使用蛋白质印迹法研究神经元K-Cl共转运蛋白KCC2的功能和活性
Summary
本协议重点介绍了蛋白质印迹技术在研究神经元K-Cl共转运蛋白KCC2的功能和活性方面的应用。该方案描述了通过蛋白质印迹 在 激酶调节位点Thr906/1007处KCC2磷酸化的研究。此外，本文还简要强调了确认KCC2活性的其他方法。
Abstract
氯化钾共转运蛋白2（KCC2）是阳离子氯化共转运蛋白（CCCs）溶质载体家族12（SLC12）的成员，仅在神经元中发现，对于Cl– 稳态的正常功能以及因此具有功能性GABA能抑制至关重要。KCC2的适当调节失败是有害的，并且与包括癫痫在内的几种神经系统疾病的流行有关。在了解KCC2调节所涉及的机制方面取得了相当大的进展，KCC2被认可为开发使研究人员能够研究其功能和活动的技术;通过直接（评估激酶调控位点磷酸化）或间接（观察和监测GABA活性） 调查 。在这里，该协议重点介绍了如何使用蛋白质印迹技术研究激酶调节位点 – Thr906 和 Thr1007 – 的 KCC2 磷酸化。还有其他经典方法可用于直接测量KCC2活性，例如铷离子和铊离子摄取测定。进一步的技术如膜片钳电生理学用于测量GABA活性;因此，间接反映活化和/或失活的KCC2，如细胞内氯离子稳态的评估所告知的。本文将简要讨论其中一些附加技术。
Introduction
氯化钾共转运蛋白2（KCC2）是阳离子氯化共转运蛋白（CCCs）溶质载体家族12（SLC12）的成员，仅在神经元中发现，对于Cl–稳态的正常功能以及因此功能性GABA能抑制1，2，3，4至关重要。KCC2将低神经元内Cl-浓度（[Cl-]i）维持在4-6mM，有助于大脑和脊髓中的γ-氨基丁酸（GABA）/甘氨酸超极化和突触抑制5。KCC2的适当调节失败与几种神经系统疾病的流行有关，包括癫痫4。此外，KCC2介导的Cl−挤出减少和超极化GABAA和/或甘氨酸受体介导的电流受损与癫痫，神经性疼痛和痉挛有关6，7。神经元 KCC2 通过无赖氨酸 （WNK）-STE20/SPS1 相关脯氨酸/富丙氨酸 （SPAK）/氧化应激响应 （OSR） 激酶信号传导复合物1 在其 C 末端细胞内结构域内的关键调节残基磷酸化而负调节，这有助于维持未成熟神经元中去极化的 GABA 活性2，8，9.WNK-SPAK/OSR1磷酸化苏氨酸残基906和1007（Thr906/Thr1007），随后下调KCC2的mRNA基因表达，导致其生理功能随之恶化8，10。然而，更重要的是，已知WNK-SPAK/OSR1激酶复合物磷酸化并抑制KCC2表达1，2，4，11，12已经是一个事实，并且抑制磷酸化Thr906 / Thr1007的激酶复合物信号通路与KCC2 mRNA基因13，14，15的表达增加有关。.重要的是要注意，通过蛋白质磷酸化调节神经元KCC2和Na + -K+-2Cl–共转运蛋白1（NKCC1）表达同时并以相反的模式1，4，16起作用。
在了解KCC2调节机制方面取得了持续和相当大的进展，认可开发使研究人员能够研究其功能和活动的技术;通过直接（评估激酶调控位点磷酸化）或间接（观察和监测GABA活性） 调查 。这里介绍的方案重点介绍了蛋白质印迹技术的应用，通过研究共转运蛋白在激酶调控位点Thr906/1007的磷酸化来研究神经元K+-Cl– 共转运蛋白KCC2的功能和活性。
蛋白质印迹是一种用于从组织或细胞样本中检测特定目标蛋白的方法。该方法首先通过电泳按大小分离蛋白质。然后将蛋白质电泳转移到固体载体（通常是膜）上，然后使用特异性抗体标记目标蛋白质。将抗体与使用比色法、化学发光法或荧光法检测的不同标签或荧光团偶联抗体偶联。这允许从蛋白质混合物中检测特定的目标蛋白质。该技术已用于表征KCCs1 的磷酸化特异性位点，并已用于鉴定抑制KCC3 Thr991 / Thr1048磷酸化的激酶抑制剂17。通过遵循该协议，可以特异性地检测细胞/组织裂解物中的总和磷酸化KCC2。原则上，通过该技术检测蛋白质偶联抗体具有很强的帮助性，因为它有助于提高对KCC2磷酸化位点合作活性的理解，从而阐明了参与其生理调节的分子机制。总蛋白表达的定量分析代表了KCC2的功能和活性。还有其他经典方法用于直接测量KCC2活性，例如铷离子和铊离子摄取测定。进一步的技术如膜片钳电生理学用于测量GABA活性;因此，间接反映活化和/或失活的KCC2，如细胞内氯离子稳态的评估所告知的。
Protocol
注意：该方案描述了用于检测特定目标蛋白质的蛋白质印迹方法。 1. 细胞培养和转染 在细胞培养过程之前加热珠浴（37°C）中的所有试剂。准备培养基，Dulbecco的改良鹰培养基（DMEM），补充有10%胎牛血清，1%的2mM L-谷氨酰胺，100x非必需氨基酸，100mM丙酮酸钠和100单位/ mL青霉素链霉素。 解冻稳定转染的大鼠KCC2b人胚胎肾293细胞（HEK293rnKCC2b…
Representative Results
在这里， 图1 中给出的代表性结果研究了星形孢菌素和NEM对使用蛋白质印迹技术稳定表达KCC2b（HEKrnKCC2b）18 的HEK293细胞系中WNK-SPAK/OSR1介导的KCC2和NKCC1磷酸化的影响。有关代表性结果的全面细节在Zhang等人15中进行了讨论。与NEM类似，星形孢菌素是一种广泛的激酶抑制剂，可以增强KCC2转运活性并抑制NKCC1活性15</su…
Discussion
许多方法已被用于测量在神经元中表达的CCC的SLC12的活性，包括KCC2。其中许多技术已被证明可以增强分析这些转运蛋白的功能相关性及其在不同疾病相关突变中的结构 – 功能模式的科学知识。至关重要的是，各种方法都有优点和注意事项21.然而，上面解释的方案概述了如何使用蛋白质印迹评估激酶调控位点Thr906和Thr1007的KCC2磷酸化，这有助于研究KCC2的功能和活性。
<p class="jove…Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了英国皇家学会（Grant no. IEC\NSFC\201094）和英联邦博士奖学金的支持。
Materials
