Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

Two-electrode Voltage-clamp Recordings in Xenopus laevis Oocytes：Reconstitution of Abscisic Acid Activation of SLAC1 Anion Channel via PYL9 ABA Receptor

非洲爪蟾卵母细胞中的双电极电压钳记录技术：脱落酸通过ABA受体PYL9激活离子通道SLAC1过程的重建

Cun Wang Cun Wang
Jingbo Zhang Jingbo Zhang
Julian I. Schroeder Julian I. Schroeder

发布: 2017年01月20日第7卷第2期 DOI: 10.21769/BioProtoc.2114 浏览次数: 12653

评审: Marisa RosaKathrin SutterAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of The Plant Cell, featuring study using the protocol.

Feb 2016

Advertisement

Special Issue on Protocols for Organoid Research
Technologies for RNA Detection
“科研可重复性与创新”系列网络研讨会
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

利用免疫荧光法原位检测光合生物中TOR激酶活性

利用免疫荧光法原位检测光合生物中TOR激酶活性

Ana P. Lando [...] Giselle M. A. Martínez-Noël

2024年12月20日 2018 阅读

利用体外类囊体实验评估硫氧还蛋白介导的 CFoCF1 还原

利用体外类囊体实验评估硫氧还蛋白介导的 CFoCF1 还原

Takatoshi Sekiguchi [...] Toru Hisabori

2026年06月05日 256 阅读

利用酶标仪测定 OsAIM1 的 NADH 依赖性氧化还原酶活性

利用酶标仪测定 OsAIM1 的 NADH 依赖性氧化还原酶活性

Yuan Hu [...] Jianmin Wan

2026年07月05日 186 阅读

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 GHR1 (GUARD CELL HYDROGEN PEROXIDE-RESISTANT 1) transmembrane receptor-like protein. Data are presented showing reconstitution of abscisic acid activation of the SLAC1 anion channel by the ‘monomeric’ ABA (abscisic acid) receptor RCAR1/PYL9 (PYRABACT INRESISTANCE1 [PYR1]/PYR1-LIKE [PYL]/REGULATORYCOMPONENTS OF ABA RECEPTORS [RCAR]) by co-expressing four components of the abscisic acid signaling core. This protocol is also suitable for studying other ion channel functions and regulation mechanisms, as well as transporter proteins.

Keywords: Ion channel (离子通道) search Voltage-clamp (电压钳) search Oocytes (卵母细胞) search SLAC1 (SLAC1) search ABA receptor (ABA受体) search Slow-type Anion Channel (慢流动型阴离子通道) search

Background

Ion channels expressed in Xenopus laevis oocytes can be studied using two-electrode voltage-clamping. This protocol provides a method to measure ion channel or transporter currents expressed in oocytes, including plant ion channels. In this protocol, we not only summarize how to prepare cRNA, isolate oocytes, inject cRNA and record currents, but also provide information on how to succeed in completing experiments upon co-expressing a signal transduction cascade from receptor to ion channel.

Materials and Reagents

  1. Borosilicate glass capillaries (World Precision Instruments, catalog number: 1B100F-4 )
  2. Parafilm (Sigma-Aldrich, catalog number: P7793-1EA )
  3. Xenopus laevis oocytes (Ecocyte Bioscience, catalog number: 0-100-2 )
  4. Vector: pNB1 oocyte expression vector harboring the cDNA of interest using the USER method (Nour-Eldin et al., 2006), or other oocytes expression vector like
  5. mMESSAGE mMACHINE® T7 Kit (Thermo Fisher Scientific, AmbionTM, catalog number: AM1344 )
  6. Collagenase D (Roche Diagnostics, catalog number: 11088882001 )
  7. Mineral oil (Sigma-Aldrich, catalog number: M5904 )
  8. MES hydrate (Sigma-Aldrich, catalog number: M2933 )
  9. Tris-base (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP152-5 )
  10. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670 )
  11. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  12. Sodium chloride (NaCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S271-10 )
  13. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  14. Na-gluconate (Sigma-Aldrich, catalog number: S2054 )
  15. D-sorbitol (Sigma-Aldrich, catalog number: S1876 )
  16. Gentamicin solution (Sigma-Aldrich, catalog number: G1272 )
  17. ND96 buffer (see Recipes)
  18. Recording buffer (see Recipes)

Equipment

  1. Two-electrode voltage clamp amplifier (e.g., Warner Instrument, model: Oocyte Clamp OC-725C )
  2. Digidata 1440A low-noise data acquisition system (Molecular Devices, model: Digidata 1440A)
  3. P-87 flaming/brown microelectrode micropipette puller (Sutter Instrument, model: P-87)
  4. Osmometer (e.g., Wescor, model: Vapor Pressure Osmometer 5500 )
  5. Microdispenser (Drummond Scientific, catalog number: 3-000-510 )
  6. Custom glass tubing (Drummond Scientific, catalog number: 3-000-210-G8 )

Software

  1. pCLAMP 10 Electrophysiology Data Acquisition and Analysis Software (Molecular Devices).

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Wang, C., Zhang, J. and Schroeder, J. I. (2017). Two-electrode Voltage-clamp Recordings in Xenopus laevis Oocytes：Reconstitution of Abscisic Acid Activation of SLAC1 Anion Channel via PYL9 ABA Receptor. Bio-protocol 7(2): e2114. DOI: 10.21769/BioProtoc.2114.

ris ris Download Citation in RIS Format

分类

植物科学 > 植物生物化学 > 蛋白质 > 活性

细胞生物学 > 基于细胞的分析方法 > 离子分析

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件