Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

Podsumowanie

The protocol here describes the interactions of purified hEAG1 ion channel protein with the small molecule lipid ligand phosphatidylinositol 4, 5-bisphosphate (PIP₂). The measurement demonstrates that BLI could be a potential method for novel small-molecule ion channel ligand screening.

Streszczenie

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP₂. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purified hEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP₂ by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP₂ demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

Wprowadzenie

Targeting the cell surface-accessible ion channel proteins with small molecules offers a tremendous potential for the ligand screening and biological drug discovery^1,2,3. Thus, an appropriate tool is needed for studying the interaction between ion channel and small molecules and their corresponding function. The patch-clamp recording has been demonstrated to be a unique and irreplaceable technique in ion channel functional assay. However, determining whether the small molecules directly target ion channels require other technologies. Traditionally, the radioactive ligand binding assay was used to observe the kinetics of binding between small molecule and its target ion channel protein. However, the usage of this technique is limited because of its requirement in radioactive labeling and detection. Moreover, the prerequisite step to label the small ligand in the study prevents its using in many types of ion channels without known specific ligand. Some label-free techniques such as NMR spectroscopy, X-ray diffraction, microscale thermophoresis (MST)⁴ and surface plasmon resonance (SPR) have been used to measure the protein-small molecule interactions. But these types of assays usually cannot provide sufficient information because of the difficulty to get the full-length protein, low resolution of dynamics, low throughput, and high cost⁵. In contrast with these techniques, bio-layer Interferometry (BLI) is emerging as a novel label-free methodology to overcome these drawbacks for detecting protein-small molecule interactions by immobilizing a tiny amounts of protein sample on the surfaces of biosensor and measuring the optical changing signals^6,7. As a promising biosensor platform, BLI technique is already performed to observe the interaction of small molecules with natural water soluble proteins such as a human monoclonal antibody CR8020⁸ and the detailed assay procedure has been reported in a previous article⁹. Although the key role of ion channel protein for new therapeutic targets discovery has been recognized, the ion channel protein-small molecule interaction assay based on BLI has not been described.

The human Ether à go-go channels (hEAG1) are expressed in various types of cancer cells and central nervous system which makes the channel a potential therapeutic target of many cancers and neuronal disorders^{10,11,12,13,14}. The electrophysiological study in our lab has confirmed the inhibitory effect of phosphatidylinositol 4, 5-bisphosphate (PIP₂) on hEAG1 channel¹⁵. Based on our results, testing PIP₂ directly interaction with the hEAG1 by using BLI technique can be as a model for other types of ion channel protein-small molecule compound interaction especially for those channels lacking specific ligands. According to the instructions of BLI assay, we prepared biotinylated hEAG1 proteins and immobilized them on the surface of streptavidin (SA) biosensor tips followed by interaction them to PIP₂ solutions to observe their direct binding between the protein and the lipid. After the attachment of PIP₂ to the hEAG1 protein coated surface, the thickness of the layer on the surface increases, which directly correlates the spectral shift and can be measured in real-time¹⁶. The binding kinetics can be determined due to a positive shift in association step and a negative shift in dissociation step. According to this principle, we purified the functional hEAG1 ion channel protein from HEK-239T stable expression system by using affinity purification method to maintain the in vitro functional state, then measured the kinetics of binding of different concentration PIP₂, and yielded a semblable kinetic data as observed in electrophysiological measurements¹⁵. The close correspondence between the results from the BLI and electrophysiological measurements demonstrate for the first time the suitability of BLI as an appropriate analytical tool for ion channel membrane protein-small molecule interaction.

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Protokół

NOTE: The HEK-293T cell line continuously expressing FLAG-tagged hEAG1 channel protein is constructed by transfecting a pCDH lentiviral plasmid containing the DNA sequence of hEAG1 with a FLAG at the distal C-terminus into HEK-293T cells followed by the puromycin-resistant selection as previously described¹⁵.

