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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The present protocol highlights the application of western blotting technique to study the functions and activities of neuronal K-Cl co-transporter KCC2. The protocol describes the investigation of KCC2 phosphorylation at kinase regulatory sites Thr906/1007 via western blotting. Also, additional methods to confirm KCC2 activity are briefly highlighted in this text.

Abstract

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively in the neuron and is essential for the proper functioning of Cl- homeostasis and consequently functional GABAergic inhibition. Failure in proper regulation of KCC2 is deleterious and has been associated with the prevalence of several neurological diseases, including epilepsy. There has been considerable progress with regard to understanding the mechanisms involved in the regulation of KCC2, accredited to the development of techniques that enable researchers to study its functions and activities; either via direct (assessing kinase regulatory sites phosphorylation) or indirect (observing and monitoring GABA activity) investigations. Here, the protocol highlights how to investigate KCC2 phosphorylation at kinase regulatory sites - Thr906 and Thr1007- using western blotting technique. There are other classic methods used to directly measure KCC2 activity, such as rubidium ion and thallium ion uptake assay. Further techniques such as patch-clamp-electrophysiology are used to measure GABA activity; hence, indirectly reflecting activated and/or inactivated KCC2 as informed by the assessment of intracellular chloride ion homeostasis. A few of these additional techniques will be briefly discussed in this manuscript.

Introduction

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively in the neuron and is essential for the proper functioning of Cl- homeostasis and consequently functional GABAergic inhibition1,2,3,4. The maintenance of low intraneuronal Cl- concentration ([Cl-]i) at 4-6 mM by KCC2 facilitates γ-aminobutyric acid (GABA)/glycine hyperpolarization and synaptic inhibition in the brain and spinal cord<....

Protocol

NOTE: The protocol describes the western blotting method to detect specific proteins of interest.

1. Cell culture and transfection

  1. Warm all the reagents in the bead bath (37 ËšC) before the cell culture procedure. Prepare culture medium, Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum, 1% each of 2mM L-glutamine, 100x non-essential amino acid, 100 mM sodium pyruvate, and 100 units/mL penicillin-streptomycin.
  2. Thaw .......

Representative Results

Here, the representative result presented in Figure 1 investigated the impact of staurosporine and NEM on WNK-SPAK/OSR1 mediated phosphorylation of KCC2 and NKCC1 in HEK293 cell lines stably expressing KCC2b (HEKrnKCC2b)18 using the western blotting technique. Comprehensive details on the representative results are discussed in Zhang et al.15. Similar to NEM, staurosporine is a broad kinase inhibitor that can enhance KCC2 tr.......

Discussion

Many methods have been used to measure the activities of SLC12 of CCCs that are expressed in the neurons, including KCC2. Many of these techniques have proven to enhance scientific knowledge on the analysis of the functional relevance of these transporters and their structure-function patterns in different disease-related mutations. Critically, there are advantages and caveats to the various methods21. However, the protocol explained above, outlined how to assess KCC2 phosphorylation at kinase reg.......

Acknowledgements

This work was supported by The Royal Society UK (Grant no. IEC\NSFC\201094), and a Commonwealth Ph.D. Scholarship.

....

