State-dependent block of Orai3 TM1 and TM3 cysteine mutants: insights into 2-APB activation.
State-dependent block of Orai3 TM1 and TM3 cysteine mutants: insights into 2-APB activation.
复制标题
DOI：
10.1085/jgp.201411171
复制
发表时间：
2014-05
期刊：
影响因子：
--
通讯作者：
Cahalan MD
中科院分区：
文献类型：
--
作者：
Amcheslavsky A;Safrina O;Cahalan MD
在跨膜螺旋3中，残基E165参与了2-APB激活的Orai3通道扩张孔的形成，但不参与更具选择性的存储操作Orai3孔的形成。内质网钙库耗尽后，质膜上的ORAI通道直接被内质网基质相互作用分子(STIM)蛋白激活，形成钙离子选择性钙释放激活钙通道(CRAC)。在三个人Orai通道同源物中，只有Orai3能被高浓度(>；50微米)的2-氨基乙基二苯基硼酸酯(2-APB)激活。Orai3的2-APB激活在没有STIM1-Orai3相互作用或存储耗尽的情况下发生，并导致阳离子的非选择性电流，其特征是两相向内和向外整流。在这里，我们使用半胱氨酸扫描突变、硫醇反应试剂和膜片钳分析来定义帮助形成2-APB激活的Orai3孔的残基。将跨膜(TM)1残基Q83、V77和L70突变为半胱氨酸会导致镉离子(Cd+)的增强阻断。TM1突变体E81C、G73A、G73C和R66C形成对2-APB激活不敏感的通道。我们还发现Orai3突变体V77C对2-氨乙基甲烷硫磺酸盐(MTSEA)敏感，但不能被2-(三甲基氨基)乙基甲烷硫磺酸盐(MTSET)阻断。与MTSEA反应引起的阻断是状态依赖的，因为只有当Orai3-V77C通道被2-APB或通过与STIM1共转染并同时被动存储耗尽时才会发生。我们还分析了TM3残基E165。Orai3基因E165A突变导致2-APB激活电流减弱。然而，它对存储操作电流密度的影响很小。此外，突变E165C导致Cd~(2+)诱导的状态依赖的阻断：Cd~(2+)只阻断2-APB激活的突变通道，而不是存储操作的突变通道。我们的数据表明，2-APB激活的Orai3的扩张孔被TM1残基排列，但也允许TM3 E165接近形成传导途径或孔的通道的中心轴。
Residue E165, in transmembrane helix 3, participates in formation of the dilated pore of the 2-APB–activated Orai3 channel but not that of the more selective store-operated Orai3 pore. After endoplasmic reticulum (ER) Ca2+ store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident stromal interacting molecule (STIM) proteins to form the Ca2+-selective Ca2+ release-activated Ca2+ (CRAC) channel. Of the three human Orai channel homologues, only Orai3 can be activated by high concentrations (>50 µM) of 2-aminoethyl diphenylborinate (2-APB). 2-APB activation of Orai3 occurs without STIM1–Orai3 interaction or store depletion, and results in a cationic, nonselective current characterized by biphasic inward and outward rectification. Here we use cysteine scanning mutagenesis, thiol-reactive reagents, and patch-clamp analysis to define the residues that assist in formation of the 2-APB–activated Orai3 pore. Mutating transmembrane (TM) 1 residues Q83, V77, and L70 to cysteine results in potentiated block by cadmium ions (Cd2+). TM1 mutants E81C, G73A, G73C, and R66C form channels that are not sensitive to 2-APB activation. We also find that Orai3 mutant V77C is sensitive to block by 2-aminoethyl methanethiosulfonate (MTSEA), but not 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). Block induced by reaction with MTSEA is state dependent, as it occurs only when Orai3-V77C channels are opened by either 2-APB or by cotransfection with STIM1 and concurrent passive store depletion. We also analyzed TM3 residue E165. Mutation E165A in Orai3 results in diminished 2-APB–activated currents. However, it has little effect on store-operated current density. Furthermore, mutation E165C results in Cd2+-induced block that is state dependent: Cd2+ only blocks 2-APB–activated, not store-operated, mutant channels. Our data suggest that the dilated pore of 2-APB–activated Orai3 is lined by TM1 residues, but also allows for TM3 E165 to approach the central axis of the channel that forms the conducting pathway, or pore.
登录
查看更多内容
DOI：
10.1126/science.1228757
发表时间：
2012-12-07
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Hou X;Pedi L;Diver MM;Long SB
通讯作者：
Long SB
影响因子：
64.8
作者：
McNally, Beth A.;Somasundaram, Agila;Yamashita, Megumi;Prakriya, Murali
通讯作者：
Prakriya, Murali
影响因子：
64.8
作者：
Prakriya, Murali;Feske, Stefan;Hogan, Patrick G.
通讯作者：
Hogan, Patrick G.
影响因子：
64.8
作者：
Yeromin, Andriy V.;Zhang, Shenyuan L.;Cahalan, Michael D.
通讯作者：
Cahalan, Michael D.
影响因子：
4.8
作者：
Schindl, Rainer;Bergsmann, Judith;Romanin, Christoph
通讯作者：
Romanin, Christoph