1. Affinity Purification of FLAG-tagged hEAG1 Channel Protein from HEK-293T Cells

1. Thaw cells stably expressing hEAG1 channels from liquid nitrogen into the warm water (37 °C) quickly. Seed the cells (about 5 x 10⁶ frozen cells) in a 10 cm dish. Grow the cell overnight in Dulbecco's Modified Eagle's (DMEM) medium supplemented with 8 mL 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 μg/mL streptomycin, 4 mM L-glutamine and changed the medium the next day.
2. Check the GFP fluorescence of these HEK-293T cells by using a fluorescent microscope to make sure the high percentage of cells stably express hEAG1 channels in the culturing system. Exponentially trypsinize the growing cells with 1 mL of 0.25% trypsin for 1 min at room temperature and thenadd 2 mL of serum-containing culture fluid to terminate the digestion. Transfer 400 μL of cell suspension to a 15 cm dish containing 15 mL of complete DMEM medium. Prepare four 15 cm dishes in total.
3. Harvest these cells after 2–3 days culture when the cells reach at about 90% confluency.
   1. Remove the growth medium from the cells and wash them twice with 4 mL of phosphate buffered saline (1x PBS, pH = 7.4).
   2. Discard PBS after washing.
   3. Scrape the cells into 2 mL 1x PBS for each dish by using cell scrape and transfer the scraped cells with 1 mL pipette into a 15 mL tube.
   4. Centrifuge the cell suspension for 5 min at 420 x g at 4 °C.
   5. Decant and discard the supernatant.
   6. Resuspend the cell pellet in total 4 mL of lysis buffer (10 mM HEPES, 1.5 mM MgCl₂, 10 mM NaCl, 1% NP-40, pH 7.0, contained complete protease inhibitor) for 30 min on ice, and vortex thoroughly the lysate every 10 min.
   7. Centrifuge the cell lysate for 10 min at 12,000 x g at 4 °C.
   8. Transfer the supernatant to a 5 mL tube and keep it on ice for immediate use.
4. Prepare the anti-FLAG M2 affinity resin.
   1. Thoroughly suspend the resin (supplied as a 50% suspension in store buffer) by gentle inversion to make sure the bottle of anti-FLAG M2 affinity gel is a uniform suspension of gel beads.
   2. Immediately transfer 400 μL of suspension to a chilled 1.5 mL tube.
   3. Centrifuge the suspension for 30 s at 8, 000 x g at 4 °C and discard the supernatant carefully to wash out the store buffer.
   4. Add 500 μL 1x PBS to the resin and suspend the pellet with 1 mL pipette. Then centrifuge the suspension for 30 s at 8, 000 x g at 4 °C and discard the supernatant PBS carefully. Repeat these wash step to clear the stored buffer.
5. Add 500 μL protein extract supernatant prepared at step 1.3.8 to the resin pellet to suspend the pellet and transfer the suspension to a new chilled 5 mL tube. Repeat this step again to make sure no gel beads left. Add the left protein extract to the mixture.
6. Incubate the mixture overnight on a shaker at 8 rpm at 4 °C to capture the FLAG fusion protein.
7. Centrifuge the mixture after 12 h incubation for 10 min at 1, 000 x g at 4 °C.
8. Discard the supernatant and wash the pellet three times with 500 μL of 1x PBS. Keep it on ice for immediate use.
9. Elution the FLAG hEAG1 protein with 3x FLAG peptide.
   1. Prepare 3X FLAG elution solution. Dissolve 3X FLAG peptide in 500 μL stock solution (0.5 M Tris-HCl, 1 M NaCl, pH = 7.5) at a concentration of 8 μg/μL.
   2. Add 10 μL 3x FLAG elution solution to 390 μL of PBS to be a 200 ng/μL final concentration solution.
   3. Add 400 μL of 3x FLAG elution solution to the gel beads prepared at step 1.8.
   4. Incubate the sample at the shaker at 8 rpm for 2 h at 4 °C.
   5. Centrifuge the resin for 30 s at 8, 000 x g.
   6. Transfer the supernatant to a fresh 1.5 mL tube and store it at 4 °C for immediate use.

2. Concentration Assay and Confirmation of Purified FLAG Fusion hEAG1 by BCA Protein Assay Kit and Western Blotting

1. Determine the concentration of purified protein using the BCA protein assay kit according the manufacture's instruction.
2. Use 30 μL sample for western analysis to confirm the interest protein had been purified by using an ANTI-FLAG antibody as previously described¹⁵.