Materials

NameCompanyCatalog NumberComments
40% acrylamideSigma-AldrichA2917Used to make seperating and stacking gel for SDS-PAGE 
Ammonium Per SulfateSigma-Aldrich248614Used to make seperating and stacking gel for SDS-PAGE 
anti pSPAKDundee UniversityS670BUsed as primary antibody for western blotting
anti-KCC2Dundee UniversityS700CUsed as primary antibody for western blotting
anti-KCC2 pSer940Thermo Fisher ScientificPA5-95678Used as primary antibody for western blotting
anti-KCC2 pThr1007Dundee UniversityS961CUsed as primary antibody for western blotting
anti-KCC2 pThr906Dundee UniversityS959CUsed as primary antibody for western blotting
anti-mouseCell Signalling technology66002Used as secondary antibody for western blotting
anti-NKCC1Dundee UniversityS841BUsed as primary antibody for western blotting
anti-NKCC1 pThr203/207/212Dundee UniversityS763BUsed as primary antibody for western blotting
anti-rabbitCell Signalling technologyC29F4Used as secondary antibody for western blotting
anti-sheepabcamab6900Used as secondary antibody for western blotting
anti-SPAKDundee UniversityS669DUsed as primary antibody for western blotting
anti-β-Tubulin IIISigma-AldrichT8578Used as primary antibody for western blotting
BenzamineMerck UK135828Used as component of lysis buffer
Beta-mercaptoethanolSigma-AldrichM3148Used as component of loading buffer and lysis buffer
Bradford CoomasieThermo Scientific1856209Used for lysate protein quantification
Casting apparatusAtto WSE-1165WUsed to run SDS-page electrophoresis
CentrifugeEppendorf5804Used in lysate preparation
CentrifugeVWRMicroStar 17RUsed for spinning samples
Dimethyl sulfoxide (DMSO)Sigma-AldrichD2650-100MLUsed for cell culture experiment
Dried Skimmed MilkMarvelN/AUsed to make blocking buffer
Dulbecco's Modified Eagle's Medium - high glucoseSigma-AldrichD6429Used for cell culture
ECL reagentPerkin ElmerORTT755/2655Used to develop image for western blotting
EDTAFisher ScientificD/0700/53Used as component of lysis buffer
EGTASigma-Aldriche4378Used as component of lysis buffer
Electrophoresis Power SupplyBioRadPowerPAC HCTo supply power to run SDS-page electrophoresis
EthanolThermoFisherE/0650DF/17Used for preparing sterilized equipments and environment
Fetal Bovine Serum -  heat inactivatedMerck Life Sciences UKF9665Used for cell culture
FumehoodWalkerA7277Used for cell culture
Gel Blotting - WhatmanGE Healthcare 10426981Used in western blotting to make transfer sandwich
GlycineSigma-Aldrich15527Used to make buffers
GraphPad Prism SoftwareGraphPad Software, Inc., USAVersion 6.0Used for plotting graphs and analysing data for  western blotting
HClAcros Organics10647282Used to make seperating and stacking gel for SDS-PAGE 
Heating blockGrantQBT1Used to heat WB loading samples
HEK293 cellsMerck UK12022001-1VLCell line for culture experiment
ImageJ SoftwareWayne Rasband and Contributors; NIH, USA ImageJ 1.53eUsed to measure band intensities from western blotting images
Imaging systemBioRadChemiDoc MPUsed to take western blotting images
IncubatorLEECLEEC precision 190DUsed for cell culture
IsopropanolHoneywell24137Used in casting gel for electrophoresis
L-glutamine solutionSigma-AldrichG7513Used for cell culture
Lithium dodecyl sulfate (LDS)NovexNP0008Used as loading buffer for western blotting
MEM Non-essential amino acid Merck Life Sciences UKM7145Used for cell culture
MicrocentrifugeEppendorf5418Used for preparing lysates for WB
Microplate readerBioRadiMarkUsed for lysate protein concentration readout
Microsoft PowerpointMicrosoft, USAPowerPoint2016Used to edit western blotting images
Molecular Weight MarkerBioRad1610373Used for western blotting
N-ethylmaleimideThermo Fisher Scientific23030Used for cell culture experiment
Nitrocellulose membraneFisher Scientific45004091Used for western blotting
Penicillin-StreptomycinGibco15140122Used for cell culture
pH MeterMettler ToledoSeven compact s210Used to monitor pH of buffer solutions
Phenylmethylsulfonylfluoride (PMSF)Sigma-AldrichP7626Used as component of lysis buffer
Phosphate Buffer SalineSigma-AldrichD8537Used for cell culture
PKCδ pThr505Cell Signalling technology9374Used as primary antibody for western blotting
Sepharose Protein GGeneronPG50-00-0002Used for immunoprecipitation
Sodium chlorideSigma-AldrichS7653Used as component of wash buffer
Sodium ChlorideSigma-AldrichS7653Used to prepare TBS-T buffer
Sodium Dodecyl SulfateSigma-AldrichL5750Used to make seperating and stacking gel for SDS-PAGE 
sodium orthovanadateSigma-AldrichS6508Used as component of lysis buffer
Sodium PyruvateSigma-AldrichS8636Used for cell culture
sodium-β-glycerophosphateMerck UKG9422Used as component of lysis buffer
Staurosporine (from Streptomyces sp.)Scientific Laboratory Supplies, UKS4400-1MGUsed for cell culture experiment
SucroseScientifc Laboratory SuppliesS0389Used as component of lysis buffer
TEMEDSigma-AldrichT7024Used to make seperating and stacking gel for SDS-PAGE 
Transfer ChamberBioRad1658005EDUUsed in western blotting to transfer protein on membrane
TrisSigma-AldrichT6066Used to make seperating and stacking gel for SDS-PAGE 
Triton-X100Sigma-AldrichT8787Used as component of lysis buffer
Trypsin-EDTA SolutionMerck Life Sciences UKT4049Used for cell culture
Tween-20Sigma-AldrichP3179Used as make TBS-T buffer
Vacuum pumpCharles AustenDymax 5Used for cell culture
VortexScientific IndustriesK-550-GEUsed in sample preparation
Vortex mixerScientific Industries LtdVortex-Genie  K-550-GEUsed of mixing resolved sample
Water bathGrant Instruments Ltd. (JB Academy)JBA5Used to incubate solutions

References

  1. 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).
  2. Heubl, M., et al.

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