵入2个或多个强干扰信号发生相互调制，并产生新的频率成分，这种现象称为互调。互调干扰不仅能降低有用信号的功率，引起信号失真，降低系统选择性，还能破坏邻近信道的性能。因此，互调性能是系统常检指标，通常用IP3来表示。

IP3是工作频率信号在理想线性系统中的输出信号与三阶互调分量幅值相等时的交点，是一个固定点。如图1所示[1]。该点是虚交点，实际系统中无法直接测出，但可以通过相关的测量值计算出来。下面将简单介绍IP3计算式的原理。

虽然侵入系统的强信号可能有2个或2个以上，但为了测试的方便，假设只有2个强的等幅单音信号侵入了系统。若用一个幂级数来表示器件的非线性作用，并假设单音信号的频率分别为f1和f2，那么不难推出三阶互调分量的频率为(2f1-f2)或(2f2-f1)。IP3(IIP3，OIP3)的计算式为[2]：

其中：IIP3为输入IP3，是IP3的横坐标;

OIP3为输出IP3，是IP3的纵坐标;

Pin为单音信号的输入功率电平;

Pout为单音信号的输出功率电平;

G为被测件(Device Under Test - DUT)的小信号增益。

IMD3为三阶互调失真，他等于干扰信号的输出功率电平减去三阶互调量功率电平的值，即：

式(2)中各元素的关系如图2所示。由式(1)和(2)可知，如果测出单音信号的输入/输出功率和三阶互调分量的电平值，就可求出输入/输出IIP3的值。

2　高IP3的测试方法

IP3的一般测试方法是按照图3搭建测试环境，向DUT输入2个强的单音信号，测出DUT输出端单音信号的电平和三阶互调产物的电平，再利用式(1)和式(2)计算出IP3的大小。

当DUT的线性度较好时，其IP3较高。测试这种IP3有2个特点：一是输入的单音信号很强;二是产生的三阶互调分量很弱。由于强信号输入容易使测试系统其他器件也进入非线性状态，产生同频的互调分量或其他杂波;弱互调分量容易被大信号掩盖，所以高IP3的测试工作不能简单按照一般测试方法进行，需做一些改进：

(1)选用高质量的信号源

信号源本身具有非线性，有一定的动态范围。当信号源输出大功率信号时，一些器件进入非线性状态，使得输出信号质量大大降低，如含有各种杂波或多次谐波。因此需选用高质量的信号源，如合成信号源，他的线性度较高，噪声比较低。

(2)隔离2个信号源，减小他们的相互作用　　如果不隔离2个信号源，他们的自适应逻辑电路会相互作用产生互调分量[3]，影响DUT弱互调分量的测试。因此最好在每个信号源与功率合成器之间加一个隔离器。铁氧体磁性材料隔离器是较理想的选择，因为他的隔离度高，差损小。也可以选用10～20 dB的固定衰减器来隔离，但他们的隔离度不高，为了补偿衰减器的衰减量需要加大信号源的输出功率，因此采用固定衰减器不是理想选择。

(3)选用线性好的功率合成器

功率合成器也有一定的非线性，遭遇强信号时也会产生同频互调分量，如果他的互调分量较大，就会掩盖DUT产生的弱互调分量。因此，需采用易匹配且线性度高的功率合成器，如阻性功率合成器。他基本上完全线性，自己不会产生互调分量，并且各个端口具有良好的匹配性。

(4)增强测试系统的匹配性

系统的匹配性非常重要，为确保系统的良好匹配，可在功率合成器与DUT之间和频谱仪与DUT之间分别加一个6～10 dB的固定衰减器[3]。系统统一采用50Ω匹配。

(5)选用动态范围大的频谱分析仪

频谱仪的动态范围是指在能以给定不确定度测量较小信号的频谱分析仪输入端同时存在的最大信号与最小信号之比。当测试高IP3时，输入频谱仪的单音信号幅度很大而三阶互调分量幅度又很小，如果频谱仪的动态范围不够将无法同时测出这2种信号的大小，因此需选用大动态范围的频谱分析仪。

(6)需判别测试结果的有效性

频谱分析仪的前端结构如图4所示。频谱分析仪的IP3通常不高，如安捷伦PSA系列频谱仪(E444xA)在混频器输入电平为-30 dBm时，其IIP3小于+20 dBm。所以测试高IP3时不能忽略频谱仪的非线性，输入DUT的强单音信号也会在频谱仪中相互调制产生同频的互调分量。当该互调分量较大时就需判断频谱仪上显示的互调分量主要是DUT产生的还是频谱仪自身产生的，即判断测试结果是否有效。下面总结了3种判断方法：

①改变频谱仪射频输入衰减器的衰减量(如加大或减小10 dB)，观察互调分量的电平值是否相应减少或增加。如果该电平值改变了，则说明频谱仪产生的互调分量电平值不能忽略，测试结果无效。这是最简单的判断方法。

②在其他条件不变的情况下，比较加上DUT和不加DUT测得的互调分量电平值。如果后者的电平值比前者的小得多则说明所测结果是DUT产生的互调分量;否则，测试结果无效。

