卵母细胞K<sup>+</sup>释放量及K<sup>+</sup>水平测定

Hong Li

doi:10.21769/BioProtoc.3802

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Peer-reviewed

K⁺ Release Assay and K⁺ Measurement in Oocyte Assay

卵母细胞K⁺释放量及K⁺水平测定

HL Hong Li email

发布: 2020年11月05日第10卷第21期 DOI: 10.21769/BioProtoc.3802 浏览次数: 4080

评审: Dheeraj Singh RathoreAnonymous reviewer(s)

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

Aug 2017

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Abstract

The Xenopus oocyte is a powerful system for the exogenous expression and functional characterization of plant membrane transport proteins. Until now, a number of potassium transporters and channels have been identified in oocytes expression system by the two-electrode voltage clamp technology. It is difficult to characterize K⁺/H⁺ anti-transporters, especially, electroneutral transporter. The K⁺ efflux assay system enables easy, fast, large-scale measurement of the transporters activity without two-electrode voltage clamp technology. This protocol describes a technique to measure the efflux activity of potassium transporter in oocytes expressing system.

Keywords: Xenopus oocyte (爪蟾卵母细胞)

K⁺ release (K⁺ 释放)

Potassium transporter (钾转运蛋白)

K⁺ measurement (K⁺ 测定)

Membrane transport proteins (膜转运蛋白)

Background

Xenopus oocytes have accumulated stores of enzymes, proteins and organelles, all of which can be exploited to produce large quantities, correctly post-translation modified, correctly destinated foreign protein (Miller and Zhou, 2000). Thus, oocytes offer an effective expression system to functionally characterize plant membrane proteins by using two-electrode voltage clamp technology. The first plant potassium transport proteins expressed in oocytes were K⁺ channels (Cao et al., 1992). Since then, a majority of potassium channels and transporters have been studied in oocyte (Schachtman et al., 1994; Véry et al., 1994; Wang and Wu, 2013). But some K⁺/H⁺ anti-transporters are electroneutral, the two-electrode voltage clamp technology unable to characterize these transporters. To overcome this problem, we have used a new approach to identify the activity of these transporters by measuring the change of potassium content in medium before and after incubation. The intracellular K⁺ concentration is about 70-150 mM (Weber, 1999). K⁺ efflux, mediated by expressed K⁺ transport-proteins, may cause the changes of both intracellular and extracellular K⁺ concentrations. To test whether a transporter is able to release K⁺, measure the changes of intracellular and extracellular K⁺ concentrations. This K⁺ efflux measurement system is recognized as an effective assay for studying K⁺ efflux activity of NRT1.5 (Li et al., 2017). This method is easy, fast, convenient measurement of the transporter activity independent on two-electrode voltage clamp. In this protocol, we describe how to measure the K⁺ transport activity in oocyte.

Materials and Reagents

Pipette tips (Axygen, catalog numbers: T-300, T-200-Y, T-1000-B )
Scalpel blade (GLOBALMED, catalog number: BD001 )
1.5 ml microcentrifuge tubes (Axygen, catalog number: MCT-150-C )
Petri dish (Nunc, catalog number: 150466 )
24-well plate (Nunc, catalog number: 142475 )
Capillary glass (Sutter Instrument Company, catalog number: B100-58-10 )
Parafilm (Bemis, catalog number: PM-996 )
Micropipette tip (SUTTER, catalog number: BF100-50-10 )
3-ml plastic pipettes dropper ( KEHUA, catalog number: KH145 )
0.22 μm Syringe filter (GREEN MALL, catalog number: RSF02 )
Competent E. coli DH5α (TIANGEN, catalog number: CB101-02 )
pT7TS (provided by Xiaorong Fan, Nanjing Agricultural University)
Plasmid Purification Midi Kit (QIAGEN, catalog number: 12143 )
KOD polymerase (TOYOBO, catalog number: KOD-201B )
Agarose (BIOWEST, catalog number: B21033 )
DNA Gel Extraction Kit (AxyGEN, catalog number: AP-GX-250 )
Restriction enzyme Bgl II (Takara Bio, catalog number: 1021A )
Restriction enzyme Spe I (Takara Bio, catalog number: D1086a )
Restriction enzyme BamH I (Takara Bio, catalog number: 1010 )
T4 Ligase (STRATAGENE, catalog number: M0202V )
Plasmid DNA Extraction Mini Kit (Axygen, catalog number: AP-MN-P-250 )
Plasmid purification Midi (QIAGEN, catalog number: 12145 )
AxyPrep DNA Gel Extraction Kit (Axygen, catalog number: AP-GX-250 )
Isopropanol (SCRC, catalog number: 80109218 )
Sodium acetate (Sigma-Aldrich, catalog number: S2889 )
Proteinase K (Sigma-Aldrich, catalog number: P2308 )
Chloroform (SCRC, catalog number: 100068720 )
mMESSAGE mMACHINE^® Kit High Yield Capped RNA Transcription Kit (Ambion, catalog number: AM1344 )
Xenopus (sh-maosheng Co.)
Collagenase (Sigma-Aldrich, catalog number: C0130 )
Trypsin inhibitor (Sigma-Aldrich, catalog number: T2011 )
KCl (Sigma-Aldrich, catalog number: P3911 )
NaCl (Sigma-Aldrich, catalog number: S7653 )
MgCl₂·6H₂O (Sigma-Aldrich, catalog number: M2670 )
HEPES (Sigma-Aldrich, catalog number: PHR1428 )
CaCl₂ (Sigma-Aldrich, catalog number: C1016 )
NaHCO₃ (Sigma-Aldrich, catalog number: S5761 )
MgSO₄·7H₂O (Sigma-Aldrich, catalog number: M2773 )
MES (Sigma-Aldrich, catalog number: M3671 )
Streptomycin (Sigma-Aldrich, catalog number: 5711 )
Ampicillin (Sigma-Aldrich, catalog number: A5345 )
Gentamicin sulfate (Sigma-Aldrich, catalog number: G1914 )
Inorganic salt stock solution (see Recipes)
Antibiotic stock solutions (see Recipes)
LB medium (see Recipes)
LB agar (see Recipes)
ND96 solution (Ca²⁺-free) (see Recipes)
ND96 solution (see Recipes)
MBS solution (see Recipes)
K⁺-Free MBS solution (see Recipes)
Oocytes isolation solution (see Recipes)

Equipment

Pipettes (Eppendorf, model: Research^® plus , 0.5-10 μl, 10-100 μl, 100-1,000 μl)
Centrifuge (Eppendorf, model: Centrifuge 5242R )
Thermal Cycler (Eppendorf, model: Mastercycler^® nexus gradient)
Water Bath Shaker (BIOER, model: N1-13S )
Micropipette Puller (Sutter Instrument Company, model: P-2000 )
Microinjection (Harvard Apparatus, model: PLI-100 )
Nano Photometer (Implen, model: P-360 )
pH Meter (Mettler Toledo, model: FE20K )
ICP-AES (Agilent, model: 4100MP-AES )
Dissecting microscope (Olympus, model: SZX16 )