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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   

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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), Voltage-clamp(电压钳), Oocytes(卵母细胞), SLAC1(SLAC1), ABA receptor(ABA受体), 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

  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

  1. Prepare cRNA
    1. All constructs were cloned into the pNB1 oocyte expression vector using the USER method (Nour-Eldin et al., 2006).
    2. cRNAs were synthesized from 0.5-1 μg of linearized plasmid DNA template using the mMESSAGE mMACHINE® T7 Kit from Thermo Fisher Scientific.

  2. Isolation of oocytes
    1. Individual Xenopus laevis oocytes can be ordered from Ecocyte Bioscience and arrives the next day on dry ice. Alternatively, ovary lobes can be surgically extracted as described in previous reports (Miledi, 1982; Gundersen et al., 1983; Stühmer and Parekh, 1995).
    2. To isolate oocytes from ovary lobes, wash ovary lobes with ND96 buffer 3 times, incubate it in ND96 buffer at 16 °C overnight.
    3. Place 2-3 ovary lobes in ND96 buffer containing 30 mg/ml collagenase D, shake 2-3 h to remove follicle cell layer at room temperature (~23 °C) (Figure 1A).
    4. Wash oocytes with ND96 buffer 5 times, sort stage IV and V oocytes (approximate diameter 1 mm, Wasserman et al., 1984) in ND96 buffer. Incubate sorted oocytes in ND96 buffer at 16 °C overnight before injecting cRNA (Figure 1B).

  3. Injection of cRNA
    1. Use stage IV and V oocytes, for which the follicle cell layer has been removed (Wasserman et al., 1984). Inject more than 30 oocytes with each mRNA combination that is to be investigated.
    2. Pull injection glass needles on P-87 flaming/brown microelectrode micropipette puller using borosilicate glass capillaries to produce injection needles (Figure 1C).
    3. Break off the needle tip with forceps so that it is easier to inject cRNA into oocytes (Figure 1D).
    4. Fill pipette tip with mineral oil to about one-third to two-thirds.
    5. Mount pipette to the microdispenser.
    6. Deposit cRNA sample onto Parafilm, to a total volume of cRNA > 1 μl.
    7. Fill the pipette with RNA solution with microdispenser by applying negative pressure to the pipette.
    8. Inject cRNA into selected oocytes. The volume of cRNA injected into oocytes is about 50 nl (oocyte volume ~500 nl). The concentration of injected cRNA is better more than 2 ng/μl (Figure 1E).
    9. Incubate oocytes in ND96 buffer at 16 °C for 2-3 days before recording currents.


      Figure 1. Oocytes and glass needle at different steps. A. Oocyte ovary lobes; B. Oocytes after isolation; C. Injection glass needle before the tip is ‘broken’; D. ‘Broken’ needle; E. An oocyte being injected with cRNA. Note that injection pipette is out of focus. F. An oocyte impaled with two electrodes for voltage clamping.

  4. Recording
    1. Pull recording glass electrodes on P-87 flaming/brown microelectrode micropipette puller using custom glass tubing.
    2. In some case, slightly break the needle tip so that it is easier to insert the micropipette into oocytes since needle tips can be too soft for multiple sequential injections into oocytes. Alternatively, press the electrode against the oocyte and tap the recording table. Vibrations aid in impaling the oocyte.
    3. Fill the micropipette with 3 M KCl. Place one electrode into each of the two holders, making sure that Ag/AgCl electrode wire contacts the KCl solution in the micropipette. The resistances of the filled electrodes were 0.5-1.5 M (Figure 1F).
    4. For anion channel recordings, steady state currents are recorded starting from a holding potential of 0 mV and ranging from +40 to -160 mV in -20 mV decrements, followed by a -120 mV voltage ‘tail’ pulse (Figure 2).
      Note: The time-dependent properties of SLAC1 channel currents in Xenopus laevis oocytes vary among individual oocytes. This may depend on posttranslational modification of the channel protein. This property is also known from guard cell recordings of the SLAC1-encoding S-type anion channels (Schmidt and Schroeder, 1994).