③一般的频谱仪手则上都会给出在混频器输入信号电平为某个值(如-30 dBm)时各个频段三阶互调失真的大小或直接给出各个频段IIP3的值。因此，可利用式(1)和式(2)计算频谱分析仪产生的三阶互调分量大小。比较计算结果与测试结果，如果计算值比测试结果小得多，则测试结果为有效值。

当测试结果无效时，解决办法之一是减小2个单音信号的输入电平或加大频谱仪输入衰减器的衰减量。另一种是用测试结果(dBm转化为mW)减去利用判断方法③得出的频谱仪互调分量大小(mW)，从而得到DUT互调分量的大小(mW)。

在测试过程中还需注意：

(1)IP3的计算式(1)是在假设输入DUT的2个干扰信号电平相等的前提下得到的。如果2个干扰信号电平不等，计算公式需调整[1]：

(2)一般情况下，当2个单音信号的幅度均减少1 dB时，三阶互调分量的电平值会减少3 dB，IMD3将相应增加2dB。可见，减少单音信号的输入幅度可大大减少三阶互调分量的幅度。

因此，要减少测试环境中其他配件的非线性对测试结果的影响，最行之有效的方法是尽可能地减小单音信号的电平值。

(3)测试环境中的连接电缆应尽量不要弯曲(特别是在接头处)，以防止增加信号反射，产生过多的互调产物，影响测试准确性。为保持测试系统互调特性的稳定，测试环境不要轻易挪动，每个端口的接头都要拧紧。

3　实验

根据该测试方法，对CDMA2000基站接收通道射频输入部分(从低噪声放大器输出端到第一混频器输出端)的IP3进行了测试。其测试原理图如图5所示。其中，E4432B和E4440A均为Agilent公司的测试仪器。信号源输出的单音信号频率是根据3GPP2协议要求来确定的：分别偏离中心频率(454 MHz)+900 kHz和+1700 kHz。

在混频器输出端的信号频率分别为70.9 MHz和71.7 MHz，即分别偏离中频频率(70 MHz)+900 kHz和+1 700 kHz，用E4440A测得DUT信号经衰减器后的电平值均为-17.8 dBm。表1是测试结果。

如果采用一般的测试方法，得到的IIP3值为28.7 dBm。由此可见，采用上面介绍的高IP3测试方法，大大提高了高IP3的测试准确度。

4　结　语

随着无线通信的快速发展，通信产品需达到的指标要求越来越高，精确测量产品性能愈为重要。线性度是影响系统性能提高的重要因素，做好IP3的准确测试工作是研究并提高系统线性度的一个重要前提。本高IP3测试方法已在3G基站射频部分的IP3测试中得到较好应用，希望能对其他产品的IP3测试工作有所帮助。

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Structural basis for activation and gating of IP3 receptors | Nature Communications

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Structural basis for activation and gating of IP3 receptors

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Structural basis for activation and gating of IP3 receptors

Emily A. Schmitz

ORCID: orcid.org/0000-0002-5122-89911,2 na1, Hirohide Takahashi

ORCID: orcid.org/0000-0002-2553-88061,2 na1 & Erkan Karakas

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Calcium channelsCryoelectron microscopyMembrane proteinsMolecular conformationPermeation and transport

AbstractA pivotal component of the calcium (Ca2+) signaling toolbox in cells is the inositol 1,4,5-triphosphate (IP3) receptor (IP3R), which mediates Ca2+ release from the endoplasmic reticulum (ER), controlling cytoplasmic and organellar Ca2+ concentrations. IP3Rs are co-activated by IP3 and Ca2+, inhibited by Ca2+ at high concentrations, and potentiated by ATP. However, the underlying molecular mechanisms are unclear. Here we report cryo-electron microscopy (cryo-EM) structures of human type-3 IP3R obtained from a single dataset in multiple gating conformations: IP3-ATP bound pre-active states with closed channels, IP3-ATP-Ca2+ bound active state with an open channel, and IP3-ATP-Ca2+ bound inactive state with a closed channel. The structures demonstrate how IP3-induced conformational changes prime the receptor for activation by Ca2+, how Ca2+ binding leads to channel opening, and how ATP modulates the activity, providing insights into the long-sought questions regarding the molecular mechanism underpinning receptor activation and gating.