| 40% acrylamide | Sigma-Aldrich | A2917 | Used to make seperating and stacking gel for SDS-PAGE |
| Ammonium Per Sulfate | Sigma-Aldrich | 248614 | Used to make seperating and stacking gel for SDS-PAGE |
| anti pSPAK | Dundee University | S670B | Used as primary antibody for western blotting |
| anti-KCC2 | Dundee University | S700C | Used as primary antibody for western blotting |
| anti-KCC2 pSer940 | Thermo Fisher Scientific | PA5-95678 | Used as primary antibody for western blotting |
| anti-KCC2 pThr1007 | Dundee University | S961C | Used as primary antibody for western blotting |
| anti-KCC2 pThr906 | Dundee University | S959C | Used as primary antibody for western blotting |
| anti-mouse | Cell Signalling technology | 66002 | Used as secondary antibody for western blotting |
| anti-NKCC1 | Dundee University | S841B | Used as primary antibody for western blotting |
| anti-NKCC1 pThr203/207/212 | Dundee University | S763B | Used as primary antibody for western blotting |
| anti-rabbit | Cell Signalling technology | C29F4 | Used as secondary antibody for western blotting |
| anti-sheep | abcam | ab6900 | Used as secondary antibody for western blotting |
| anti-SPAK | Dundee University | S669D | Used as primary antibody for western blotting |
| anti-β-Tubulin III | Sigma-Aldrich | T8578 | Used as primary antibody for western blotting |
| Benzamine | Merck UK | 135828 | Used as component of lysis buffer |
| Beta-mercaptoethanol | Sigma-Aldrich | M3148 | Used as component of loading buffer and lysis buffer |
| Bradford Coomasie | Thermo Scientific | 1856209 | Used for lysate protein quantification |
| Casting apparatus | Atto | WSE-1165W | Used to run SDS-page electrophoresis |
| Centrifuge | Eppendorf | 5804 | Used in lysate preparation |
| Centrifuge | VWR | MicroStar 17R | Used for spinning samples |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D2650-100ML | Used for cell culture experiment |
| Dried Skimmed Milk | Marvel | N/A | Used to make blocking buffer |
| Dulbecco's Modified Eagle's Medium – high glucose | Sigma-Aldrich | D6429 | Used for cell culture |
| ECL reagent | Perkin Elmer | ORTT755/2655 | Used to develop image for western blotting |
| EDTA | Fisher Scientific | D/0700/53 | Used as component of lysis buffer |
| EGTA | Sigma-Aldrich | e4378 | Used as component of lysis buffer |
| Electrophoresis Power Supply | BioRad | PowerPAC HC | To supply power to run SDS-page electrophoresis |
| Ethanol | ThermoFisher | E/0650DF/17 | Used for preparing sterilized equipments and environment |
| Fetal Bovine Serum - heat inactivated | Merck Life Sciences UK | F9665 | Used for cell culture |
| Fumehood | Walker | A7277 | Used for cell culture |
