Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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^1,2,3,4. 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 cord⁵. Failure in the proper regulation of KCC2 has been associated with the prevalence of several neurological diseases, including epilepsy⁴. Furthermore, decreased KCC2-mediated Cl⁻ extrusion and impaired hyperpolarizing GABA_A and/or glycine receptor-mediated currents have been implicated in epilepsy, neuropathic pain, and spasticity^6,7. Neuronal KCC2 is negatively modulated via phosphorylation of key regulatory residues within its C-terminal intracellular domain by the with-no-lysine (WNK)-STE20/SPS1-related proline/alanine-rich (SPAK)/Oxidative stress-responsive (OSR) kinase signaling complex¹, which facilitates the maintenance of depolarized GABA activity in immature neurons^2,8,9. The WNK-SPAK/OSR1 phosphorylates threonine residues 906 and 1007 (Thr906/Thr1007) and subsequently downregulates mRNA gene expression of KCC2, leading to a consequent deterioration in its physiological function^8,10. More importantly, however, it is already a fact that the WNK-SPAK/OSR1 kinase complex is known to phosphorylate and inhibit KCC2 expression^1,2,4,11,12, and that the inhibition of the kinase complex signaling pathways to phosphorylate Thr906/Thr1007 has been linked with the increased expression of the KCC2 mRNA gene^13,14,15. It is important to note that the regulation of neuronal KCC2 and Na⁺-K⁺-2Cl^- cotransporters 1 (NKCC1) expression via protein phosphorylation works concomitantly and in converse patterns^1,4,16.

There has been consistent and considerable progress with regard to the understanding of mechanisms involved in the regulation of KCC2, accredited to the development of techniques that enables researchers to study its functions and activities; either via direct (assessing kinase regulatory sites phosphorylation) or indirect (observing and monitoring GABA activity) investigations. The protocol presented here highlights the application of western blotting techniques to study the functions and activities of neuronal K⁺-Cl^- co-transporter KCC2 by investigating the phosphorylation of the cotransporter at kinase regulatory sites Thr906/1007.

Western blot is a method used to detect specific proteins of interest from a sample of tissue or cell. This method first separates the proteins by size through electrophoresis. The proteins are then electrophoretically transferred to a solid support (usually a membrane) before the target protein is marked using a specific antibody. The antibodies are conjugated to different tags or fluorophore-conjugated antibodies that are detected using either colorimetric, chemiluminescence, or fluorescence methods. This allows for a specific target protein to be detected from a mixture of proteins. This technique has been used to characterize phosphospecific sites of KCCs¹ and has been used to identify kinase inhibitors that inhibit KCC3 Thr991/Thr1048 phosphorylation¹⁷.By following this protocol, one can specifically detect total and phosphorylated KCC2 from cell/tissue lysates. In principle, the detection of protein-conjugated antibodies by this technique is highly instrumental as it helps to improve the understanding of cooperative activities at the phospho-sites of KCC2, which sheds light on the molecular mechanisms involved in their physiological regulations. The quantitative analysis of the total protein expression is representative of the function and activity of KCC2. There are other classical 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.

Protokół

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 ˚C) 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 stably transfected rat KCC2b human embryonic kidney 293 cells (HEK293^rnKCC2b)¹⁸ completely in the bead bath (37 ˚C). Transfer the cells from the cryovial tube into a centrifuge tube containing 5 mL of fresh media. Spin the cell at 1200 ×g for 3-5 min.
3. Use an aspirator pipette (fixed to a vacuum pump) to aspirate the supernatant and add 10 mL of fresh media to re-suspend the cells. Transfer the cell suspension to a 10 cm dish plate.
4. Place the dish into the incubator and allow it to grow for 48 h at 37 °С in a humidified 5% CO₂ atmosphere. Monitor the incubation period to ensure healthy cell growth. Further split the cells when they have attained over 90% confluence after the incubation period.
5. Aspirate the old media out from the cells dish and gently rinse cells with 2 mL of phosphate buffer saline (PBS). Add 2 mL of trypsin and incubate for about 1-2 min at room temperature.
6. Use 2 mL of complete media to gently wash the trypsinized cells in the dish. Add 9 mL of fresh media to new dishes and add 1 mL solution from the old dish to each of the new ones. Transfer the split cell dishes back to the incubator for 48 h to attain ≥ 90% confluency.
7. Treat the cells with either dimethyl sulfoxide (DMSO) as control, 8 µM staurosporine, or 0.5 mM N-ethylmaleimide (NEM) for 15 min before harvesting the cells in lysis buffer.