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IntroductionIP3Rs are intracellular Ca2+ channels, predominantly localized to the ER and activated by the binding of IP3 generated in response to external stimulation of G-protein coupled receptors1,2,3. Opening of the IP3Rs results in the rapid release of Ca2+ from the ER lumen into the cytoplasm, triggering diverse signaling cascades that regulate physiological processes such as learning, fertilization, gene expression, and apoptosis. Dysfunctional IP3Rs cause abnormal Ca2+ signaling and are associated with many diseases, including diabetes, cancer, and neurological disorders4,5. There are three IP3R subtypes (IP3R-1, -2, and -3) that share 60–70% sequence identity, form homo- or hetero-tetramers, exhibit different spatial expression profiles, and are involved in different signaling pathways1,2,3. Each IP3R subunit is about 2700 amino acids in length and contains a transmembrane domain (TMD) and a large cytoplasmic region comprising two β-trefold domains (βTF1 and βTF2), three Armadillo repeat domains (ARM1, ARM2, and ARM3), a central linker domain (CLD), a juxtamembrane domain (JD), and a short C-terminal domain (CTD)6,7,8,9,10 (Fig. 1).Fig. 1: Cryo-EM structures of hIP3R-3 in multiple conformations.a Domain boundaries of hIP3R-3. b–d Composite maps of hIP3R-3 in pre-active A (b), active (c), inactive (d) conformations. Each domain in one of the subunits is colored as in (a). Maps within the boxes, shown transparent, are close-up views of the Ca2+ binding site (red) and the pore (blue) with ribbon representation of hIP3R-3. Select residues are shown in the sticks. Dashed circles indicate opening through the gate.Full size imageIn addition to IP3, the receptor activation requires Ca2+ at nanomolar concentrations, whereas Ca2+ at higher concentrations is inhibitory, causing the receptor to be tightly regulated by Ca2+, 11,12,13,14,15. The cryo-EM structure of human IP3R-3 (hIP3R-3) in the presence of the inhibitory Ca2+ concentrations (2 mM) revealed two binding sites6. However, their role in channel activation and inhibition has remained uncertain. Furthermore, although ATP binding potentiates the receptor by increasing the open probability and duration of the channel openings, the underlying molecular mechanism has not been uncovered16,17. In this study, we illuminate the structural framework of receptor activation and channel opening by analyzing five cryo-EM structures of hIP3R-3 in the closed-pre-activated, open-activated, and closed-inactivated conformations.Results and discussionCryo-EM structures of hIP3R-3 gating conformationsBimodal regulation of IP3R activity by Ca2+ complicates sample preparation because of the requirement for fine adjustment of Ca2+ concentration to trap the channel in the open conformation. Although free Ca2+ concentrations in solutions can easily be controlled by using Ca2+ buffers such as EGTA or BAPTA, it becomes challenging during sample preparation for cryo-EM due to the small volumes used. Typically, a 2–3 µl protein sample is applied to a cryo-grid, but more than 99% of the sample volume is lost during grid preparation due to extensive blotting with filter paper18. During this time, the samples contact filter paper and cryo-grids, containing various amounts of Ca2+. Small sample volumes and short time frames may reduce these buffers’ efficiency, causing the free Ca2+ concentration to increase to inhibitory levels prior to sample freezing. In order to maximize the chances of obtaining particles in the active state, we prepared the sample in: (1) EDTA, which has ~200 fold faster binding kinetics to Ca2+ than EGTA19, a common Ca2+ chelator, and is more likely to chelate excess Ca2+ and other divalent cations within the short period prior to the sample plunging, (2) ATP, which increases the open probability of IP3Rs and dampens the inhibitory effect of Ca2+,16,17, and (3) high concentrations of IP3.The final hIP3R-3 sample was purified in the presence of 1 mM EDTA and supplemented with 0.5 mM IP3, 5 mM ATP, and 0.1 mM CaCl2 before preparing cryo-grids. Although the free Ca2+ concentration was calculated around 100 nM under these conditions using Maxchelator20, the actual free Ca2+ concentration may be higher due to potential leakage of Ca2+ during the cryo-grid preparation as mentioned above. We performed a cryo-EM analysis on a large dataset by employing exhaustive 3D classification strategies to separate particles belonging to different functional states resulting in five high resolution (3.2–3.8 Å) structures (Supplementary Figs. 1–6; Supplementary Table 1). The pore region in all structures resolved to 3.5 Å or better, allowing us to build side chains and determine if the channel was open or closed (Fig. 1; Supplementary Figs. 2–6).Three structures have closed pores with well-resolved densities for IP3 and ATP and are referred to as pre-active A, B, and C (Fig. 1b; Supplementary Figs. 1–7). The structure named “active” displays drastic conformational changes at the TMD, leading to pore opening (Fig. 1c). In addition to the well-resolved densities for IP3 and ATP, the active structure reveals substantial density, interpreted as Ca2+, at the ARM3-JD interface, referred to as the activatory Ca2+ binding site (Fig. 1c; Supplementary Figs. 5, 7). In the fifth structure, the channel is closed, the activatory Ca2+ binding site is occupied, and the intersubunit interactions of the cytoplasmic domains are lost (Fig. 1d; Supplementary Fig. 6). The structure is highly similar to the hIP3R-3 structures obtained in the presence of inhibitory Ca2+ concentrations6, except for βTF1, which moves closer to the ARM1 (Supplementary Fig. 8). Most notably, ARM2 adopted the same conformation relative to ARM1 and CLD, creating the binding site for the second Ca2+ observed at high Ca2+ concentrations (Supplementary Fig. 8). While these similarities suggest a Ca2+ ion occupies this site and the structure represents the Ca2+ inhibited state, the quality of the map around the region did not allow accurate inspection of the presence of Ca2+ (Supplementary Fig. 8). Therefore, we refer to the structure as “inactive” while it remains unclear if it represents a desensitized state that hIP3R-3 adopts without additional Ca2+ binding or an inhibited state forced by binding of additional Ca2+ to an inhibitory site.It is