Biomed Microdevices (2015) 17:12 DOI 10.1007/s10544-014-9919-4
Single channel and ensemble hERG conductance measured in droplet bilayers Viksita Vijayvergiya & Shiv Acharya & Jason Poulos & Jacob Schmidt
# Springer Science+Business Media New York 2015
Abstract The human ether-a-go-go related gene (hERG) encodes the potassium channel Kv11.1, which plays a key role in the cardiac action potential and has been implicated in cardiac disorders as well as a number of off-target pharmaceutical interactions. The electrophysiology of this channel has been predominantly studied using patch clamp, but lipid bilayers have the potential to offer some advantages, including apparatus simplicity, ease of use, and the ability to control the membrane and solution compositions. We made membrane preparations from hERG-expressing cells and measured them using droplet bilayers, allowing measurement of channel ensemble currents and 13.5 pS single channel currents. These currents were ion selective and were blockable by E-4031 and dofetilide in a dose-dependent manner, allowing determination of IC50 values of 17 nM and 9.65 μM for E-4031 and dofetilide, respectively. We also observed time- and voltagedependent currents following step changes in applied potential that were similar to previously reported patch clamp measurements.
Keywords Ion channel conductance . hERG . Lipid bilayer . Single channel
Electronic supplementary material The online version of this article (doi:10.1007/s10544-014-9919-4) contains supplementary material, which is available to authorized users. V. Vijayvergiya : S. Acharya : J. Schmidt (*) Department of Bioengineering, University of California Los Angeles, Los Angeles, CA 90095, USA e-mail: [email protected] J. Poulos Librede Inc, Sherman Oaks, CA, USA
1 Introduction The human ether-a-go-go related gene (hERG) encodes the pore-forming subunit of the rapid component of the delayed rectifier K+ channel Kv11.1, which is expressed in the heart, various brain regions, smooth muscle cells, endocrine cells, and a wide range of tumor cell lines. Kv11.1 is involved in long QT syndrome, an inherited disorder associated with a markedly increased risk of ventricular arrhythmia and sudden cardiac death (Algra et al. 1993). Blockade of Kv11.1 by a wide range of prescription medications can cause drug-induced QT prolongation with an increase in risk of sudden cardiac arrest (Curran et al. 1995; Sanguinetti et al. 1995). Patch clamp has been the ‘gold standard’ for characterization of hERG (Kiehn et al. 1996; Smith et al. 1996; Zou et al. 1997) and is the basis for development of high-throughput electrophysiological assays (Gillie et al. 2013). These measurements show that the channel opens from a closed state at depolarizing potentials followed by rapid inactivation. Single channel measurements in eukaryotic cellular systems, (Zhou et al. 1998) cardiac myocytes, (Liu et al. 2004) and Xenopus oocytes (Zou et al. 1997) show a 10–14 pS conductance. A number of compounds have been found as hERG activators (Asayama et al. 2013) or inhibitors, (Zhou et al. 1998; Huang et al. 2010; Jiménez-Vargas et al. 2012; Long et al. 2013) including E-4031, astemizole, and dofetilide as well as quaternary ammonium compounds. Measurement of drug potency and determination of IC50 values is important for drug discovery and also safety screening, (Hancox et al. 2008) due to hERG’s involvement in QT prolongation. hERG channels have also been studied in various artificial lipid bilayer platforms such as tethered bilayers (Becucci et al. 2008) and droplet interface bilayers, (Leptihn et al. 2011) as well as bilayers on Teflon, (Zhang et al. 2012) alumina, (Hirano-Iwata et al. 2012) and microfabricated silicon chips
