• 主题
高级搜索  >
历史搜索 清空历史
首页> 美国卫生研究院文献>other >Probing the Structure of Affinity-Purified and Lipid-Reconstituted Torpedo Nicotinic Acetylcholine Receptor
【2h】

Probing the Structure of Affinity-Purified and Lipid-Reconstituted Torpedo Nicotinic Acetylcholine Receptor

机译：亲和纯化和脂质重构鱼雷烟气乙酰胆碱受体的结构的探讨。

代理获取
本网站仅为用户提供外文OA文献查询和代理获取服务，本网站没有原文。下单后我们将采用程序或人工为您竭诚获取高质量的原文，但由于OA文献来源多样且变更频繁，仍可能出现获取不到、文献不完整或与标题不符等情况，如果获取不到我们将提供退款服务。请知悉。
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

The Torpedo nicotinic acetylcholine receptor (nAChR) is the only member of the Cys-loop superfamily of ligand gated ion channels (LGIC) that is available in high abundance in a native membrane preparation. To study the structure of the other LGICs using biochemical/biophysical techniques, detergent-solubilization, purification, and lipid-reconstitution are usually required. To assess the effects of purification on receptor structure, we used the hydrophobic photoreactive probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) to compare the state-dependent photolabeling of Torpedo nAChR before and after purification and reincorporation into lipid. For purified nAChR, the agonist-sensitive photolabeling within the M2 ion channel domain of positions M2-6, M2-9 and M2-13, the agonist-enhanced labeling of δThr274 (δM2-18) within the δ subunit helix bundle, and the labeling at the lipid-protein interface (αM4) were the same as for nAChR in native membranes. However, addition of agonist did not enhance [125I]TID photolabeling of δIle288 within the δM2-M3 loop. These results indicate that after purification and reconstitution of Torpedo nAChR, the difference in structure between the resting and desensitized states within the M2 ion channel domain was preserved, but not the agonist-dependent change of structure of the δM2-M3 loop. To further characterize the pharmacology of [125I]TID binding sites in the nAChR in the desensitized state, we examined the effect of phencyclidine (PCP) on [125I]TID photolabeling. PCP inhibited [125I]TID labeling of amino acids at the cytoplasmic end of the ion channel (M2-2 and M2-6), while potentiating labeling at M2-9 and M2-13 and allosterically modulating the labeling of amino acids within the δ subunit helix bundle.
机译：鱼雷烟碱型乙酰胆碱受体（nAChR）是配体门控离子通道（LGIC）的Cys环超家族的唯一成员，可在天然膜制剂中大量使用。为了使用生化/生物物理技术研究其他LGIC的结构，通常需要去污剂溶解，纯化和脂质重构。为了评估纯化对受体结构的影响，我们使用了疏水性光反应性探针3-三氟甲基-3-（m-[ 125 I]碘苯基）二嗪（[ 125 I ] TID）比较鱼雷nAChR在纯化和重新掺入脂质之前和之后的状态依赖性光标记。对于纯化的nAChR，在位置M2-6，M2-9和M2-13的M2离子通道域内对激动剂敏感的光标记，在δ亚基螺旋束内的δThr274（δM2-18）的激动剂增强标记以及脂质-蛋白质界面（αM4）的标记与天然膜中的nAChR相同。但是，添加激动剂并不能增强δM2-M3环中δIle288的[ 125 I] TID光标记。这些结果表明，在纯化和重建鱼雷nAChR之后，保留了M2离子通道域内静止状态和脱敏状态之间的结构差异，但没有保留依赖激动剂的δM2-M3环结构变化。为了进一步表征脱敏状态下nAChR中[ 125 I] TID结合位点的药理作用，我们研究了苯环利定（PCP）对[ 125 I] TID的作用光标签。 PCP抑制离子通道（M2-2和M2-6）的胞质末端氨基酸的[ 125 I] TID标记，同时增强M2-9和M2-13的标记并变构调节δ亚基螺旋束中氨基酸的标记。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
代理获取

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号