      Figure 2. OST1 activates SLAC1 anion channel currents in Xenopus laevis oocytes. A. The voltage protocol used for recording SLAC1 anion channel currents. B and C. Example of whole-cell current traces recorded from oocytes injected with cRNA of (B) SLAC1yc only and (C) SLAC1yc+OST1yn (in SLAC1yc, SLAC1 is tagged with the C-terminal ‘half’ of YFP and in OST1yn, OST1 is tagged with N-terminal ‘half’ of YFP. Constructs for similar experiments see Geiger et al., 2009; Lee et al., 2009; Hua et al., 2012; Brandt et al., 2015; Wang et al., 2016).

    5. To assess whether ‘monomeric’ abscisic acid ABA receptor PYL9 (Dupeux et al., 2011; Hao et al., 2011) activation of SLAC1 can be reconstituted, we co-injected cRNAs of the SLAC1yc channel, ABI1 (ABA-INSENSITIVE1) protein phosphatase, the PYL9 receptor, and the OST1yn protein kinase into oocytes (Geiger et al., 2009; Brandt et al., 2012). Without ABA, PYL9 did not activate the anion channel currents in oocytes expressing the ABI1 with SLAC1yc and OST1yn. When ABA was injected into oocytes 30 min before measuring of oocyte currents, SLAC1 anion channels activity was dramatically enhanced in oocytes expressing SLAC1yc, OST1yn, PYL9 and ABI1 (Figure 3). Thus, the ‘monomeric’ ABA receptor PYL9 enables ABA signaling reconstitution in oocytes.


      Figure 3. Reconstitution of ABA activation of SLAC1 anion channels by PYL9 ABA receptor. Average current of SLAC1 anion channels recorded at -140 mV. In the absence of ABA, the ABA receptor PYL9 is unable to enhance SLAC1 anion channels currents. However, in the presence of injected ABA, SLAC1 anion channels currents are greatly increased. Data are mean ± SEM (SLACyc, n = 9; SLAC1yc+OST1yn, n = 13; SLAC1yc+OST1yn+ABI1, n = 10; SLAC1yc+OST1yn+ABI1+PYL9, n = 12; SLAC1yc+OST1yn+ABI1+PYL9+ABA, n = 15).

Recipes

  1. ND96 buffer
    10 mM MES/Tris (pH 7.5)
    1 mM CaCl2
    1 mM MgCl2
    96 mM NaCl
    Osmolality is adjusted to 220 mM using D-sorbitol (Geiger et al., 2009; Wang et al., 2016)
  2. Recording buffer
    10 mM MES/Tris (pH 7.5)
    1 mM MgCl2
    1 mM CaCl2
    2 mM KCl
    24 mM NaCl
    70 mM Na-gluconate
    Osmolality is adjusted to 220 mM using D-sorbitol (Geiger et al., 2009; Wang et al., 2016)

Acknowledgments

This research was surpported by grants from the National Institutes of Health (GM060396) and the National Science Foundation (MCB1616236) to J.I.S.