2. Preparation of cell lysates and loading samples

1. Aspirate the media on the culture dish (from transfection procedures). Place the cell culture on ice and wash the cells with ice-cold PBS.
2. Aspirate the PBS, then add 1.0 mL of ice-cold lysis buffer containing 50 mM tris-HCl (pH 7.5), 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 10 mM sodium-β-glycerophosphate, 1 mM sodium orthovanadate, 1% (w/v) Triton X-100, 0.27 M sucrose, 1 mM benzamine, 0.1% (v/v) 2-mercaptoethanol, and 2 mM phenylmethylsulfonylfluoride (PMSF) to the dish.
   NOTE: The volume of lysis buffer during cell harvest varies with dish sizes, for example, 1 mL of lysis buffer is suitable per 1 x 10⁷ cells/100 mm dish/150 cm² flask, while 0.5 mL is suitable per 5 x 10⁶ cells/60 mm dish/75 cm² flask.
3. Use a cold plastic cell scraper to scrape the cells off the bottom of the dish, then gently use a pipette to transfer the cell suspension into a micro-centrifuge tube already on ice. Constantly agitate the tube for 30 min at 4 °C.
4. Spin the cell lysates in a cold centrifuge (4 °C) at 16,000 x g for 20 min, remove the tube gently from the centrifuge, and place it on ice. Collect the supernatant into a pre-cooled fresh tube and discard the pellet.
   NOTE: It may be necessary to vary the centrifugation force and time depending on the cell type. Though, general guidelines encourage centrifugation at 16,000 x g for 20 min. However, this must be determined for every experiment. For instance, delicate cells like leukocytes need a very light centrifugation speed.

3. Preparation of immunoprecipitants from cell lysates

1. Pipette 300 µL of protein-G-sepharose into a microcentrifuge tube. Spin the solution at 500 x g for 2 min and discard the supernatant.
2. Add 500 µL of PBS and vortex the solution well. Spin the solution at 500 x g for 2 min and discard the supernatant. Repeat this step.
3. Mix 1 mg of anti-KCC2 Thr906 and anti-KCC2 Thr1007 antibodies with 200 µL of protein G-sepharose beads and make up the volume to 500 µL with PBS. Shake on a vibrating platform or rotating wheel for 2 h at 4 °C. Wash 2x with PBS.
4. Carry out protein quantification on the whole cell lysates and add 1 mg of the cell lysate to the washed beads. Gently incubate in an end-to-end rotator for 2 h at 4 °C. Spin down the beads and wash them 3x with PBS containing 150 mM sodium chloride (NaCl).
5. Wash immunoprecipitants (beads) 3x with 200 µL of PBS. Resuspend the final pellet in 100 μL of 1x lithium dodecyl sulfate (LDS) sample buffer.
6. Shake the tubes in a rotor shaker at room temperature for 5 min and incubate them in a heating block at 75 °C for 10 min. Centrifuge the loading sample at 11,000 x g for 2 min and use the supernatant for gel loading.