3. Labeling the Purified Channel Protein with Biotin for the BLI Assay

1. Prepare 5 mg/mL biotin stock solution in PBS. For each test (two biosensors), add a 3-fold molar excess of biotin to 20 μg purified protein to achieve a preferable N-terminal biotinylation of the purified protein in PBS.
2. Incubate the sample in the dark on ice for at least 30 min.
3. Prepare the sample dilution (SD) buffer: PBS with 0.02% polysorbate 20 and 0.1% bovine serum albumin (BSA, pH 7.4).
4. Perform ultrafiltration to change the buffer of the purified channel protein to SD buffer. Remove the unbound biotin by using ultrafiltration device with molecular weight cutoff of 30 kDa, adding the SD buffer and centrifuging the sample at 12,000 x g, for 10 min at 4 °C.
5. Remove the ultrafiltrate from the centrifuge tube of ultrafiltration device, add 200 μL SD buffer into the filter device and centrifuging the sample at 12,000 x g, for 10 min at 4°C. Repeat this operation at least three times.
6. To collect the buffer exchanged sample, reversed insert the filter device into a 1.5 mL tube and centrifuging them at 2,000 x g, for 5 min at 4 °C. Keep the sample on ice for immediate use.

4. Preparation of PIP₂ Solution for Assay

1. Prepare the stock solution of PIP₂ (1 mM) in deionized H₂O by sonicating for 30 min on ice as previously described¹⁷. Store the solution in glass vials at -20 °C and dilute it to the final concentrations immediately before experiments by vigorous vortexing.

5. BLI Assay

1. Turn on the equipment and check the "instrument status" window to confirm the machine is at "ready" state to prewarm the equipment at least for 30 min before the BLI study.
2. Make sure the door of the instrument is closed before opening the Data Acquisition software and choose the "New Kinetics Experiment" in the Experiment Wizard.
3. Define the wells to be used on the 96-well plate by right click to choose buffer, load, and sample. For sample wells, the unit of concentration of biotinylated FLAG fusion hEAG1 protein should be input as molar (10 μg, 0.09 μM).
4. Define the assay steps including baseline, loading, association and dissociation. Choose an assay step and double click on the respective column. A duplicate of assay definition is set for control sensors. Set the rpm as 1,000. Choose 1 min for baseline step and 5–10 min for loading, association, and dissociation, respectively. Perform the test at room temperature (about 24 °C).
   NOTE: There are two main procedures: loading the biotinylated channel protein to the sensors and assaying the interaction with small molecule compounds. They can be proceeded continuously (baseline, loading, baseline, association and dissociation). Alternatively, they can be proceeded separately to avoid wasting the test compounds when the first loading part unsuccessful.
5. Click the columns which contain the sensors and click the "Fill" to indicate the locations of sensors in the sensor tray.
6. Review all planned steps to check for mistakes and go back to correct them.
7. Set the location of data files and click "Go" to start the assay.
   NOTE: The sensors need prewetted for at least 10 min in SD buffer, if this step has been done, then the "Delayed experiment start" setting should be skipped. If not, set a 600 s delay before prewetting the sensors.
8. Put a black 96-well plate in the bottom of the tray and insert the A1 corner of the plate into the notch on the tray to seat the plate. For the wells to load the sensors, 200 μL of assay buffer per well add into 2 wells in row A and 2 wells in row B of the 96-well plate.
9. Prepare another black 96-well plate as the sample plate and fill the wells with 200 μL SD buffer in row B as control or biotinylated hEAG1 protein solution (10 μg) in row A as assigned during programming in step 5.3.
   NOTE: Avoid introducing bubbles.
10. Open the door of the instrument and insert the sensor tray and sample plate into the left and right plate holder, respectively. Check that the sensor tray and sample plate are positioned correctly based on the shape of left plate holder and the "A1" marker on the top right corner of right plate holder. Close the door and start the assay.