| Gel Blotting – Whatman | GE Healthcare | 10426981 | Used in western blotting to make transfer sandwich |
| Glycine | Sigma-Aldrich | 15527 | Used to make buffers |
| GraphPad Prism Software | GraphPad Software, Inc., USA | Version 6.0 | Used for plotting graphs and analysing data for western blotting |
| HCl | Acros Organics | 10647282 | Used to make seperating and stacking gel for SDS-PAGE |
| Heating block | Grant | QBT1 | Used to heat WB loading samples |
| HEK293 cells | Merck UK | 12022001-1VL | Cell line for culture experiment |
| ImageJ Software | Wayne Rasband and Contributors; NIH, USA | ImageJ 1.53e | Used to measure band intensities from western blotting images |
| Imaging system | BioRad | ChemiDoc MP | Used to take western blotting images |
| Incubator | LEEC | LEEC precision 190D | Used for cell culture |
| Isopropanol | Honeywell | 24137 | Used in casting gel for electrophoresis |
| L-glutamine solution | Sigma-Aldrich | G7513 | Used for cell culture |
| Lithium dodecyl sulfate (LDS) | Novex | NP0008 | Used as loading buffer for western blotting |
| MEM Non-essential amino acid | Merck Life Sciences UK | M7145 | Used for cell culture |
| Microcentrifuge | Eppendorf | 5418 | Used for preparing lysates for WB |
| Microplate reader | BioRad | iMark | Used for lysate protein concentration readout |
| Microsoft Powerpoint | Microsoft, USA | PowerPoint2016 | Used to edit western blotting images |
| Molecular Weight Marker | BioRad | 1610373 | Used for western blotting |
| N-ethylmaleimide | Thermo Fisher Scientific | 23030 | Used for cell culture experiment |
| Nitrocellulose membrane | Fisher Scientific | 45004091 | Used for western blotting |
| Penicillin-Streptomycin | Gibco | 15140122 | Used for cell culture |
| pH Meter | Mettler Toledo | Seven compact s210 | Used to monitor pH of buffer solutions |
| Phenylmethylsulfonylfluoride (PMSF) | Sigma-Aldrich | P7626 | Used as component of lysis buffer |
| Phosphate Buffer Saline | Sigma-Aldrich | D8537 | Used for cell culture |
| PKCδ pThr505 | Cell Signalling technology | 9374 | Used as primary antibody for western blotting |
| Sepharose Protein G | Generon | PG50-00-0002 | Used for immunoprecipitation |
| Sodium chloride | Sigma-Aldrich | S7653 | Used as component of wash buffer |
| Sodium Chloride | Sigma-Aldrich | S7653 | Used to prepare TBS-T buffer |
| Sodium Dodecyl Sulfate | Sigma-Aldrich | L5750 | Used to make seperating and stacking gel for SDS-PAGE |
| sodium orthovanadate | Sigma-Aldrich | S6508 | Used as component of lysis buffer |
| Sodium Pyruvate | Sigma-Aldrich | S8636 | Used for cell culture |
