| Expression and Purification of the Human Cation-chloride Cotransporter KCC1 from HEK293F Cells for Structural Studies
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Author:
Date:
2021-04-05
[Abstract] Cation-chloride cotransporters (CCCs) mediate the coupled, electroneutral symport of cations such as Na+ and/or K+ with chloride across membrane. Among CCCs family, K-Cl cotransporters (KCC1-KCC4) extrude intracellular Cl- by the transmembrane K+ gradient. In humans, these KCCs play vital roles in the physiology of the nervous system and kidney. However, mechanisms underlying the KCCs specific properties remain poorly understood, partly because purification of membrane proteins is challenging. Here, we present the protocol for purifying the full-length KCC1 from HEK293F cells used in our ...
[摘要] [摘要]阳离子-氯化物共转运蛋白（CCC）介导诸如Na +和/或K +的阳离子与氯离子在膜上的耦合，电中性共价。间幼儿中心家庭，K-CL协同转运蛋白（KCC1-KCC4）抽UDE细胞内氯-通过跨膜ķ +梯度。在人类中，这些KCC在神经系统和肾脏的生理中起着至关重要的作用。然而，特定的KCC性质保持基本机制知之甚少，部分是因为膜蛋白的纯化是具有挑战性的。在这里，我们介绍了从我们最近的出版物中使用的HEK293F细胞中纯化全长KCC1的方案（Liu等人，2019）。该程序可适用于功能和结构研究。
[背景]人类溶质载体12（SLC12 ）基因家族编码阳离子的氯化物协同转运蛋白（CCCS）介导Cl组成的电中性同向转运-和阳离子的Na +或（和）K +跨越质膜。根据其转运特性和氨基酸序列定义，CCC可分为几个分支，包括两个Na-K-2Cl协同转运蛋白（NKCC1和NKCC2），一个Na-Cl协同转运蛋白（NCC）和四个K-Cl协同转运蛋白（KCC1-KCC4 ）。CCC在细胞体积调节，肾脏盐分重吸收和神经元GABA能调节中起重要作用。CCC的结构，生化和生物物理研究涉及在去污剂溶解状态下蛋白质生产和稳定方面的挑战。杆状病毒转导HEK293F细胞（BacMam）系统是异源表达由Eric ...
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| A Protocol to Measure the Cytoplasmic Calcium in Arabidopsis Guard Cells
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Author:
Date:
2015-05-05
[Abstract] Cytoplasmic calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in multiple signal transduction cascades (Allen et al., 1999). In plant cells, a dramatic and readily assayed response to stimulus is the change of stomatal aperture. Changes in [Ca2+]cyt of stomatal guard cells were involved in stomatal movement in response to various stimuli and cellular processes. In general, there are two available ways to measure [Ca2+]cyt in guard cells, i.e., loading of calcium-sensitive fluorescence dyes such ...
[摘要] 细胞质钙（[Ca 2+] + cyt）在多个信号转导级联中用作刺激诱导的第二信使（Allen等人， 1999）。在植物细胞中，对刺激的戏剧性和容易测定的响应是气孔孔径的变化。气孔保卫细胞的[Ca + ] 细胞的变化参与响应于各种刺激和细胞过程的气孔运动。一般来说，有两种可用的方法来测量保护细胞中的钙离子荧光染料的加载量，即 例如fluo-3AM和fura-2或表达遗传编码的钙指示剂例如黄色卡梅伦（Krebs等人，2012）。在该方案中，我们旨在描述在ABA或PA处理时加载fluo-3AM的保卫细胞中记录[Ca 2 cyT 用共聚焦激光扫描显微镜进行荧光成像。
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