6. Data Analysis

1. Open the Data Analysis software and load the folder containing the assay data. Click "Processing" to get into the processing menu interface and we can see the colorful raw kinetic curves.
2. Under Step1: "Data Selection", click "Sensor Selection". On the "Sensor Tray #1", click the sensor wells only wetted with SD buffer and right click to "Change Sensor Type" to "Reference Sensor". On the 'Sample Plate Map", designate the all the non-specific binding wells and right click to "Change Well Type" to "Reference Well".
3. Tick in the box before "Subtraction" of Step 2 and point "Double Reference".
4. In Step 3: "Align Y Axis", select "Baseline" as the alignment step. For "Time Range", enter the last 10 s of that baseline (i.e. From: 0.1 To: 59.8).
5. In Step 4: "Inter-step Correction", select "align to Baseline" to minimize signal shifts between the association and dissociation steps.
6. In Step 5: "Process", select Savitzky-Golay filtering function in most cases and proceed "Process Data".
7. Save Raw Data for further data analysis using other software in Step 7: "Save Results".
8. Click "Analysis | Curve Fitting".
   1. For "Step to Analyze", choose "Association | Dissociation". For "Model", select 1:1.
      NOTE: we choose 1:1 model because it fitted well on our original data and avoided the possibility of over fitting under 1:2 or 2:1 model due to their high freedom. But other options are available here and can be suitably chosen for other fitting analysis for different binding models of analyte.
   2. For "Fitting", choose Global (Full). For "Group By", select "Color". Select "Rmax Unlinked By Sensor" to allow independent fitting of maximal signal response (Rmax).
   3. Click "Fit Curves!" to start the nonlinear regression analysis. Hill equation and single exponential function were used in our study.
   4. Click "Data Export |Save Report" to save fitting results. Or click "Data Export | Export Fitting Results" to save the raw data for further graphing and data analysis with other software.

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Wyniki

We purified the FLAG fusion hEAG1 channel protein from HEK-293T cells stably overexpressed hEAG1. The function of this fusion protein has been demonstrated by using the patch-clamp method and the quality and specificity of purified protein are confirmed by Western blot (Figure 1). The purified channel protein is biotinylated to perform an interaction assay with the lipids (PIP₂) by using the real-time BLI assay. The BLI binding assay configura...

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Dyskusje

Membrane ion channels have been verified as the primary therapeutic targets of over 13% of currently known drugs for the treatment of a variety of human diseases, including cardiovascular and neurological disorders¹⁸. Patch-clamp recording, the golden standard for measuring the functional of ion channels with small molecules, has been widely used for ion channel ligands screening. However, such electrophysiological approaches cannot demonstrate whether the small molecules binds to the channel dire...

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Materiały

Name	Company	Catalog Number	Comments
DMEM/High Glucose Medium	HyClone	SH30243.01
Phosphate Buffered Saline (1x)	HyClone	SH30256.01
Fetal Bovine Serum	Gibco	10270
Penicillin/Streptomycin	Gibco	1697550
Cell Culture Dish	Corning	430599	150 mm X 25 mm
Nonidet P-40 Substitute	Amresco	E109
Sodium chloride	BBI Life Sciences	A610476
Potassium chloride	BBI Life Sciences	A610440
Bovine Serum Albumin	BBI Life Sciences	A600332
Polyoxyethylene-20-Sorbitan Monolaurate	BBI Life Sciences	A600560
ANTI-FLAG M2 Affinity Gel	Sigma	A2220
3x FLAG peptide	Sigma	F4799
Octet-RED96	Pall/FortéBio	30-5048
Data Acquisition software	Pall/FortéBio	Version 7.1
Data Analysis software	Pall/FortéBio	Version 7.1
Biosensor/Streptavidin	Pall/FortéBio	18-5019
Microtiter plate	Greiner Bio-one	655209
Sulfo-NHS-LC-LC-Biotin	ThermoFisher	21338
Centrifugal Machine	ThermoFisher	75004250
PageRuler Prestained Protein Ladder	ThermoScientific	318120
Ultrafiltration device	MILLIPORE	UFC503008	NMWL of 30 kDa
phosphatidylinositol 4, 5-bisphosphate (PIP2)	Sigma	P9763
Monoclonal ANTI-FLAG M2 antibody	Sigma	F1804	1:2000 dilution
goat anti-mouse HRP-conjugated secondary antibody	Santa Cruz Biotechnology	sc-2005	1:5000 dilution
Enhanced BCA Protein Assay Kit	Beyotime	P0010
Protease Inhibitor Cocktail Tablets	Roche	04693159001
Amersham Imager 600 Imaging System	GE Healthcare Bio-Sciences
Western blot system	BIO-RAD