| sodium-β-glycerophosphate | Merck UK | G9422 | Used as component of lysis buffer |
| Staurosporine (from Streptomyces sp.) | Scientific Laboratory Supplies, UK | S4400-1MG | Used for cell culture experiment |
| Sucrose | Scientifc Laboratory Supplies | S0389 | Used as component of lysis buffer |
| TEMED | Sigma-Aldrich | T7024 | Used to make seperating and stacking gel for SDS-PAGE |
| Transfer Chamber | BioRad | 1658005EDU | Used in western blotting to transfer protein on membrane |
| Tris | Sigma-Aldrich | T6066 | Used to make seperating and stacking gel for SDS-PAGE |
| Triton-X100 | Sigma-Aldrich | T8787 | Used as component of lysis buffer |
| Trypsin-EDTA Solution | Merck Life Sciences UK | T4049 | Used for cell culture |
| Tween-20 | Sigma-Aldrich | P3179 | Used as make TBS-T buffer |
| Vacuum pump | Charles Austen | Dymax 5 | Used for cell culture |
| Vortex | Scientific Industries | K-550-GE | Used in sample preparation |
| Vortex mixer | Scientific Industries Ltd | Vortex-Genie K-550-GE | Used of mixing resolved sample |
| Water bath | Grant Instruments Ltd. (JB Academy) | JBA5 | Used to incubate solutions |
Referências
- de Los Heros, P., et al. The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K+-Cl- co-transporters. Biochemical Journal. 458 (3), 559-573 (2014).
- Heubl, M., et al. GABAA receptor dependent synaptic inhibition rapidly tunes KCC2 activity via the Cl(-)-sensitive WNK1 kinase. Nature Communications. 8 (-), 1776 (2017).
- Schulte, J. T., Wierenga, C. J., Bruining, H. Chloride transporters and GABA polarity in developmental, neurological and psychiatric conditions. Neuroscience & Biobehavioral Reviews. 90, 260-271 (2018).
- Shekarabi, M., et al. WNK Kinase Signaling in Ion Homeostasis and Human Disease. Cell Metabolism. 25 (2), 285-299 (2017).
- Rivera, C., et al. The K+/Cl- co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature. 397 (6716), 251-255 (1999).
- Kahle, K. T., et al. Modulation of neuronal activity by phosphorylation of the K-Cl cotransporter KCC2. Trends in Neuroscience. 36 (12), 726-737 (2013).
- Andrews, K., Josiah, S. S., Zhang, J. The Therapeutic Potential of Neuronal K-Cl Co-Transporter KCC2 in Huntington’s Disease and Its Comorbidities. International Journal of Molecular Sciences. 21 (23), 9142 (2020).
- Friedel, P., et al. WNK1-regulated inhibitory phosphorylation of the KCC2 cotransporter maintains the depolarizing action of GABA in immature neurons. Science Signaling. 8 (383), 65 (2015).
- Watanabe, M., et al. Developmentally regulated KCC2 phosphorylation is essential for dynamic GABA-mediated inhibition and survival. Science Signaling. 12 (603), (2019).
