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 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.
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 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 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 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.
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
2. Concentration Assay and Confirmation of Purified FLAG Fusion hEAG1 by BCA Protein Assay Kit and Western Blotting
3. Labeling the Purified Channel Protein with Biotin for the BLI Assay
4. Preparation of PIP2 Solution for Assay
5. BLI Assay
6. Data Analysis
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 is shown in Figure 2. A typical binding curve between hEAG1 and PIP2 is shown in Figure 3. In this case, 3 μM PIP2 is dissolved in PBS buffer (the configuration of PIP2 is shown in Supplementary Figure 1), and the signal is analyzed using a double reference subtraction protocol to subtract the non-specific binding (the binding between sensor and PIP2), background (the interaction between biotinylated hEAG1 protein and PBS), and signal drift (the binding between sensor and PBS) caused by sensor variability. And the binding trace is globally fit and shown a well-fitting overlay (Supplementary Figure 2). Also, we measure the kinetics of binding of PIP2 to the purified hEAG1 channel complex by incubating the proteins at different concentrations of PIP2. After analysis, we get a dissociation constant (Kd) value of 0.35 ±0.04 μM, which is similar to the IC50 value obtained from the electrophysiological measurements15. These results demonstrated that the BLI assay is appropriate for ion channel membrane protein and lipids interaction analysis.
Figure 1: Identification of the expression, function, and specificity of recombinant hEAG1 protein from HEK-293T cells by GFP imaging, patch clamp, and western blot, respectively. (A) The stable expression hEAG1 HEK-293T system is successfully established by monoclonal puromycin-resistant selection after transfection with hEAG1-pCDH lentivirus system as evidenced by GFP expression in almost all cells. (B) Pulse protocol (top) and superimposed current traces from a representative whole-cell patch-clamp recording from hEAG1 channels in a stable cell in A. The current is elicited by depolarizing voltages from the holding voltage of -80 mV to 70 mV with the step of 10 mV followed by repolarization to -80 mV. The cells are incubated in the normal K+ channel recording solutions as described previously15. The voltage-dependent outward potassium currents suggest that functional hEAG1 channels are highly expressed in HEK293T cells. (C) Western blot of hEAG1 channel protein from purified protein samples. The anti-FLAG antibody recognizes a single protein band of ~110 kDa, demonstrating a full-length of FLAG-tagged hEAG1 channel expression. This Figure 1C has been modified from Han et al.15. Please click here to view a larger version of this figure.
Figure 2: A schematic diagram showing the BLI binding assay protocol. Four sensors are used in parallel in biotinylated-channel proteins or their dissolved SD buffer to load the channel proteins and the references. After that, these four sensors are transferred to assay phase to detect the association and disassociation with phospholipids or its solution buffer. The positions of channel proteins, phospholipids, and buffer are colored as indicated. The horizontal red dotted line indicates the two major steps of BLI study: loading phase and interaction assay phase. This Figure 2 has been modified from Han et al.15. Please click here to view a larger version of this figure.
Figure 3: Screen captures showing the raw data and processed data in a typical BLI study. (A) Typical loading and equilibration curves showing the equilibration step (60 s) with SD-buffer (baseline), the loading step with hEAG1 proteins (loading) and the reference curve equilibrated and loaded with hEAG1 proteins (loading), simultaneous measurement of two individual sensor tips. (B) The vertical red lines indicate the transferring of sensors from the lipid solution to the buffer solution during assay operation. (C) The original optical signals at association and dissociation phases after processing the double reference subtraction to subtract the non-specific binding signals. Please click here to view a larger version of this figure.
Figure 4: Results from BLI assay showing hEAG1 channel protein directly interacting with PIP2. (A) Screen capture showing the raw data of hEAG1 protein binding with PIP2 at concentration-dependence manner (0.03–3 μM). The accumulated concentrations of PIP2 denoted corresponding to the biosensors' data traces. (B) The raw data processing showing the changes in optical interference in different concentrations of PIP2 in a representative assay. (C) Curve fit with Hill equation obtained from the peak value of the optical interference signal measured at different PIP2 concentrations for determination of the equilibrium dissociation constants (Kd) of the interaction between the hEAG1 channel protein and PIP2 (n = 3).The Figure 4B and 4C have been modified from Han et al.15. Please click here to view a larger version of this figure.