4. Performing the western blot

1. Assemble the casting apparatus and pour freshly prepared 8% separating gel (see Table 1 for recipe/preparation) to cast the gel allowing about 2 cm of space from the top of the casting glass. Add 200 µL of absolute isopropanol to the setup and allow it to stand at room temperature for 60 min.
   NOTE: The polyacrylamide gel recipe for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) depends on the size of the protein of interest (Table 2). Hence, make a note of the protein size before determining the desired gel percentage.
2. Use a pipette to remove the isopropanol and carefully rinse the gel with about 200 µL of distilled water. Add freshly prepared 6% stacking gel (see Table 1 for recipe/preparation) to fill up the approximately 2 cm space on the casting setup. Gently fit in the well comb and allow to stand at room temperature for 30 min.
3. Fix the casted gel into the electrophoresis tank.
4. Dissolve 30.3 g of Tris base, 144.1 g of glycine, and 10 g of SDS in 1000 mL of distilled water to prepare 10x transfer buffer. Prepare 1x running buffer by adding 10 mL of 10% SDS and 100 mL of 10x transfer buffer to 890 mL of distilled water.
5. Pour 1x running buffer into the tank. Load 5 µL of molecular weight marker into the first well and an equal amount of protein into each well of the SDS-PAGE gel (between 18-30 µL, depending on the comb size used). Fill up empty wells with 1x LDS and run the gel for about 90-120 min at 120 V.
6. Dissolve 58.2 g of Tris base and 29.3 g of glycine in 1000 mL of distilled water to prepare 10x transfer buffer. Activate nitrocellulose membrane with 1x transfer buffer containing 20% methanol. Rinse the gel and membrane with the transfer buffer and gently spread them out on the preparing stack.
   NOTE: There is no need to adjust the pH of the running and transfer buffers as they should be at the optimum pH required. Also, it is advisable to prepare these buffers and store them at room temperature before the experiment to save time.
7. Arrange the sandwich to be transferred in this order: negative electrode (black frame/end) - sandwich foam - filter paper - rinsed SDS-PAGE gel - rinsed nitrocellulose membrane - filter paper - sandwich foam - positive electrode (red frame). Stack the assembled sandwich in the transfer tank and run at 90 V for 90 min or 30 V for 360 min.

5. Antibody staining and image development

1. Remove the membrane and let it dry. Block the membrane for 1 h at room temperature using a blocking buffer made from 5% skimmed milk in Tris-buffered saline containing 0.1% 1x Tween 20 (1x TBS-T).
2. Incubate the membranes with appropriate dilutions of primary antibody^8,15,19 and beta-actin (loading control) in blocking buffer for 1 h at room temperature or overnight at 4 °C. Wash the membrane in three washes of 1x TBS-T for 5 min each.
   NOTE: The incubation of the separate membrane with loading controls such as beta-actin, alpha-tubulin, or glyceraldehyde 3-phosphate dehydrogenase antibodies is to normalize the other western blotting results during subsequent quantification. The recommended dilution for each primary antibody is available in the manufacturer's manual.
3. Incubate the washed membrane with secondary antibody diluted 5000-fold in blocking buffer for 60 min at room temperature. Again, wash the membrane 3x in 1x TBS-T for 5 min each.
   NOTE: It is highly recommended that the secondary antibody must be raised against the species in which the primary antibody is raised. For example, if the primary antibody of total KCC2 is mouse anti-KCC2, then the secondary antibody for anti-mouse must be used.
4. Place the washed membrane on the imaging board. Mix equal volumes of each enhanced chemiluminescence (ECL) reagent and gently spread the mixed solution on the membrane to develop signals.

6. Image acquisition using an imaging system and data quantification

1. Transfer the imaging board to the appropriate compartment on the imaging system for imaging. Open the imaging software on the computer for image processing.
2. On the toolbar, click New Protocol and choose Single Channel. Click Select under gel imaging/application dialog box, scroll to Blot and select either Chemi Hi Sensitivity or Chemi Hi Resolution. Then click on Signal Accumulation Setup to set the duration and number of images to acquire.
3. Click Position Gel under protocol setup and adjust the gel in the imaging system if necessary. Click Run Protocol to acquire the images.
4. Right-click on one of the acquired images under the imaging catalog box to save the images on the computer. Navigate to the desired image and click on Select Image and Continue under the run dialog box.
5. Click on the Image Transform icon to adjust the contrast and the pixel saturation of the image. Click on the Screenshot icon on the general toolbar to save the image to the computer depending on the desired image format.
6. Measure the band densities using ImageJ software and analyze using appropriate statistical tools.

Wyniki

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 (HEK^rnKCC2b)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...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
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