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(Oshima et al. 2013). These artificial bilayer platforms are attractive for their ease of use and ability to allow direct control of temperature, pH, and lipid composition, among other parameters. Droplet bilayers, in which the bilayer is formed by contacting lipid monolayers self-assembled at two aqueous/oil interphases, (Funakoshi et al. 2006; Holden et al. 2007) are particularly advantageous in that pure protein or cellular membrane preparations may be used and measurement of single channels or channel ensembles may be controlled simply through dilution (Leptihn et al. 2011; El-Arabi et al. 2012; Portonovo et al. 2013). The small droplet sizes (~μl or smaller) minimize use of protein and pharmaceutical compounds and their measurement can be parallelized and automated (Kawano et al. 2013). Using droplet bilayers we previously measured concentration-dependent changes in conductance of TRPM8 (purified from engineered E. coli and reconstituted into liposomes) and hERG channels (commercial membrane preparations) with several pharmaceutical compounds (El-Arabi et al. 2012; Portonovo et al. 2013). The TRPM8 measurements were not able to resolve the sub-10 ms temporal changes in channel conductance following voltage steps observed in patch clamp studies (Voets et al. 2004). It is unclear whether these rapid changes were unresolvable as a result of the large transient capacitive charging currents arising from step changes in voltage that are characteristic of the ~100– 400 pF capacitance of large area lipid bilayer membranes. In contrast to the rapid kinetics of the TRPM8 channel, hERG channels have longer time constants associated with channel activation (~1 s), which should be observable even after the lipid bilayer membrane’s capacitive charging or discharging is complete. However, our previous measurements of hERG did not result in any observations of the expected temporal or voltage dependent conductance of ion channel ensembles,{Portonovo et al. 2013 #34} similar to others’ measurements, {Zhang et al. 2012 #38} although Oshima and coworkers reported voltage dependent single channel conductance.{Oshima et al. 2013 #33} Temporal and voltage dependence of conductance for purified ion channels reconstituted in lipid bilayers has been previously reported by Tao and MacKinnon (Tao and MacKinnon 2008) and Schmidt and MacKinnon (Schmidt et al. 2009) for K v1.2 and K vAP channels, respectively. In the work described here, we used droplet bilayers to measure ion channel currents from membrane preparations of hERG-expressing cells at the single channel and ensemble levels, demonstrating ion selectivity and dose-dependent inhibition by E-4031 and dofetilide, with IC50 values of 17 nM and 9.65 μM. Time and voltage dependent currents were also observed similar to previous reported patch clamp measurements.
Biomed Microdevices (2015) 17:12
2 Materials and methods 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was obtained from Avanti Polar Lipids. E-4031, dofetilide, and all commonly used chemicals were purchased from SigmaAldrich. Human embryonic kidney (HEK) cells expressing hERG were obtained from Invitrogen, and mouse L cells expressing hERG were a gift (Dr. Eckhard Ficker, Rammelkamp Center, Cleveland OH). Aqueous solutions used were buffer K130 (130 mM KCl, 1 mM MgCl2, 5 mM EGTA and 10 mM HEPES (pH 7.4)) and buffer K4 (4 mM KCl, 137 mM NaCl 1.8 mM CaCl2 1 mM MgCl2, and 10 mM HEPES (pH 7.4)) similar to those described previously (Zhou et al. 1998). 2.1 Formation of lipid bilayers and conductance measurements Droplet bilayer formation used the same apparatus and methods as previously described (Fig. 1) (El-Arabi et al. 2012; Portonovo and Schmidt 2012; Portonovo et al. 2013). Briefly, the lower chamber was filled with aqueous solution, followed by addition of 80 μl of DPhPC in hexadecane (10 mg/ml) to the middle chamber. A droplet of solution containing diluted membrane vesicles (described below) was placed on the end of a Ag/AgCl electrode mounted on a micromanipulator and lowered into the hexadecane solution in the center well. A waiting time of 30 min allowed for formation of lipid monolayers on the hexadecane/aqueous interfaces of the lower aqueous solution and the droplet. The droplet was lowered further to bring the two monolayers into contact, with the contact area bounded by a 100 μm diameter circular aperture in a 75 μm thick Delrin sheet (McMaster-Carr). Experiments were performed at room temperature (23 °C). Currents were recorded with an Axopatch 200B patch clamp amplifier (Axon Instruments). Signals were filtered with a 1 kHz low pass Bessel filter and digitized at 5 kHz with Digidata 1322A (Axon Instruments). pClamp9 software (Axon Instruments) was used to generate voltage clamp commands, acquire membrane currents, and analyze digitized data. The currents were recorded without leak subtraction or capacitance compensation and low-pass filtered in software with a cut-off frequency of 60–90 Hz after data acquisition. Measurement of hERG inhibition by E-4031 and dofetilide was achieved by addition of aliquots of concentrated E-4031 and dofetilide to the lower chamber in the same buffer. 