This work was supported by Bio-ID Center and SJTU Cross-Disciplinary Research Fund in Medicine and Engineering (YG2016QN66), National Natural Science Foundation of China (31271217), and National Basic Research Program of China (2014CB910304).

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
<ol>
	<li>Thaw cells stably expressing hEAG1 channels from liquid nitrogen into...

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A., Stuhmer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eag1:+An+emerging+oncological+target.">Eag1: An emerging oncological target.</a> Cancer. Res. 68 (6), 1611-1613 (2008).</li><li>Simons, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+the+voltage-gated+potassium+channel+gene+KCNH1+cause+Temple-Baraitser+syndrome+and+epilepsy.">Mutations in the voltage-gated potassium channel gene KCNH1 cause Temple-Baraitser syndrome and epilepsy.</a> Nat. Genet. 47 (1), 73-77 (2015).</li><li>Kortum, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+KCNH1+and+ATP6V1B2+cause+Zimmermann-Laband+syndrome.">Mutations in KCNH1 and ATP6V1B2 cause Zimmermann-Laband syndrome.</a> Nat. 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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...

Targeting the cell surface-accessible ion channel proteins with small molecules offers a tremendous potential for the ligand screening and biological drug discovery1,2,3. Thus, an appropriate tool is needed for studying the interaction between ion channel and&#160;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 signals6,7. 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 &#224; 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 disorders10,11,12,13,14. 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.

<table>
	<tbody>
		<tr>
			<td>DMEM/High Glucose Medium</td>
			<td>HyClone</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (1x)</td>
			<td>HyClone</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>1697550</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>Corning</td>
			<td>430599</td>
			<td>150 mm X 25 mm</td>
		</tr>
		<tr>
			<td>Nonidet P-40 Substitute</td>
			<td>Amresco</td>
			<td>E109</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>BBI Life Sciences</td>
			<td>A610476</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>BBI Life Sciences</td>
			<td>A610440</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>BBI Life Sciences</td>
			<td>A600332</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene-20-Sorbitan Monolaurate</td>
			<td>BBI Life Sciences</td>
			<td>A600560</td>
			<td></td>
		</tr>
		<tr>
			<td>ANTI-FLAG M2 Affinity Gel</td>
			<td>Sigma</td>
			<td>A2220</td>
			<td></td>
		</tr>
		<tr>
			<td>3x FLAG peptide</td>
			<td>Sigma</td>
			<td>F4799</td>
			<td></td>
		</tr>
		<tr>
			<td>Octet-RED96</td>
			<td>Pall/Fort&#233;Bio</td>
			<td>30-5048</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition software</td>
			<td>Pall/Fort&#233;Bio</td>
			<td>Version 7.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Analysis software</td>
			<td>Pall/Fort&#233;Bio</td>
			<td>Version 7.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosensor/Streptavidin</td>
			<td>Pall/Fort&#233;Bio</td>
			<td>18-5019</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtiter plate</td>
			<td>Greiner Bio-one</td>
			<td>655209</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-NHS-LC-LC-Biotin</td>
			<td>ThermoFisher</td>
			<td>21338</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugal Machine</td>
			<td>ThermoFisher</td>
			<td>75004250</td>
			<td></td>
		</tr>
		<tr>
			<td>PageRuler Prestained Protein Ladder</td>
			<td>ThermoScientific</td>
			<td>318120</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrafiltration device</td>
			<td>MILLIPORE</td>
			<td>UFC503008</td>
			<td>NMWL of 30 kDa</td>
		</tr>
		<tr>
			<td>phosphatidylinositol 4, 5-bisphosphate (PIP2)</td>
			<td>Sigma</td>
			<td>P9763</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal ANTI-FLAG M2 antibody</td>
			<td>Sigma</td>
			<td>F1804</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr>
			<td>goat anti-mouse HRP-conjugated secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2005</td>
			<td>1:5000 dilution</td>
		</tr>
		<tr>
			<td>Enhanced BCA Protein Assay Kit</td>
			<td>Beyotime</td>
			<td>P0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail Tablets</td>
			<td>Roche</td>
			<td>04693159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham Imager 600 Imaging System</td>
			<td>GE Healthcare Bio-Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Western blot system</td>
			<td>BIO-RAD</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

capturing the interaction kinetics of an ion channel protein with small molecules by the bio-layer interferometry assay

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&#160;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.

We purified&#160;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...

Watch this Scientific Journal Video about Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay at JoVE.com

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

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.