References

  1. Brandt, B., Brodsky, D. E., Xue, S., Negi, J., Iba, K., Kangasjarvi, J., Ghassemian, M., Stephan, A. B., Hu, H. and Schroeder, J. I. (2012). Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action. Proc Natl Acad Sci USA 109(26): 10593-10598.
  2. Brandt, B., Munemasa, S., Wang, C., Nguyen, D., Yong, T., Yang, P. G., Poretsky, E., Belknap, T. F., Waadt, R., Aleman, F. and Schroeder, J. I. (2015). Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells. Elife 4: e03599.
  3. Dupeux, F., Santiago, J., Betz, K., Twycross, J., Park, S. Y., Rodriguez, L., Gonzalez-Guzman, M., Jensen, M. R., Krasnogor, N., Blackledge, M., Holdsworth, M., Cutler, S. R., Rodriguez, P. L. and Marquez, J. A. (2011). A thermodynamic switch modulates abscisic acid receptor sensitivity. EMBO J 30(20): 4171-4184.
  4. Geiger, D., Scherzer, S., Mumm, P., Stange, A., Marten, I., Bauer, H., Ache, P., Matschi, S., Liese, A., Al-Rasheid, K. A., Romeis, T. and Hedrich, R. (2009). Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair. Proc Natl Acad Sci USA 106(50): 21425-21430.
  5. Gundersen, C. B., Miledi, R. and Parker, I. (1983). Voltage-operated channels induced by foreign messenger RNA in Xenopus oocytes. Proc R Soc Lond B Biol Sci 220(1218): 131-140.
  6. Hao, Q., Yin, P., Li, W., Wang, L., Yan, C., Lin, Z., Wu, J. Z., Wang, J., Yan, S. F. and Yan, N. (2011). The molecular basis of ABA-independent inhibition of PP2Cs by a subclass of PYL proteins. Mol Cell 42(5): 662-672.
  7. Hua, D., Wang, C., He, J., Liao, H., Duan, Y., Zhu, Z., Guo, Y., Chen, Z. and Gong, Z. (2012). A plasma membrane receptor kinase, GHR1, mediates abscisic acid- and hydrogen peroxide-regulated stomatal movement in Arabidopsis. Plant Cell 24(6): 2546-2561
  8. Lee, S. C., Lan, W., Buchanan, B. B. and Luan, S. (2009). A protein kinase-phosphatase pair interacts with an ion channel to regulate ABA signaling in plant guard cells. Proc Natl Acad Sci U S A 106(50): 21419-21424.
  9. Miledi, R. (1982). A calcium-dependent transient outward current in Xenopus laevis oocytes. Proc R Soc Lond B Biol Sci 215(1201): 491-497.
  10. Nour-Eldin, H. H., Hansen, B. G., Norholm, M. H., Jensen, J. K. and Halkier, B. A. (2006). Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. Nucleic Acids Res 34(18): e122.
  11. Schmidt, C. and Schroeder, J. I. (1994). Anion selectivity of slow anion channels in the plasma membrane of guard cells (large nitrate permeability). Plant Physiol 106(1): 383-391.
  12. Stühmer, W. and Parekh, A. B. (1995). Electrophysiological recordings from Xenopus oocytes. In: Bert S. and Erwin N. (Eds.). Single-Channel Recording. Springer pp: 341-356.
  13. Wang, C., Hu, H., Qin, X., Zeise, B., Xu, D., Rappel, W. J., Boron, W. F. and Schroeder, J. I. (2016). Reconstitution of CO2 regulation of SLAC1 anion channel and function of CO2-permeable PIP2;1aquaporin as CARBONIC ANHYDRASE4 interactor. Plant Cell 28(2): 568-582.
  14. Wasserman, W. J., Houle, J. G. and Samuel, D. (1984). The maturation response of stage IV, V, and VI Xenopus oocytes to progesterone stimulation in vitro. Dev Biol 105(2): 315-324.

简介

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 可以使用双电极电压钳位来研究在非洲爪蟾卵母细胞中表达的离子通道。该方案提供了一种测量在卵母细胞中表达的离子通道或转运蛋白电流的方法,包括植物离子通道。在本协议中,我们不仅总结了如何制备cRNA,分离卵母细胞,注射cRNA和记录电流,还提供了如何在从受体到离子通道共表达信号转导级联的过程中成功完成实验的信息。