2.2 Preparation of hERG-enriched membrane vesicles Two million HEK or mouse L cells were washed with phosphate buffered saline (PBS) and resuspended in 5 ml of 10 mM HEPES buffer (pH 7.4) containing 250 mM sucrose and protease inhibitors (Halt Protease inhibitor cocktail, Thermo Scientific), sonicated in a bath sonicator (VWR) at
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Fig. 1 Bilayer chip schematic. (a) Side wells are loaded with lower aqueous solution and the central well is loaded with hexadecane containing 10 mg/ml DPhPC. The lower aqueous solution and center well are separated by a 75 μm thick Delrin film containing a 100 μm aperture. (b) A Ag/AgCl electrode with sessile aqueous droplet
containing hERG membrane preparation is lowered into the center well from above. Lipid monolayers spontaneously form at the aqueous/ hexadecane interfaces (insets). A lipid bilayer is formed by lowering the sessile droplet into contact with the lower aqueous solution, bounded by the Delrin aperture
100 % power for 30 s, and centrifuged at 1000 g at 4 °C. The pellet was removed and the supernatant was centrifuged again at 7000 g at 4 °C to remove cell debris. The resulting supernatant was centrifuged at 22,000 g for 30 min at 4 °C to collect the membrane vesicles. The membrane vesicles were resuspended in buffer K130. The total protein concentration in the membrane preparation was determined by the Bradford method (Bio-Rad 500–0002). The total protein in the droplet as described above varied from 100 to 1000 ng in the experiments described here. Membrane vesicles were stored at −80 °C and used within one week of preparation by diluting them into buffer K130 in ratios of 1:100 to 1:10000 before use. The membrane samples containing protein were further characterized by Western blot. The membrane samples were boiled in sample buffer (0.12 M Tris–HCl pH 6.8, 2 % SDS, 2 % mercaptoethanol, 20 % glycerol, and 0.001 % bromophenol blue) and electrophoresed on a 4–15 % MiniProtean TGX pre-cast polyacrylamide gel (Bio-Rad 456– 1083). The membrane proteins were then transferred into nitrocellulose filter using a Mini Trans-Blot Module (Bio-Rad 170–3935). After transfer, the filters were blocked with 5 % nonfat dry milk and 0.2 % Tween-20 in PBS for 1 hr. The filters were washed with PBS and mixed with hERG C-20 (Santa Cruz Biotech SC-15968) and incubated for 1 hr. The filters were incubated for 30 min with purified rabbit anti-goat IgG-HRP Antibody (Santa Cruz Biotech, SC-2768) at a 1:1000 dilution at room temperature, washed, and developed. The Western blot showed one primary band near 150 kD (Fig. S1, Supplementary Material), expected for the presence of hERG (Zhou et al. 1998).
measurement of a small constant current (~pA) with 100 mV applied trans-membrane potential, corresponding to a stable low conductance bilayer. However, when the hERGcontaining membrane vesicles were present in the hanging droplet, currents ranging from tens to thousands of pA were observed 30–40 % of the time, with pA currents similar to the control observed the remainder of the time. At a 1:1000– 1:10000 dilution of the membrane preparation, small ~ pA fluctuating currents corresponding to hERG single channels could be observed. At smaller dilutions (1:100–1:1000), we observed larger currents of magnitude hundreds of pA to nA, of two types. The first type consisted of currents for which the measured conductance was constant and independent of applied voltage and independent of time following step changes in voltage. The second type was currents for which the conductance did show voltage dependence and temporal variation following step changes in the applied voltage. Both of these types of macroscopic currents were blockable by E-4031, a hERG-specific ion channel blocker (Kamiya et al. 2006). Below we discuss both types of current measurement in more detail.