important to note that our initial 3D classification runs resulted in two major classes grouping the pre-active and active structures into one class and the inactive structure into another (Supplementary Fig. 1). It was essential to perform another round of 3D classification focusing only on the core of the protein to separate the particles in the active state from the pre-active states, potentially due to subtle differences in the overall structures and the much fewer number of particles in the active state (20,039 particles compared to 346,684 particles in the pre-active states) (Supplementary Fig. 1; Supplementary Table 1).Priming of hIP3R-3 for activationTo compare the structures presented here, we aligned their selectivity filters and pore helices (residues 2460-2481), which reside at the luminal side of the TMD and are virtually identical in all classes. The pre-active A structure is almost identical to the previously published IP3-bound hIP3R-3 structure6 (Supplementary Fig. 9a) and reveals that IP3 binding causes the ARM1 to rotate about 23° relative to the βTF-2, causing global conformational changes within the cytoplasmic domains, as observed in previous cryo-EM and X-ray crystallography experiments6,8,21,22,23,24 (Supplementary Fig. 9b, c). The pre-active B and C structures adopt distinct conformations that are intermediates between the pre-active A and open state structures. Based on these conformational changes, we propose a sequential transition from pre-active A to B, then C, although the alternative transitions cannot be ruled out entirely. During the transition to the pre-active B state, the N-terminal domain (NTD) of each protomer comprising βTF1, βTF2, ARM1, ARM2, and CLD rotates about 4° counter-clockwise relative to the TMD and moves about 2 Å closer to the membrane plane (Fig. 2a; Supplementary Movie 1). In the pre-active C state, the NTDs remain primarily unchanged compared to the pre-active B state, while the ARM3 and JD are rotated by 7°, causing mild distortions at the cytoplasmic side of the TMD without opening the channel (Fig. 2b; Supplementary Movie 1). Compared to the ligand-free conformation, the βTFs move about 7 Å closer to the membrane plane, and ARM3-JD rotates about 11° in the pre-active C conformation.Fig. 2: Conformational changes in the pre-active states.a, b Ribbon representations of hIP3R structures superposed on the residues forming the selectivity filter and P-helix of the TMDs, emphasizing the conformational changes between the states indicated above. Domains with substantial conformational changes are shown in full colors only on one subunit, while the rest of the protein is transparent. Curved and straight red arrows indicate the rotation and translation of the domains with red labels relative to the rotation axis (black bars), respectively.Full size imageCa2+-mediated conformational changes leading to pore openingIn the absence of Ca2+, the ARM3 and JD act as a rigid body, where there are no significant conformational changes relative to each other (Fig. 2a, b). When Ca2+ is bound, the JD rotates about 11° relative to the ARM3 (Fig. 3a), resembling a clamshell closure, which leads to global conformational changes in the whole receptor, including the movement of the NTD closer to the membrane plane by 2 Å (Fig. 3b; Supplementary Movie 1). In contrast to the limited rotation of the ARM3 (about 5°), the JD rotates about 14° around an axis roughly perpendicular to the membrane plane, leading to conformational changes at the TMD and resulting in pore opening in the active state (Fig. 3b).Fig. 3: Conformational changes coupling Ca2+ binding to pore opening.a Comparison of the JD (shown in full colors) orientation relative to the ARM3 (shown transparent) in the pre-active-C (orange) and active (blue) structures. The black bar indicates the axis for the rotation of the JD. Ca2+ and ATP are shown as spheres. b Global conformational changes induced by Ca2+ binding are depicted similar to Fig. 2. c Close-up view of the Ca2+ binding site in the active conformation. Domains are colored as in Fig. 1.Full size imageCa2+ is coordinated by E1882 and E1946 on the ARM3 and the main-chain carboxyl group of T2581 on the JD (Fig. 3c). H1884 and Q1949 are also close and may interact with Ca2+ through water molecules (Fig. 3c). These residues are highly conserved in the homologous ion channel family, ryanodine receptors (RyRs), suggesting a common activation mechanism in IP3Rs and RyRs25 (Supplementary Fig. 10a, b). Mutation of the corresponding residues in RyRs markedly reduced the sensitivity to Ca2+, further supporting this site’s involvement in the Ca2+ induced activation26,27,28.ATP binding siteWithin the JD, we observed a well-resolved cryo-EM density for ATP in all the structures (Supplementary Fig. 7). The quality of the maps obtained through local refinement allowed unambiguous modeling of ATP, revealing its key interactions with the protein residues (Fig. 4a, b). The adenosine base intercalates into a cavity surrounded by F2156, F2539, I2559, M2565, and W2566 near the zinc finger motif and forms hydrogen bonds with the sulfur of C2538, the backbone amide group of F2539, and the carbonyl groups of H2563 and I2559 (Fig. 4b). The phosphate moieties interact with K2152, K2560, and N2564 (Fig. 4b). There are no apparent structural changes around the binding site upon ATP binding, suggesting that ATP’s potentiating effect is likely due to the increased rigidity of the JD (Supplementary Fig. 9d). ATP binding site is highly conserved among the subtypes except for E2149 which corresponds to lysine and arginine in IP3R-1 and IP3R-2 (Fig. 4c). A positively charged residue instead of E2149 in the proximity of the phosphate moieties may cause tighter interaction of ATP with IP3R-1 and IP3R-2, explaining the low binding affinity of ATP to IP3R-3 compared to IP3R-1 and IP3R-217.Fig. 4: ATP binds to the JD.a The cryo-EM density of ATP (red mesh) from the composite map of the pre-active A state and the modeled ATP molecule. b Close-up view of the ATP binding site in the pre-active A state. Dashed lines indicate hydrogen bonding. c Sequence alignment of hIP3R subtypes around the residues forming the ATP binding site. Residues shown in (b) are highlighted. Select residues are indicated by arrows, and E2149 is labeled in red.Full size imageATP binds to a similar