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

The overall goal of this bio-layer interferometry or BLI assay, is to measure the potential interaction between purified hEAG1 ion channel protein and small molecule lipids. This method can help answer key questions in the ion channel pharmacology field, such as whether the small molecule of interest can directly interact with this ion channel and what are the kinetics of the interaction? The main advantage of this technique is that it is a labor free and high throughput method.Only a small amount of immobilized protein is needed. Generally, individuals who are new to this method will struggle because a successful assay involves multiple steps, including protein expression, purification, labeling, accurately tuned sensor, and data analysis. After culturing HEK293 T cells stabling expressing hEAG1 for two to three days, harvest the cells by removing the growth medium, and use four milliliters of PBS to wash the cells twice.Discard the PBS. Scape the cells into two milliliters of PBS per dish, and use a one milliliter pipette to transfer to cells into a 15 milliliter tube. Centrifuge the cell suspension at 420 times gravity and four degrees Celsius for five minutes.Then, decant the supernatant and use four milliliters of lysis buffer to resuspend the pellet. Incubate the suspension on ice for 30 minutes, thoroughly vortexing the lysate every 10 minutes. Next, centrifuge the cell lysate at 12, 000 times gravity and four degrees Celsius for 10 minutes.Then, transfer the supernatant to a five milliliter tube and keep it on ice for immediate use. To prepare the anti-FLAG M2 affinity resin thoroughly suspend the resin by gentle inversion to make sure the bottle of anti-FLAG M2 ffinity gel is a uniform suspension of gel beads. Immediately transfer 400 microliters of suspension to a chilled 1.5 milliliter tube.Then, centrifuge the suspension at 8, 000 times gravity and four degrees Celsius for 30 seconds, And carefully discard the supernatant to wash out the storage buffer. To resuspend the resin pellet, add 500 microliters of protein extract supernatant to the resin, and transfer the suspension to a new, chilled, five milliliter tube. Pipette an additional 500 microliters of protein extract into the original tube to collect any remaining beads, and transfer the extract to the five milliliter tube.Incubate the mixture on a shaker at eight rpm and four degrees Celsius overnight to bind the FLAG fusion protein to the resin. Then, centrifuge the mixture at 1, 000 times gravity at four degrees Celsius for 10 minutes. Discard the supernatant and wash the pellet three times with 500 microliters of one x PBS.Keep it on ice for immediate use. Add 10 microliters of three x FLAG elution solution to 390 microliters of PBS for a final concentration of 200 nanograms per microliter. Then, add 400 microliters of three x FLAG elution solution to the previously prepared gel beads.Incubate the sample on a shaking incubator at eight rpm and four degrees Celsius for two hours. Then, centrifuge the resin at 8000 times gravity for 30 seconds. Transfer the supernatant to a fresh 1.5 milliliter tube, and store it at four degrees Celsius for immediate use.Use the BCA protein assay according to the manufacturer's instructions to determine the concentration of the purified protein. Then, use a 30 microliter sample for Western Blot analysis with an anti-Flag antibody to confirm that the protein of interest has been purified. To label the purified protein with biotin, prepare five milligrams per milliliter of biotin stock solution in PBS.For each test of two biosensors add a three fold molar excess of biotin to 20 micrograms of purified protein to achieve a preferable N-terminal biotinylation of the purified protein in PBS. Incubate the sample in the dark on ice for at least 30 minutes. To remove the unbound biotin and buffer, exchange the protein into SD buffer.Transfer the protein to an ultrafiltration device with a molecular cut off of 30 kilodaltons. Then, add SD buffer and centrifuge the sample at 12, 000 times gravity and four degrees Celsius for 10 minutes. Remove the ultrafiltrate from the centrifuge tube.Add 200 microliters of SD buffer into the filter device and centrifuge the sample again. Repeat the process at least three times. Collect the buffer exchanged sample by flipping the filtration device and place it into a 1.5 milliliter tube.Centrifuge the sample at 2, 000 times gravity and four degrees Celsius for five minutes. Keep the sample on ice for immediate use. To carry out the BLI assay, 30 minutes before the study, prewarm the equipment by turning on the instrument.Check the instrument status window to confirm the machine is at ready state before beginning. Make sure the door of the instrument is closed before opening the data acquisition software and in the experiment wizard, choose the New Kinetics Experiment. Define the wells to be used on the 96 well plate by right clicking to choose the buffer, the load, and the sample.For sample wells, the unit of concentration of PIP2 should be input as molars. To define the assay steps, choose a step, and double click on the respective column. A duplicate of the assay definition is set for the control sensors.Set the rpm to 1, 000. Choose one minute for the baseline step, and five to 10 minutes for loading, association, and dissociation respectively. Perform the test at room temperature.Click the columns which contain the sensors and click fill to indicate the location of sensors in the sensor tray. Review all planned steps to check for mistakes and go back to correct them. In the entire operation of BLI assay the step of reviewing all planned steps could be neglected by the researcher.However, it's very important to check that all the planned steps are correct. Set the location of data files and click go to start the assay. Run the assay and analyze the data according to the text protocol.As seen in this patch clamp experiment in an HEK293 T cell, stably expressing hEAG1, the voltage dependent outward potassium currents suggests that functional hEAG1 channels are highly expressed in the cells. Using the anti-FLAG antibody this Western Blot of hEAG1 from purified protein samples indicates the quality and specificity of the purified protein. The purified channel protein was biotinylated to perform an interaction assay with the lipid PIP2 by using the real time BLI assay.A typical binding curve between hEAG1 and PIP2 is shown here. hEAG1 is bound to PIP2 in a concentration dependent manner. The accumulated concentrations of PIP2 correspond to the biosensor's data traces.This representative assay shows the changes in optical interference in different concentrations of PIP2. Finally, a curve fit with the Hill equation was obtained from the peak value of the optical interference signal measured at different PIP2 concentrations and determined the equilibrium dissociation constants between hEAG1 and PIP2. Once mastered, this technique can be done in about one hour if it is performed properly.When attempting this procedure, it's very important to remember to review all planned steps, to make sure this procedure is correct. Following this procedure, other methods like patch clamp can be performed in order to answer additional questions like identifying the function of this ion channel after binding the small molecule. After its development, this technique paved the way for researchers in the field of ligand screening of ion channels to explore the novel ligands in ion channel drug discovery.After watching this video, you should have a good understanding of how to prepare the ion channel protein and how to test its interaction with small molecules, with the BLI method.

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Research

JoVE Journal

Biochemistry

8.1K visualizações.  Shanghai Jiao Tong University School of Medicine. The overall goal of this bio-layer interferometry or BLI assay, is to measure the potential interaction between purified hEAG1 ion channel protein and small molecule lipids. This method can help answer key questions in the ion channel pharmacology field, such as whether the small molecule of interest can directly interact with this ion channel and what are the kinetics of the interaction? The main advantage of this technique is that it is a labor free and high throughput method.Only a ...