3 Results and discussion Control experiments, in which the droplet hanging from the electrode contained only buffer K130 or K130 and membrane preparations of non-transfected HEK cells, resulted in
3.1 Single channel measurements Using membrane preparation dilution ratios of 1:1000 and 1:10000, ion selective single channel currents were observed (Fig. 2). With buffer K4 in the lower solution and the droplet containing K130 with hERG membrane preparations from mouse L cells, potassium selective single channel currents were observed while applying −80 mV for 1 s followed by +60 mV for 2 s and steps to −100 to 40 mV in 10 mV increments for 4 s (Fig. 2a). The single channel conductance was obtained from histogram analysis of step changes in the measured currents (for example Fig. S2, Supplementary Material) plotted against the applied potential (Fig. 2b). A linear regression of the single channel steps in Fig. 2b yielded a single channel conductance of 12 pS, consistent with previous reports in bilayers (Leptihn et al. 2011; Oshima et al. 2013; Portonovo et al. 2013) and patch clamp (Zhou et al. 1998;
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Biomed Microdevices (2015) 17:12
Fig. 2 (a) Measured currents from membrane preparations of hERGexpressing mouse L cells in asymmetric buffers K130 and K4 using applied voltages: −80 mV for 1 s, +60 mV for 2 and 4 s steps from +40 to −100 mV in 10 mV increments. Histograms of the currents measured during the step voltages were fit to a double Gaussian function to find the
single channel current as a function of voltage (B). The average current measured during each step voltage was also plotted as a function of voltage (B). The traces shown were electronically filtered at 1 kHz, acquired at 5 kHz, and notch-filtered in software at 60 Hz
Kiehn et al. 1999). The single channel currents indicated potassium selectivity and were blocked following addition of E4031 (Fig. S3, Supplementary Material).
At dilution ratios between 1:100 and 1:1000, larger currents ranging from tens of pA to tens of nA were measured (Fig. 3). These currents were blockable by 10 μM E-4031. These currents were predominantly representative of a voltage- and time-independent constant conductance similar to previously reported bilayer measurements of hERG (Zhang et al. 2012; Portonovo et al. 2013). These voltage- and time-independent constant conductance currents were also observed to be potassium selective, with Erev =−84 mV for the trace average currents in Fig. 2b (also Fig. S4, Supplementary Material).
Measurement of these currents as the concentration of E4031 was increased in the surrounding solution showed a concentration-dependent reduction in amplitude, similar to our previous measurements (Portonovo et al. 2013). Sometimes these currents were not completely blocked following application of 10 μM E-4031, possibly as a result of the presence of other channels in the membrane preparation or bilayer leak currents. This is commonly seen in patch clamp experiments on over-expressing cells which have some endogenous channels in the membrane that are not responsive to hERG specific blockers (Kiehn et al. 1996). Likewise with those experiments we have subtracted these unblockable currents from the total measured current when determining the concentration-dependent drug inhibition. These currents, normalized to the zero concentration value, were plotted versus E-4031 concentration and fit to the Hill equation with an
Fig. 3 Measurement of macroscopic currents from HEK cell membrane preparations using a dilution ratio of 1:100. Measurement solutions were K130 in both the droplet and lower solution. Voltage protocol, Top: The applied voltage was held at −80 mV for 1 s followed by a constant voltage
step progressively increasing from −60 to +60 mV in steps of 20 mV for 1 s, followed by −50 mV for 1 s. Middle: The resultant measured current. Bottom: The measured current measured from the same sample with the same applied potentials but following the addition of 10 μM E-4031
3.2 Macroscopic current measurements
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Fig. 4 Concentration dependent inhibition of measured currents in symmetric K130 solutions. (Left) I/I max plotted versus E-4031 concentration (points) with a fit of this data to the Hill equation (lines) for two experiments, shown in red and blue. IC50 values from each fit
were 14.5±1.1 and 19.6±1.5 nM. (Right) I/Imax plotted versus dofetilide concentration (points) with a fit of this data to the Hill equation (lines) for two experiments. IC50 values from each fit were 9.4±0.5 and 9.9± 0.3 μM