关键字:离子通道, 电压钳, 卵母细胞, SLAC1, ABA受体, 慢流动型阴离子通道

材料和试剂

  1. 硼硅玻璃毛细管(World Precision Instruments,目录号:1B100F-4)
  2. 石蜡膜(Sigma-Aldrich,目录号:P7793-1EA)
  3. 非洲爪蟾卵母细胞(Ecocyte Bioscience,目录号:0-100-2)
  4. 载体:使用USER方法(Nour-Eldin等人,2006)携带感兴趣的cDNA的pNB1卵母细胞表达载体。或其他卵母细胞表达载体,如
  5. mMESSAGE mMACHINE T7试剂盒(Thermo Fisher Scientific,Ambion TM,目录号:AM1344)
  6. 胶原酶D(Roche Diagnostics,目录号:11088882001)
  7. 矿物油(Sigma-Aldrich,目录号:M5904)
  8. MES水合物(Sigma-Aldrich,目录号:M2933)
  9. Tris-base(Thermo Fisher Scientific,Fisher Scientific,目录号:BP152-5)
  10. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C5670)
  11. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  12. 氯化钠(NaCl)(Thermo Fisher Scientific,Fisher Scientific,目录号:S271-10)
  13. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  14. 葡萄糖酸钠(Sigma-Aldrich,目录号:S2054)
  15. D-山梨糖醇(Sigma-Aldrich,目录号:S1876)
  16. 庆大霉素溶液(Sigma-Aldrich,目录号:G1272)
  17. ND96缓冲液(参见食谱)
  18. 录音缓冲(见配方)

设备

  1. 双电极电压钳位放大器(例如,Warner Instrument,型号:Oocyte Clamp OC-725C)
  2. Digidata 1440A低噪声数据采集系统(Molecular Devices,型号:Digidata 1440A)
  3. P-87火焰/棕色微电极微量吸管拔出器(Sutter Instrument,型号:P-87)
  4. 渗透计(例如,,Wescor,型号:蒸气压渗透计5500)
  5. 微分子(Drummond Scientific,目录号:3-000-510)
  6. 定制玻璃管(Drummond Scientific,目录号:3-000-210-G8)

软件

  1. pCLAMP 10电生理学数据采集和分析软件(Molecular Devices)。

程序

  1. 准备cRNA
    1. 使用USER方法(Nour-Eldin等人,2006)将所有构建体克隆到pNB1卵母细胞表达载体中。
    2. 使用来自Thermo Fisher Scientific的mMESSAGE mMACHINE T7试剂盒,从0.5-1μg线性化质粒DNA模板合成cRNA。

  2. 卵母细胞的分离
    1. 个体的非洲爪蟾卵母细胞可以从Ecocyte Bioscience订购,并在第二天到达干冰。或者,可以如先前报道(Miledi,1982; Gundersen等人,1983;Stühmer和Parekh,1995)所述手术提取卵巢瓣。
    2. 为了从卵巢叶中分离卵母细胞,用ND96缓冲液洗涤卵巢瓣3次,在ND96缓冲液中于16℃孵育过夜。
    3. 在含有30mg/ml胶原酶D的ND96缓冲液中放置2-3个卵巢,摇动2-3小时以在室温(〜23℃)下除去卵泡细胞层(图1A)。
    4. 用ND96缓冲液洗涤卵母细胞5次,对ND96缓冲液中的IV期和V卵母细胞(大约直径1mm,Wasserman等人,1984)进行分类。在注射cRNA前,在16℃孵育ND96缓冲液中的分选卵母细胞过夜,(图1B)

  3. 注射cRNA
    1. 使用已经去除卵泡细胞层的阶段IV和V卵母细胞(Wasserman等人,1984)。每个mRNA组合注射超过30个卵母细胞。
    2. 使用硼硅酸盐玻璃毛细管在P-87火焰/棕色微电极微量吸管拔出器上拉注射玻璃针,以生产注射针(图1C)。
    3. 用镊子切断针尖,以便更容易将cRNA注入卵母细胞(图1D)。
    4. 用矿物油填充吸管头至约三分之一至三分之二。
    5. 将移液器安装到微型分配器。
    6. 将cRNA样品沉积到石蜡膜上,达到总体积的cRNA> 1μl。
    7. 通过向移液管施加负压,用微量分散器将移液管与RNA溶液填充。
    8. 将cRNA注入选定的卵母细胞。注入卵母细胞的cRNA体积约为50 nl(卵母细胞体积〜500 nl)。注射的cRNA的浓度优于2ng /μl(图1E)。
    9. 在记录电流之前,在16℃下将ND96缓冲液中的卵母细胞孵育2-3天

      图1.不同步骤的卵母细胞和玻璃针。 A。卵母细胞瓣; B.卵母细胞分离后; C.注射玻璃针前尖端"断"; D."破"针E.注射cRNA的卵母细胞。注意注射移液器不在焦点。 F.用两个电极刺激卵母细胞进行电压钳位。

