Monitoring voltage-dependent charge displacement of Shaker B-IR K+ ion channels using radio frequency interrogation.
Monitoring voltage-dependent charge displacement of Shaker B-IR K+ ion channels using radio frequency interrogation.
复制标题
DOI：
10.1371/journal.pone.0017363
复制
发表时间：
2011-02-28
期刊：
影响因子：
3.7
通讯作者：
Rabbitt RD
中科院分区：
文献类型：
--
作者：
Dharia S;Rabbitt RD
在这里，我们介绍了一种新技术，该技术使用细胞外施加的射频（RF，500 kHz）电场来探测可兴奋膜结合蛋白的电压依赖性电荷位移。非洲爪蟾卵母细胞被用作这些实验的模型细胞，并被注射了编码 Shaker B-IR (ShB-IR) K+ 离子通道的 cRNA，以在卵母细胞膜中表达高密度的该蛋白质。应用两电极电压钳 (TEVC) 来控制全细胞膜电位并测量通道依赖性膜电流。同时，应用射频电场扰动 TEVC 水平附近的膜电位，并测量电压相关的射频位移电流。表达 ShB-IR 的卵母细胞在膜去极化时表现出比对照卵母细胞显着更大的 RF 位移电流变化。在外部浴中添加~120 µM Cu2+ 后，表达 ShB-IR 的卵母细胞中 RF 位移电流的电压依赖性变化进一步增加。已知 Cu2+ 与 ShB-IR 离子通道结合并抑制 Shaker K+ 电导，表明此处报告的 RF 位移电流的变化与 ShB-IR 蛋白的 Cu2+ 连接的移动结构域的 RF 振动相关。结果表明，使用细胞外射频电极来询问带电移动蛋白域的电压依赖性运动，这种能力可能能够检测与完整膜蛋白构象和/或药物-蛋白质相互作用相关的电荷分布的微小变化。
Here we introduce a new technique that probes voltage-dependent charge displacements of excitable membrane-bound proteins using extracellularly applied radio frequency (RF, 500 kHz) electric fields. Xenopus oocytes were used as a model cell for these experiments, and were injected with cRNA encoding Shaker B-IR (ShB-IR) K+ ion channels to express large densities of this protein in the oocyte membranes. Two-electrode voltage clamp (TEVC) was applied to command whole-cell membrane potential and to measure channel-dependent membrane currents. Simultaneously, RF electric fields were applied to perturb the membrane potential about the TEVC level and to measure voltage-dependent RF displacement currents. ShB-IR expressing oocytes showed significantly larger changes in RF displacement currents upon membrane depolarization than control oocytes. Voltage-dependent changes in RF displacement currents further increased in ShB-IR expressing oocytes after ∼120 µM Cu2+ addition to the external bath. Cu2+ is known to bind to the ShB-IR ion channel and inhibit Shaker K+ conductance, indicating that changes in the RF displacement current reported here were associated with RF vibration of the Cu2+-linked mobile domain of the ShB-IR protein. Results demonstrate the use of extracellular RF electrodes to interrogate voltage-dependent movement of charged mobile protein domains — capabilities that might enable detection of small changes in charge distribution associated with integral membrane protein conformation and/or drug–protein interactions.
登录
查看更多内容
每通道总充电移动。门控电荷位移与激活的电压灵敏度之间的关系。
影响因子：
3.8
作者：
Sigg, D;Bezanilla, F
通讯作者：
Bezanilla, F
影响因子：
3.7
作者：
Cheung, K;Gawad, S;Renaud, P
通讯作者：
Renaud, P
影响因子：
2.7
作者：
Ayliffe, HE;Frazier, AB;Rabbitt, RD
通讯作者：
Rabbitt, RD
影响因子：
6.1
作者：
Werdich, AA;Lima, EA;Baudenbacher, FJ
通讯作者：
Baudenbacher, FJ
影响因子：
64.8
作者：
Glauner, KS;Mannuzzu, LM;Isacoff, EY
通讯作者：
Isacoff, EY