location near the zinc finger motif in RyRs25,29,30. However, its binding mode differs, potentially due to the differences in the residues that form the binding pocket, most notably the basic residues interacting with the phosphate moieties (Supplementary Fig. 10a, c, d). In RyR-1s, the phosphate moieties interact with K4211, K4214, and R4215, all located on a single helix (Supplementary Fig. 10c, d). In hIP3R-3, there is only a single lysine residue (K2152) on the corresponding helix, and the phosphate moieties interact with K2560, located on the opposite side of the binding pocket. A leucine residue (L4980) occupies this position in RyR-1s. The differences in the number and location of the basic residues likely force the phosphate moieties of ATP to adopt different conformations. Furthermore, F2156 in hIP3R-3 points toward the adenosine binding pocket, prohibiting ATP from adopting the conformations observed in RyR-1s due to steric clash in hIP3R-3s (Supplementary Fig. 10c, d).Structure of the TMD in the open conformationThe TMD of IP3Rs has the same overall architecture of voltage-gated ions channels with a central pore domain, consisting of S5, S6, and pore (P) helix, surrounded by pseudo-voltage-sensor domains (pVSDs), consisting of S1, S2, S3, and S4 helices along with two IP3R/RyR specific TM helices (S1’ and S1”) (Fig. 5). In the closed channel, F2513 and I2517 of the S6 helix form two layers of hydrophobic constriction at the pore, blocking the path for the permeation of hydrated ions (Fig. 5). JD’s rotation upon Ca2+ binding pushes the pVSD’s cytoplasmic side away from the pore domain by about 7 Å and tilts the cytoplasmic side of the S6 (S6cyt) by 12° (Fig. 5a; Supplementary Movie 1). Concurrently, the S4-5 linker and S5 helix move away from the S6 helix, thereby inducing a distortion of S6 around the constriction site and moving F2513 and I2517 away from the pore. As a result, the diameter of the water-accessible pore increases to 8 Å, large enough to permeate hydrated cations (Fig. 5b). The flexibility introduced by the neighboring glycine residue (G2514), mutation of which to alanine in IP3R-1 is associated with spinocerebellar ataxia 29 (SCA29)31, is likely critical to the movement of F2513. The tilting of the S6cyt breaks the salt bridge between D2518 and R2524 of the neighboring subunits, moving D2518 towards the pore while pulling R2524 away, which creates an electronegative path on the cytoplasmic side of the pore (Supplementary Fig. 11). In contrast to the prediction of a π- to α-helix transition at the S6lum during channel opening6,10, the π-helix remains intact, and its tip acts as a pivot for the S6cyt tilting and bulging (Fig. 5).Fig. 5: Structure of the IP3R-3 in the open conformation.a Comparison of the hIP3R-3 structures in the pre-active C and active conformations aligned as in Fig. 2. b Ion permeation pathways of hIP3R-3 in pre-active C and active conformations (radii coloring: red, <0.8 Å; green, 0.8-4.0 Å; gray, >4.0 Å) along with the 1D graph of the pore radius. The JD and TMD of the active state are shown in cyan and violet, respectively. The pre-active C structure is colored orange.Full size imageAlthough the TMDs of IP3Rs and RyRs are highly similar, there are noticeable differences in their pore structures (Supplementary Fig. 10e, f). In RyRs, the constriction site is formed by glutamine and isoleucine, corresponding to F2513 and I2517 in hIP3R-3, respectively32. In the open state of RyRs, the isoleucine is positioned similarly to I2517 of hIP3R-325,33,34. On the other hand, the glutamine residue faces the pore in the open state, forming part of the hydrophilic permeation pathway, unlike F2513. Interestingly, N2510 in hIP3R-3, which corresponds to alanine in RyRs, faces the permeation pathway similar to the glutamine of RyRs, suggesting that the amide group plays an important role in the ion permeation. However, since the side chain of N2510 extends from a different position on the S6 helix than the side chain of glutamine in RyRs, the binding pocket for ryanodine25, a RyR-specific inhibitor, is not present in IP3R, potentially causing IP3Rs to be unresponsive to ryanodine32.Several missense mutations identified in the IP3R subtypes are associated with diseases, including spinocereblar ataxia, Gillespie syndrome, anhidrosis, and neck squamous cell carcinoma (Supplementary Fig. 12; reviewed in32,35,36). Perhaps not surprisingly, most of these mutations are localized around the IP3 binding site and alter IP3 binding affinity32,35,36,37. Another hot spot for these mutations is the constriction site of the pore, which undergoes conformational changes during channel opening (Supplementary Fig. 12). It is plausible that these mutations either affect the Ca2+ permeability (e.g., mutation of N251038 or I251739) or restrict conformational changes required for dilation of the pore (e.g., mutation of G251431). Two of the mutated residues (T251940 and F252041) interact with the residues on the S4-S5 linker, which couples the tilting of the pVSD to the bulging of the constriction site (Supplementary Fig. 12b). Mutations of these residues are likely to impair this coupling and thus hinder gating.The flexibility of the CTDThe CTD, extending from the JD along the symmetry axis, forms a left-handed coiled-coil motif before interacting with the βTF2 of the neighboring subunit. The density for the CTD was poorly resolved in all of the states (Supplementary Fig. 13a, b). However, the coiled-coil motifs were visible in the unsharpened maps in the pre-active and active states, enabling us to model poly-alanine peptides without assigned registries (Supplementary Fig. 13a, b). The densities for the extensions from the coiled-coil motif towards the βTF2 become visible when viewed at lower thresholds, whereas the linkers between the JD and the coiled-coil motif remain invisible, indicating higher flexibility for this region (Supplementary Fig. 13a, b). We did not observe any interpretable density for the CTD in the inactive state (Supplementary Fig. 13a, b).For IP3R-1, the CTD was proposed to transmit the conformational changes induced by IP3 at the NTD to the JD8. In IP3R-3, there are no apparent changes on the coiled-coil motif in the pre-active states, but the coiled-coil motif rotates about 20° around the symmetry axis and moves closer to the TMD by 6 Å in the