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.
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Method Article
Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay
In This Article
Summary
Abstract
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.
Introduction
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 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....
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Protocol
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
- 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'....
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Results
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 configuration .......
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Discussion
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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Disclosures
The authors declare that they have no competing financial interests.
Acknowledgements
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).
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Materials
| 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 |
References
- Peetla, C., Stine, A., Labhasetwar, V. Biophysical Interactions with Model Lipid Membranes: Applications in Drug Discovery and Drug Delivery. Mol. Pharm. 6 (5), 1264-1276 (2009).
- van de Waterbeemd, H., Smith, D. A., Beaumont, K., Walker, D. K.
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