  4. 记录
    1. 使用定制玻璃管将P-87火焰/棕色微电极微量吸管拉出器上的记录玻璃电极拉出。
    2. 在某些情况下,稍微打破针尖,以便更容易将微量移液管插入卵母细胞,因为针尖可能太软而多次连续注射入卵母细胞。或者,将电极压在卵母细胞上,然后点击记录台。振动有助于刺激卵母细胞。
    3. 用3 M KCl填充微量移液器。将一个电极放入两个支架的每一个中,确保Ag/AgCl电极线接触微量移液管中的KCl溶液。填充电极的电阻为0.5-1.5M(图1F)。
    4. 对于阴离子通道记录,稳态电流从0 mV的保持电位开始记录,在-20 mV减量范围内从+40到-160 mV范围内记录,然后是-120 mV的电压"尾"脉冲(图2)。 br /> 注意:非洲爪蟾卵母细胞中SLAC1通道电流的时间依赖性质因个体卵母细胞而异。这可能取决于通道蛋白的翻译后修饰。这种性质也从SLAC1编码S型阴离子通道的保卫细胞记录中获得(Schmidt和Schroeder,1994)。


      图2. OST1激活非洲爪蟾卵母细胞中的SLAC1阴离子通道电流。 A.用于记录SLAC1阴离子通道电流的电压方案。 (B)SLAC1yc和(C)SLAC1yc + OST1yn(在SLAC1yc,SLAC1中)注射了cRNA的卵母细胞记录的全细胞电流曲线的实例用YFP的C末端"半"和OST1yn ,OST1被标记有YFP的N末端'一半',类似实验的构建参见Geiger等人,2009; Lee等人,2009; Hua < em/et al。,2012; Brandt等人,2015; Wang等人,2016)。

    5. 为了评估"单体"脱落酸ABA受体PYL9(Dupeux等人,2011; Hao等人,2011)可以重构SLAC1的激活,将SLAC1yc通道的注射的cRNA,ABI1(ABA-INSENSITIVE1)蛋白磷酸酶,PYL9受体和OST1yn蛋白激酶注入卵母细胞(Geiger等人,2009; Brandt等人,2012)。没有ABA,PYL9没有激活表达ABI1的卵母细胞中具有SLAC1yc和OST1yn的阴离子通道电流。在测量卵母细胞电流30分钟前将ABA注射到卵母细胞中,SLAC1阴离子通道活性在表达SLAC1yc,OST1yn,PYL9和ABI1的卵母细胞中显着增强(图3)。因此,"单体"ABA受体PYL9使ABA信号在卵母细胞中重建

      图3.通过PYL9 ABA受体重建SLAC1阴离子通道的ABA活化在-140mV记录的SLAC1阴离子通道的平均电流。在没有ABA的情况下,ABA受体PYL9不能增强SLAC1阴离子通道电流。然而,在注射ABA的情况下,SLAC1阴离子通道电流大大增加。数据为平均值±SEM(SLACyc,n = 9; SLAC1yc + OST1yn,n = 13; SLAC1yc + OST1yn + ABI1,n = 10; SLAC1yc + OST1yn + ABI1 + PYL9,n = 12; SLAC1yc + OST1yn + ABI1 + PYL9 + ABA,n = 15)

食谱

  1. ND96缓冲区
    10mM MES/Tris(pH7.5)
    1mM CaCl 2
    1mM MgCl 2
    96 mM NaCl
    使用D-山梨糖醇(Geiger等人,2009; Wang等人,2016)将重量摩尔渗透压浓度调节至220mM。
  2. 录音缓冲区
    10mM MES/Tris(pH7.5)
    1mM MgCl 2
    1mM CaCl 2
    2 mM KCl
    24 mM NaCl
    70 mM葡萄糖酸钠
    使用D-山梨糖醇(Geiger等人,2009; Wang等人,2016)将重量摩尔渗透压浓度调节至220mM。

致谢

该研究由美国国立卫生研究院(GM060396)和国家科学基金会(MCB1616236)向美国科学院院士

参考文献

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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.
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