active state (Supplementary Fig. 13c, d). However, the linker between the coiled-coil motif and JD remains flexible, suggesting that the structural rearrangements of this domain are not directly enforcing the channel opening (Supplementary Fig. 13). In line with these observations, removing CTD residues interacting with the βTF2 or swapping the C-terminal region of IP3R-1 with the RyRs, which lack the extended CTD, did not diminish receptor activation21,24,42.Mechanism of hIP3R-3 activation and gatingIt has been long recognized that IP3 binding primes the receptor for activation by Ca2+,43, but how the priming is achieved has remained elusive. Our structures reveal that IP3 binding leads to several conformational changes at the NTD, ARM3, and JD, without any apparent structural changes at the activatory Ca2+ binding site, and that the ARM3 and JD adopt a new pre-gating conformation relative to the TMD with modest changes at the intersubunit interface between the JDs at the cytoplasmic side of the TMD (Fig. 6; Supplementary Fig. 14; Supplementary Movie 1). In addition, ARM3s are constrained in their pre-gating conformation by the tetrameric cage-like assembly of the NTDs, forcing the JDs to rotate upon Ca2+ binding. The NTD assembly is maintained by the βTF1-βTF2 intersubunit interactions (βTF ring), which remains intact in the pre-active and active states (Fig. 6; Supplementary Fig. 15; Supplementary Movie 1) and acts as a pivot for the conformational changes that stabilize the ARM3. On the other hand, its disruption in the inactive state leads to the loosening of the tetrameric assembly of the NTDs, relieving the ARM3 constraints and causing the JD and TMD to adopt the closed channel conformation despite the bound Ca2+ to the activatory site. Supporting this hypothesis, the removal of βTF1 or mutation of W168, which resides at the βTF1-βTF2 interface (Supplementary Fig. 15), was shown to abolish IP3R activity44,45.Fig. 6: Schematic representation of the IP3R gating cycle.A side view of the two opposing subunits (left) and cytoplasmic views of the βTFs and ARM3-JD tetramers (right) for each indicated functional state is depicted. The ARM2 and CTD are omitted for clarity. Arrows shown on the domains indicate the direction of the rotation or translation from the previous conformation.Full size imageIn conclusion, the ensemble of structures obtained from the same sample demonstrates structural heterogeneity of IP3Rs in the presence of IP3, ATP, and Ca2+. Our ability to correlate these structures with their plausible functional states allowed us to define the conformational changes at different gating states, revealing the structural features that underpin IP3R activation and gating. These structures will likely serve as foundations for future experiments addressing biophysical and functional questions related to IP3Rs. Furthermore, our study reinforces the power of cryo-EM in analyzing heterogeneous samples and highlights the importance of a thorough investigation of the data to identify physiologically relevant conformations, even when they constitute only a tiny fraction of the sample.MethodsProtein expression and purificationExpression and purification of hIP3R-3 were performed as previously described with minor modifications10. Briefly, hIP3R-3 (residues 4-2671) with a C-terminal OneStrep tag was expressed using the MultiBac expression system46. Sf9 cells (4 × 106 cells/mL) were harvested by centrifugation (4000 × g) 48 h after infection with the baculovirus. Cells resuspended in a lysis buffer of 200 mM NaCl, 40 mM Tris-HCl pH 8.0, 2 mM EDTA pH 8.0, 10 mM β-mercaptoethanol (BME), and 1 mM Phenylmethylsulfonyl fluoride (PMSF) were lysed using Avastin EmulsiFlex-C3. After centrifugation of the lysate at 7000 × g for 20 min to remove large debris, the membrane was pelleted by centrifugation at 185,000 × g (Type Ti45 rotor) for 1 h. Membrane pellets were homogenized in ice-cold resuspension buffer (200 mM NaCl, 40 mM Tris-HCl pH 8.0, 2 mM EDTA pH 8.0, 10 mM BME) using a Dounce homogenizer, and solubilized using 0.5% Lauryl maltose neopentyl glycol (LMNG) and 0.1% glyco-diosgenin (GDN) at a membrane concentration of 100 mg/mL. After 4 h of gentle mixing in the cold room, the insoluble material was pelleted by centrifugation at 185,000 × g (Type Ti45 rotor) for 1 h, and the supernatant was passed through Strep-XT resin (IBA Biotagnology) via gravity flow. The resin was washed first with 5 column volume (CV) of wash buffer composed of 200 mM NaCl, 20 mM Tris-HCl pH 8.0, 10 mM BME, 0.005% GDN, 0.005% LMNG, followed by 5 CV of wash buffer supplemented with 5 mM ATP and 20 mM MgCl2 to remove any bound chaperone proteins, and finally with 5CV of wash buffer supplemented with 1 mM EDTA. The protein was eluted using wash buffer supplemented with 1 mM EDTA and 100 mM D-Biotin (pH 8.2). The protein was further purified by size exclusion chromatography (SEC) using a Superose 6 Increase column (10/300 GL, GE Healthcare) equilibrated with the SEC buffer composed of 200 mM NaCl, 20 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 2 mM TCEP, 0.005% LMNG, and 0.005% GDN. The fractions corresponding to hIP3R-3 were combined and concentrated to 4 mg/mL using a 100 kDa centrifugal filter (Sartorius). The concentrated sample was then centrifuged at 260,000 × g using an S100AT rotor (ThermoFisher Scientific). The concentration dropped to 1.8 mg/mL.Cryo-EM sample preparation and data collectionPurified hIP3R-3 in the SEC buffer containing 1 mM EDTA was supplemented with 500 µM IP3 (from 10 mM stock in water), 0.1 mM CaCl2, and 5 mM ATP (from 100 mM stock, pH 7.2). 