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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

发布: 2020年11月05日第10卷第21期 DOI: 10.21769/BioProtoc.3802 浏览次数: 4418

评审: Dheeraj Singh RathoreAnonymous reviewer(s)

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

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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) search K+ release (K+ 释放) search Potassium transporter (钾转运蛋白) search K+ measurement (K+ 测定) search Membrane transport proteins (膜转运蛋白) search

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

  1. Pipette tips (Axygen, catalog numbers: T-300, T-200-Y, T-1000-B )

  2. Scalpel blade (GLOBALMED, catalog number: BD001 )

  3. 1.5 ml microcentrifuge tubes (Axygen, catalog number: MCT-150-C )

  4. Petri dish (Nunc, catalog number: 150466 )

  5. 24-well plate (Nunc, catalog number: 142475 )

  6. Capillary glass (Sutter Instrument Company, catalog number: B100-58-10 )

  7. Parafilm (Bemis, catalog number: PM-996 )

  8. Micropipette tip (SUTTER, catalog number: BF100-50-10 )

  9. 3-ml plastic pipettes dropper ( KEHUA, catalog number: KH145 )

  10. 0.22 μm Syringe filter (GREEN MALL, catalog number: RSF02 )

  11. Competent E. coli DH5α (TIANGEN, catalog number: CB101-02 )

  12. pT7TS (provided by Xiaorong Fan, Nanjing Agricultural University)

  13. Plasmid Purification Midi Kit (QIAGEN, catalog number: 12143 )

  14. KOD polymerase (TOYOBO, catalog number: KOD-201B )

  15. Agarose (BIOWEST, catalog number: B21033 )

  16. DNA Gel Extraction Kit (AxyGEN, catalog number: AP-GX-250 )

  17. Restriction enzyme Bgl II (Takara Bio, catalog number: 1021A )

  18. Restriction enzyme Spe I (Takara Bio, catalog number: D1086a )

  19. Restriction enzyme BamH I (Takara Bio, catalog number: 1010 )

  20. T4 Ligase (STRATAGENE, catalog number: M0202V )

  21. Plasmid DNA Extraction Mini Kit (Axygen, catalog number: AP-MN-P-250 )

  22. Plasmid purification Midi (QIAGEN, catalog number: 12145 )

  23. AxyPrep DNA Gel Extraction Kit (Axygen, catalog number: AP-GX-250 )

  24. Isopropanol (SCRC, catalog number: 80109218 )

  25. Sodium acetate (Sigma-Aldrich, catalog number: S2889 )

  26. Proteinase K (Sigma-Aldrich, catalog number: P2308 )

  27. Chloroform (SCRC, catalog number: 100068720 )

  28. mMESSAGE mMACHINE® Kit High Yield Capped RNA Transcription Kit (Ambion, catalog number: AM1344 )

  29. Xenopus (sh-maosheng Co.)

  30. Collagenase (Sigma-Aldrich, catalog number: C0130 )

  31. Trypsin inhibitor (Sigma-Aldrich, catalog number: T2011 )

  32. KCl (Sigma-Aldrich, catalog number: P3911 )

  33. NaCl (Sigma-Aldrich, catalog number: S7653 )

  34. MgCl2·6H2O (Sigma-Aldrich, catalog number: M2670 )

  35. HEPES (Sigma-Aldrich, catalog number: PHR1428 )

  36. CaCl2 (Sigma-Aldrich, catalog number: C1016 )

  37. NaHCO3 (Sigma-Aldrich, catalog number: S5761 )

  38. MgSO4·7H2O (Sigma-Aldrich, catalog number: M2773 )

  39. MES (Sigma-Aldrich, catalog number: M3671 )

  40. Streptomycin (Sigma-Aldrich, catalog number: 5711 )

  41. Ampicillin (Sigma-Aldrich, catalog number: A5345 )

  42. Gentamicin sulfate (Sigma-Aldrich, catalog number: G1914 )

  43. Inorganic salt stock solution (see Recipes)

  44. Antibiotic stock solutions (see Recipes)

  45. LB medium (see Recipes)

  46. LB agar (see Recipes)

  47. ND96 solution (Ca2+-free) (see Recipes)

  48. ND96 solution (see Recipes)

  49. MBS solution (see Recipes)

  50. K+-Free MBS solution (see Recipes)

  51. Oocytes isolation solution (see Recipes)

Equipment

  1. Pipettes (Eppendorf, model: Research® plus , 0.5-10 μl, 10-100 μl, 100-1,000 μl)

  2. Centrifuge (Eppendorf, model: Centrifuge 5242R )

  3. Thermal Cycler (Eppendorf, model: Mastercycler® nexus gradient)

  4. Water Bath Shaker (BIOER, model: N1-13S )

  5. Micropipette Puller (Sutter Instrument Company, model: P-2000 )

  6. Microinjection (Harvard Apparatus, model: PLI-100 )

  7. Nano Photometer (Implen, model: P-360 )

  8. pH Meter (Mettler Toledo, model: FE20K )

  9. ICP-AES (Agilent, model: 4100MP-AES )

  10. Dissecting microscope (Olympus, model: SZX16 )

Software

  1. Microsoft Excel (Microsoft)

  2. DNAMAN

Procedure

English
中文翻译

文章信息

版权信息

© 2020 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Li, H. (2020). K+ Release Assay and K+ Measurement in Oocyte Assay. Bio-protocol 10(21): e3802. DOI: 10.21769/BioProtoc.3802.

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分类

植物科学 > 植物生理学 > 营养

生物化学 > 其它化合物 > 离子

细胞生物学 > 基于细胞的分析方法 > 转运

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