- Rinehart, J., et al. Sites of regulated phosphorylation that control K-Cl cotransporter activity. Cell. 138 (3), 525-536 (2009).
- Lu, D. C. -. Y., et al. The role of WNK in modulation of KCl cotransport activity in red cells from normal individuals and patients with sickle cell anaemia. Pflügers Archiv-European Journal of Physiology. 471 (11-12), 1539-1549 (2019).
- Huang, H., et al. The WNK-SPAK/OSR1 Kinases and the Cation-Chloride Cotransporters as Therapeutic Targets for Neurological Diseases. Aging and Disease. 10 (3), 626-636 (2019).
- AlAmri, M. A., Kadri, H., Alderwick, L. J., Jeeves, M., Mehellou, Y. The Photosensitising Clinical Agent Verteporfin Is an Inhibitor of SPAK and OSR1 Kinases. Chembiochem. 19 (19), 2072-2080 (2018).
- Zhang, J., et al. Modulation of brain cation-Cl(-) cotransport via the SPAK kinase inhibitor ZT-1a. Nature Communications. 11 (1), 78 (2020).
- Zhang, J., et al. Staurosporine and NEM mainly impair WNK-SPAK/OSR1 mediated phosphorylation of KCC2 and NKCC1. PLoS One. 15 (5), 0232967 (2020).
- Alessi, D. R., et al. The WNK-SPAK/OSR1 pathway: master regulator of cation-chloride cotransporters. Science Signaling. 7 (334), 3 (2014).
- Zhang, J., et al. Functional kinomics establishes a critical node of volume-sensitive cation-Cl(-) cotransporter regulation in the mammalian brain. Scientific Reports. 6, 35986 (2016).
- Hartmann, A. M., et al. Opposite effect of membrane raft perturbation on transport activity of KCC2 and NKCC1. Journal of Neurochemistry. 111 (2), 321-331 (2009).
- Pisella, L. I., et al. Impaired regulation of KCC2 phosphorylation leads to neuronal network dysfunction and neurodevelopmental pathology. Science Signaling. 12 (603), (2019).
- Blaesse, P., et al. Oligomerization of KCC2 correlates with development of inhibitory neurotransmission. The Journal of Neuroscience. 26 (41), 10407-10419 (2006).
- Medina, I., Pisella, L. I. . Neuronal Chloride Transporters in Health and Disease. , 21-41 (2020).
- Thomas, P., Smart, T. G. HEK293 cell line: a vehicle for the expression of recombinant proteins. Journal of Pharmacological and Toxicological Methods. 51 (3), 187-200 (2005).
- Friedel, P., et al. A Novel View on the Role of Intracellular Tails in Surface Delivery of the Potassium-Chloride Cotransporter KCC2. eNeuro. 4 (4), (2017).
- Lee, Y. -. C., et al. Impact of detergents on membrane protein complex isolation. Journal of Proteome Research. 17 (1), 348-358 (2018).
- Vallée, B., Doudeau, M., Godin, F., Bénédetti, H. Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein. JoVE (Journal of Visualized Experiments). (148), e59820 (2019).
- Johansen, K., Svensson, L. . Molecular Diagnosis of Infectious Diseases. , 15-28 (1998).
- Mahmood, T., Yang, P. -. C. Western blot: technique, theory, and trouble shooting. North American Journal of Medical Sciences. 4 (9), 429 (2012).
- Klein, J. D., O’Neill, W. C. Volume-sensitive myosin phosphorylation in vascular endothelial cells: correlation with Na-K-2Cl cotransport. American Journal of Physiology-Cell Physiology. 269 (6), 1524-1531 (1995).
- Hannemann, A., Flatman, P. W. Phosphorylation and transport in the Na-K-2Cl cotransporters, NKCC1 and NKCC2A, compared in HEK-293 cells. PLoS One. 6 (3), 17992 (2011).