2.0 μL of the protein sample was applied to 300 mesh Cu Quantifoil 1.2/1.3 grids (Quantifoil Microtools) that were glow discharged for 20 s at 25 mA. The grids were blotted for 7 s at force 10 using single-layer Whatman ashless filter papers (Cat. #: 1442-055, GE Healthcare) and were plunged into liquid ethane using an FEI MarkIV Vitrobot at 8 °C and 100% humidity. The filter papers were not pre-treated with Ca2+ chelators or any other chemicals. Four grids prepared using the same sample were imaged using a 300 kV FEI Krios G3i microscope equipped with a Gatan K3 direct electron camera in four different data collection sessions at Case Western Reserve University. Movies containing 40–50 frames were collected at a magnification of ×105,000 in super-resolution mode with a physical pixel size of 0.828 Å/pixel and defocus values at a range of −0.8 to −1.6 µm using the automated imaging software SerialEM47 and EPU (ThermoFisher Scientific).Cryo-EM data processingDatasets from four sessions were initially processed separately using Relion 3.048. We used MotionCor249 and Gtcf50 to perform beam-induced motion correction and CTF estimations, respectively. We performed auto picking using the Laplacian-of-Gaussian option of Relion, extracted particles binned 4 × 4, and performed 2D class classification. Using the class averages with apparent features, we performed another round of particle picking. We cleaned the particles, extracted as 4 × 4 binned, through 2D classification and performed 3D classification using the hIP3R-3 map (EMD-2084910), which was converted to the appropriate box and pixel size. We observed two predominant conformations. One had a compact NTD and tight interactions between subunits as in previously published IP3R structures in the absence of Ca2+, hereafter called “compact” conformation6,7,8,10 (Supplementary Fig. 1). The other one had the NTD of each subunit tilted away from the central symmetry axis resembling hIP3R-3 structures obtained in the presence of high Ca2+ concentrations, hereafter called “loose” conformation6 (Supplementary Fig. 1). These particles were separately selected and reextracted using a box size of 480 × 480 pixels at the physical pixel size. After 3D refinements, we performed CTF refinement and Bayesian polishing51.We combined all the polished particles and performed another round of 3D classification, using one of the compact structures as a reference map. We grouped particles into “compact” and “loose” classes (Supplementary Fig. 1). Refinement of the particles in the “compact” conformation yielded a 3D reconstruction with an average resolution of 3.9 Å. Although there were slight changes at the TMD compared to the structure in the closed state, these changes were not significant enough to suggest that the channel was open. To more clearly resolve the density around the TMD, we performed another round of 3D classification using a mask that only covers the ARM3, JD, and TMD and without performing an angular or translational alignment in Relion3 (Supplementary Fig. 1)52. 3D refinements of the particles in each class were performed using non-uniform refinement in CryoSPARC, enforcing C4 symmetry and local CTF refinements (Supplementary Fig. 1)53. Five classes led to four high-resolution (better than 4 Å) 3D reconstructions, whereas the 3D refinement of the other three classes resulted in poorly resolved maps. The particles in the “loose” conformation were processed using non-uniform refinement in CryoSPARC, but without enforcing any symmetry. Local resolution estimates were calculated using CryoSPARC53 (Supplementary Figs. 2–6). Some of the data processing and refinement software was supported by SBGrid54.To improve the quality of the maps, we performed local refinements using masks covering parts of the original cryo-EM maps (Supplementary Figs. 2–6). We prepared five masks that cover distinct domains of one of the subunits for the pre-active A, B, C, and active conformations. After symmetry expansion using C4 symmetry, we performed local refinement using CryoSparc (Supplementary Figs. 2–5). For the inactive state, we prepared four masks that cover the cytoplasmic domains of each subunit and another mask that covers the tetrameric ARM3, JD, and TMD (Supplementary Fig. 6). The local refinements were performed using C1 symmetry for the cytoplasmic domains and C4 symmetry for the tetrameric ARM3, JD, and TMD. The resulting local refinement maps were aligned onto the original maps using Chimera55 and merged using the “VOP maximum” command of Chimera55 to prepare the composite maps (Supplementary Figs. 2–6).Model buildingModel building was performed using Coot56. We first placed the hIP3R-3 structure in ligand-free conformations (PDB ID: 6UQK10) into the composite map of Pre-active A, and performed rigid-body fitting of individual domains of one of the protomers. We then manually fit the residues into the density and expanded the protomer structure into a tetramer using the C4 symmetry. We performed real-space refinement using Phenix57. We repeated build-refine iterations till a satisfactory model was obtained. This model was used as a starting model for the other structures following the same workflow. Regions without interpretable densities were not built into the model. Residues without apparent density for their side chains were built without their side chains (i.e., as alanines) while maintaining their correct labeling for the amino acid type. The coiled-coil regions were modeled as poly-alanines without residue assignment using the unsharpened maps. Validations of the structural models were performed using MolProbity58 implemented in Phenix57.Figure preparationFigures were prepared using Chimera55, ChimeraX59, and The PyMOL Molecular Graphics System (Version 2.0, Schrödinger, LLC). Calculation of the pore radii was performed using the software HOLE60.Reporting summaryFurther information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The data that support this study are available from the corresponding author upon reasonable request. Cryo-EM maps and atomic coordinates are deposited to the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB) databases, respectively. The accession codes are EMD-25667 and 7T3P for pre-active A, EMD-25668 and 7T3Q for pre-active B, EMD-25669 and 7T3R for pre-active C, EMD-25670 and 7T3T for active, and EMD-25671 and 7T3U for inactive states, respectively. The following previously published datasets were used: EMD-20849, Cryo-EM structure of type 3 IP3 receptor revealing presence of a self-binding peptide10. 6UQK, Cryo-EM structure of type 3 IP3 receptor revealing presence of a self-binding peptide10. 6DRC, High IP3 Ca2+ human type 3 1,4,5-inositol trisphosphate receptor6. 6DQV Class 2 IP3-bound human type 3 1,4,5-inositol trisphosphate receptor6. 5TAL, Structure of rabbit RyR1 (Caffeine/ATP/Ca2+ dataset, class 1 and 2)25. 7M6A, High resolution structure of the membrane embedded skeletal muscle ryanodine receptor29. Reagents and other materials will be available upon request from E.K. with a completed materials transfer agreement.

