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; Jingbo Zhang; Julian I. Schroeder

doi:10.21769/BioProtoc.2114

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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

Jingbo Zhang

Julian I. Schroeder email

发布: 2017年01月20日第7卷第2期 DOI: 10.21769/BioProtoc.2114 浏览次数: 11737

评审: Marisa RosaKathrin SutterAnonymous reviewer(s)

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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)

Slow-type Anion Channel (慢流动型阴离子通道)

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

Borosilicate glass capillaries (World Precision Instruments, catalog number: 1B100F-4 )
Parafilm (Sigma-Aldrich, catalog number: P7793-1EA )
Xenopus laevis oocytes (Ecocyte Bioscience, catalog number: 0-100-2 )
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
mMESSAGE mMACHINE^® T7 Kit (Thermo Fisher Scientific, Ambion^TM, catalog number: AM1344 )
Collagenase D (Roche Diagnostics, catalog number: 11088882001 )
Mineral oil (Sigma-Aldrich, catalog number: M5904 )
MES hydrate (Sigma-Aldrich, catalog number: M2933 )
Tris-base (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP152-5 )
Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: C5670 )
Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M8266 )
Sodium chloride (NaCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S271-10 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
Na-gluconate (Sigma-Aldrich, catalog number: S2054 )
D-sorbitol (Sigma-Aldrich, catalog number: S1876 )
Gentamicin solution (Sigma-Aldrich, catalog number: G1272 )
ND96 buffer (see Recipes)
Recording buffer (see Recipes)

Equipment

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

Software

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

Procedure

English

中文翻译

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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.