Two-electrode Voltage-clamp Recordings in Xenopus laevis Oocytes:Reconstitution of Abscisic Acid Activation of SLAC1 Anion Channel via PYL9 ABA Receptor
|
Author:
Date:
2017-01-20
[Abstract] Two-Electrode Voltage-Clamp (TEVC) recording in Xenopus laevis oocytes provides a powerful method to investigate the functions and regulation of ion channel proteins. This approach provides a well-known tool to characterize ion channels or transporters expressed in Xenopus laevis oocytes. The plasma membrane of the oocyte is impaled by two microelectrodes, one for voltage sensing and the other one for current injection. Here we list a protocol that allows robust reconstitution of multi-component signaling pathways. This protocol has been used to study plant ion channels, including the SLAC1 channel (SLOW ANION CHANNEL-ASSOCIATED 1), in particular SLAC1 activation by either the protein kinase OST1 (OPEN STOMATA 1), Ca2+-dependent protein kinases (CPKs) or the ...
[摘要] 在非洲爪蟾卵母细胞中记录的双电极电压钳(TEVC)提供了一种强大的方法来研究离子通道蛋白的功能和调节。这种方法提供了一种众所周知的用于表征在非洲爪蟾卵母细胞中表达的离子通道或转运蛋白的工具。卵母细胞的质膜由两个微电极引起,一个用于电压感测,另一个用于电流注入。在这里,我们列出了一个允许多组分信号通路强制重组的协议。该方案已经用于研究植物离子通道,包括SLAC1通道(SLOW ANION CHANNEL-ASSOCIATED 1),特别是SLAC1通过蛋白激酶OST1(OPEN STOMATA 1),Ca 2 +依赖性蛋白激酶(CPK)或GHR1(GUARD细胞过氧化氢抗性1)跨膜受体样蛋白。显示了通过“单体”ABA(脱落酸)受体RCAR1 / PYL9(PYRABACT INRESISTANCE1 [PYR1] / PYR1-样[PYL] / ABA受体[RCAR]的调节因子]重建SLAC1阴离子通道的脱落酸活化的数据。通过共表达脱落酸信号核心的四个组分。该方案也适用于研究其他离子通道功能和调节机制,以及转运蛋白。背景 ...
|
|
Measurement of the Electrogenicity of Bile Salt/H+ Antiport in Escherichia coli
|
Author:
Date:
2014-11-05
[Abstract] The transmembrane proton gradient (ΔpH) is the primary source of energy exploited by secondary active substrate/H+ antiporters to drive the electroneutral transport of substrates across the Escherichia coli (E. coli) inner membrane. Such electroneutral transport results in no net movement of charges across the membrane. The charge on the transported substrate and the stoichiometry of the exchange reaction, however, can result in an electrogenic reaction which is driven by both the ΔpH and the electrical (∆Ψ) components of the proton electrochemical gradient, resulting in a net movement of electrical charges across the membrane. We have shown that the major facilitator superfamily transporter MdtM - a multidrug efflux protein from E. coli that ...
[摘要] 跨膜质子梯度(ΔpH)是由次级活性底物/H sup +反转录子开发的能量的主要来源,以驱动底物穿过大肠杆菌的电中性转运( >大肠杆菌)内膜。这种电中性转运导致电荷没有跨膜的净移动。然而,运输的底物上的电荷和交换反应的化学计量可以导致由ΔpH和质子电化学梯度的电(ΔΨ)分量驱动的电致反应,导致电移动的净移动电荷穿过膜。我们已经显示主要促进子超家族转运蛋白MdtM-来自E的多药物外排蛋白。在保护细菌细胞对抗胆汁盐中起到生理作用的大肠杆菌通过偶联外部质子(H + +)与胆汁盐的外流的交换而赋予细菌细胞对胆汁盐的抗性通过电致反应反应的细胞内部(Paul et al。,2014)。该协议使用荧光测定法描述了如何检测Ed的逆转子缺陷菌株的倒置膜囊泡中MdtM的电致逆转运蛋白活性。通过测量跨膜ΔΨ测定大肠杆菌 TO114细胞。该方法利用响应于由于MdtM催化的胆酸钠/H +交换反应引起的膜电位变化而发生在探针Oxonol V的荧光信号强度(淬灭和去淬灭)中发生的变化。该方案可以适于检测任何辅助活性反转运蛋白的活性,其将H sup +跨越生物膜的电致易位与其对应底物的电转运偶联,并且可以用于解偶联否则伪装的转运活性和生理作用。
|
|