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Download referencesAcknowledgementsWe thank Dr. Kunpeng Lee for cryo-EM data collection at Case Western Reserve University. We thank Theo Humphries and other support staff at the Pacific Northwest Center for Cryo-EM (PNCC), Drs. Elad Binshtein, Melissa Chambers, and Scott Collier at the Cryo-EM facility at Vanderbilt University for their assistance with cryo-EM sample screening. We thank Drs. Hassane Mchaourab, Terunaga Nakagawa, and Silvia Ravera for discussions and review of the manuscript. This work was conducted in part using the CPU and GPU resources of the Advanced Computing Center for Research and Education (ACCRE) at Vanderbilt University. We used the DORS storage system supported by the U.S. National Institute of Health (NIH) (S10RR031634 to Jarrod Smith). This work was supported by the NIH (R01GM141251 to E.K.), Vanderbilt University, Vanderbilt Diabetes and Research Training Center (NIH P30DK020593 to E.K.), and the Molecular Biophysics Training Program (NIH T32GM008320 to E.A.S.).Author informationAuthor notesThese authors contributed equally: Emily A. Schmitz, Hirohide Takahashi.Authors and AffiliationsDepartment of Molecular Physiology and Biophysics, Vanderbilt University, School of Medicine, Nashville, TN, 37232, USAEmily A. Schmitz, Hirohide Takahashi & Erkan KarakasCenter for Structural Biology, Vanderbilt University, Nashville, TN, 37232, USAEmily A. Schmitz, Hirohide Takahashi & Erkan KarakasAuthorsEmily A. SchmitzView author publicationsYou can also search for this author in

PubMed Google ScholarHirohide TakahashiView author publicationsYou can also search for this author in

PubMed Google ScholarErkan KarakasView author publicationsYou can also search for this author in

PubMed Google ScholarContributionsE.K. conceived the project and performed cryo-EM data analysis; E.A.S. optimized and performed protein expression and purification; H.T. performed grid preparation for cryo-EM. All authors contributed to the preparation of the manuscript.Corresponding authorCorrespondence to

Erkan Karakas.Ethics declarations

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Reprints and permissionsAbout this articleCite this articleSchmitz, E.A., Takahashi, H. & Karakas, E. Structural basis for activation and gating of IP3 receptors.

Nat Commun 13, 1408 (2022). https://doi.org/10.1038/s41467-022-29073-2Download citationReceived: 24 September 2021Accepted: 24 February 2022Published: 17 March 2022DOI: https://doi.org/10.1038/s41467-022-29073-2Share 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

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比特派钱包app下载中文版|ip3

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