Supplementary Figure 1: The configuration of long-chain phosphatidylinositol 4, 5-bisphosphate (PIP2). Please click here to download this figure.
Supplementary Figure 2: Screen capture of the fitted overlay from the BLI assay of Figure 3. The data is processed and fitted and only shown Association and Dissociation phases. The processed data curve is blue and the nonlinear fitting curve is red. Goodness of fit: R2 = 0.984789, X2 = 0.019109. The maximal binding parameter (Rmax) = 0.2875 nm (± 0.0006). Please click here to download this figure.
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 directly or not19, because the small molecule could act on other proteins or intercellular pathways that interact with the channel. Compared to widely used radioactive ligand binding assay and other commonly used label-free biosensor methods, BLI has significant advantages in terms of relative simple arrangement, the unrestricted association phase, high throughout, assay design closer to the in-vivo system and a need of small amount of immobilized protein (a few μg), and it can provide detailed insights into kinetic data5,6,20.
In order to maximally simulate the in vivo situation, we purified the functional hEAG1 channel proteins from the mammalian stably expression system. An easy and efficient protocol for both purification and identification of the overexpressed ion channel protein from adherent mammalian cells is presented. Some important tips for successful purification of the membrane proteins are: 1) The purification process can be either scaled-down or scaled-up according the expression abundance of interest proteins in mammalian expression systems; 2) We fused a FLAG tag on the C terminus of hEAG1 channel to facilitate the purification of this protein with ANTI-FLAG affinity beads using the commercial kit and purification protocol. An electrophysiological measurement demonstrated that the fusion FLAG has no effect on the function of this channel15; 3) Incubation of the mixture of ANTI-FLAG beads and protein extract overnight at 4°C with gently shaking is helpful for capturing the FLAG fusion protein; 4) Using the affinity purification, we can get enough hEAG1 ion channel protein from four 15 cm dishes with above 90% cell confluency for once assay process.
The purified hEAG1 channel proteins are biotinylated by using excess biotin (3-10 fold) and exchanging the solution buffer with SD buffer for the further BLI assay. The excess biotin can maximally modify the membrane channel protein to increase the coated efficiency on the surface of biosensor tips, and the SD assay buffer containing low concentrations of BSA and Polysorbate 20 can minimize nonspecific binding9. In spite of proceeding these key operations, the non-ideal interactions still could exist especially when performing a binding study with high concentration of small molecule, which could non-specifically bound to the surface of SA sensors and result the false-positive interaction as the cyanine curve shown in Figure 4A. Thus, a double reference subtraction protocol is still necessary to get the reliable results by subtracting the non-specific bindings including the interactions between the sensor and small molecule, biotinylated hEAG1 protein with small molecule solution, and sensors with small molecule solution. This double reference subtraction operation needs four biosensors for once assay. Two biosensors will be coated with biotinylated membrane proteins and proceeding the interaction assay with small molecule and its buffer. The other two sensors will be wetted with SD buffer and interacting with small molecule and its buffer. Only the interaction of membrane protein immobilized biosensor and small molecule shows the positive signal and the rest of three interaction signals work as the controls (Figure 2). By using this procedure, as shown in Figure 3 and Figure 4, our results clearly demonstrate the strong interaction between hEAG1 channel proteins and PIP2 and show a concentration-dependence profile. It must be pointed out that there are several limitations in our measurement. For instance,there are some other membrane lipids which could bind to the purified channel protein. Also, it's still not clear that whether the anchored purified protein keep their original conformation and activity. It's very difficult to measure the real configurations of small molecule lipids when they interact with the protein. Although the detailed processes are unknown, our study show that the binding kinetics by BLI are consistent with those derived by electrophysiological recording, which makes the novel BLI application for ion channel protein-small molecule interaction in a supplementary manner.
In summary, our study confirms that it is a reliable strategy by purifying the functional membrane protein from mammalian expression system to detect the direct interaction with its ligands. The successful application of BLI for membrane protein-small molecule interaction will facilitate the small molecule screening and mechanism exploration in ion channel drug discovery.
The authors have nothing to disclose.
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).
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 |