- Liu, J., Ma, X., Cooper, G. F., Lu, X. Explicit representation of protein activity states significantly improves causal discovery of protein phosphorylation networks. BMC Bioinformatics. 21 (13), 1-17 (2020).
- Terstappen, G. C. Nonradioactive rubidium ion efflux assay and its applications in drug discovery and development. Assay and Drug Development Technologies. 2 (5), 553-559 (2004).
- Carmosino, M., Rizzo, F., Torretta, S., Procino, G., Svelto, M. High-throughput fluorescent-based NKCC functional assay in adherent epithelial cells. BMC Cell Biology. 14 (1), 1-9 (2013).
- Adragna, N. C., et al. Regulated phosphorylation of the K-Cl cotransporter KCC3 is a molecular switch of intracellular potassium content and cell volume homeostasis. Frontiers in Cellular Neuroscience. 9, 255 (2015).
- Zhang, D., Gopalakrishnan, S. M., Freiberg, G., Surowy, C. S. A thallium transport FLIPR-based assay for the identification of KCC2-positive modulators. Journal of Biomolecular Screening. 15 (2), 177-184 (2010).
- Yu, H. B., Li, M., Wang, W. P., Wang, X. L. High throughput screening technologies for ion channels. Acta Pharmacologica Sinica. 37 (1), 34-43 (2016).
- Hill, C. L., Stephens, G. J. An Introduction to Patch Clamp Recording. Patch Clamp Electrophysiology. , 1-19 (2021).
- Conway, L. C., et al. N-Ethylmaleimide increases KCC2 cotransporter activity by modulating transporter phosphorylation. Journal of Biological Chemistry. 292 (52), 21253-21263 (2017).
- Heigele, S., Sultan, S., Toni, N., Bischofberger, J. Bidirectional GABAergic control of action potential firing in newborn hippocampal granule cells. Nature Neuroscience. 19 (2), 263-270 (2016).
- Moore, Y. E., Deeb, T. Z., Chadchankar, H., Brandon, N. J., Moss, S. J. Potentiating KCC2 activity is sufficient to limit the onset and severity of seizures. Proceedings of the National Academy of Sciences of the United States of America. 115 (40), 10166-10171 (2018).
- Kim, H. R., Rajagopal, L., Meltzer, H. Y., Martina, M. Depolarizing GABAA current in the prefrontal cortex is linked with cognitive impairment in a mouse model relevant for schizophrenia. Science Advances. 7 (14), 5032 (2021).
- Yelhekar, T. D., Druzin, M., Karlsson, U., Blomqvist, E., Johansson, S. How to properly measure a current-voltage relation?-interpolation vs. ramp methods applied to studies of GABAA receptors. Frontiers in Cellular Neuroscience. 10, 10 (2016).
- Ishibashi, H., Moorhouse, A. J., Nabekura, J. Perforated whole-cell patch-clamp technique: a user’s guide. Patch Clamp Techniques. , 71-83 (2012).
- Ebihara, S., Shirato, K., Harata, N., Akaike, N. Gramicidin-perforated patch recording: GABA response in mammalian neurones with intact intracellular chloride. The Journal of Physiology. 484 (1), 77-86 (1995).
- Kyrozis, A., Reichling, D. B. Perforated-patch recording with gramicidin avoids artifactual changes in intracellular chloride concentration. Journal of Neuroscience Methods. 57 (1), 27-35 (1995).
- Lamsa, K., Palva, J. M., Ruusuvuori, E., Kaila, K., Taira, T. Synaptic GABAA activation inhibits AMPA-kainate receptor-mediated bursting in the newborn (P0-P2) rat hippocampus. Journal of Neurophysiology. 83 (1), 359-366 (2000).
Citar este artigo
Josiah, S. S., Meor Azlan, N. F., Oguro-Ando, A., Zhang, J. Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting. J. Vis. Exp. (190), e64179, doi:10.3791/64179 (2022).