average IC50 value of 17 nM (Fig. 4), similar to our previous work (Portonovo et al. 2013) and patch clamp measurements (Zhou et al. 1998). Measurements with dofetilide also showed a concentration-dependent reduction in amplitude with an average IC50 value of 9.65 μM. This is several orders of magnitude larger than previously reported values (Kiehn et al. 1996; Ficker et al. 2001), which may be related to the lack of inactivation observed in the currents (similar to Fig. 3). Previous reports have described that non-inactivating ether a-go-go channels are relatively insensitive to block by dofetilide with IC50 in the μM range (Ficker et al. 2001). In some experiments, the observed currents exhibited timeand voltage-dependence following step changes in applied voltage. For example, in one experiment measuring hERGexpressing HEK membrane preparations with K130 buffer in the droplet and lower solution, a trans-membrane voltage of 0 mV was applied for 4 s, followed by a step to −120, −100, −80, or −60 mV for 4 s, a step to 0 mV for 4 s, and a step to 60, 80, 100, or 120 mV for 4 s, and a final step to 0 mV for 6 s
before repeating the pulse sequence and changing the step voltages (Fig. 5). The measured current showed a large amplitude that decayed in ~1 s when the voltage was stepped from 0 to −120 mV. However, when stepping from 0 mV to positive potentials, such currents were not observed. These currents were blocked by E-4031 (Fig. S5, Supplementary Material). These currents resemble the macro-patch hERG currents described by Kiehn et al. from Xenopus oocytes measured in symmetric 100 mM KCl solutions, where steps from 0 mV to negative voltages showed large transient currents which were absent following steps from 0 mV to positive voltages (Kiehn et al. 1996). Tao and Mackinnon (Tao and MacKinnon 2008) and Schmidt, Cross, and MacKinnon (Schmidt et al. 2009) have also observed time and voltage dependent ion channel conductance in lipid bilayers. In that work, Kv1.2 and KvAP channels were expressed in yeast or E. coli, purified, and reconstituted into liposomes. These vesicles were fused to freestanding lipid bilayer membranes and subsequently
Fig. 5 Measurements of droplet bilayers formed with hERG-expressing HEK cell membrane preparations at a 1:100 dilution with K130 buffer in the droplet and lower solution with the displayed voltage protocol (top) showed resultant currents (bottom) that large transient behavior
immediately following the step from 0 mV to negative voltages (bottom left). The time scale of this behavior varied with the magnitude of negative potential. Such time and voltage dependence was not observed for steps from 0 mV to positive potentials (bottom right)
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measured. Ion channel ensemble currents of several nA were observed that exhibited characteristic voltage-dependent activation and inactivation. However, there were differences in voltage activation and pharmacological inhibition of Kv1.2 measured in bilayers compared to other studies of Kv1.2 in Xenopus oocytes that they hypothesized could be due to differences in protein expression or differences in the bilayer environment compared to a normal cellular system.
4 Conclusions We have measured membrane preparations of hERGcontaining HEK cells and mouse L cells using droplet bilayers, obtaining single channel and macroscopic currents. These currents were blockable with E-4031 and dofetilide in a dose dependent manner with IC50 values close to previously reported results. Macroscopic currents with time- and voltagedependent conductance similar to previously reported patch clamp measurements were also observed. Droplet bilayer reconstitution of ion channels from membrane preparations requires much less labor and material compared to purified ion channels. The ability to use mammalian cells also lifts any restrictions on ion channel expression in yeast or bacteria. We are continuing to work with this system to extend these results to additional measurement conditions and drugs, to determine the system’s compatibility with measurement of other ion channels, and to parallelize the measurement (Lu et al. 2014). The demonstrated compatibility of droplet bilayers with automated measurement (Poulos et al. 2010) raises the possibility of its use for the performance of ion channel drug screening assays. Acknowledgments We thank Carl Salazar and Prof. Takasi Nisisako for technical support. Research reported in this publication was supported by NIGMS of the National Institutes of Health under award number R44GM097763 and R44GM088890. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Conflict of interest statement Librede Inc. has licensed intellectual property invented by Schmidt and Poulos from The Regents of the University of California. Schmidt and Poulos have a financial interest in Librede.
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Biomed Microdevices (2015) 17:12 L. Becucci, M.V. Carbone et al., Incorporation of the HERG potassium channel in a mercury supported lipid bilayer. J. Phys. Chem. B 112(4), 1315–1319 (2008) M.E. Curran, I. Splawski et al., A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 80(5), 795–803 (1995) A.M. El-Arabi, C.S. Salazar, J.J. Schmidt, Ion channel drug potency assay with an artificial bilayer chip. Lab Chip 12(13), 2409–2413 (2012) E. Ficker, W. Jarolimek, A.M. Brown, Molecular determinants of inactivation and dofetilide block in ether a-go-go (EAG) channels and EAG-related K(+) channels. Mol. Pharmacol. 60(6), 1343–1348 (2001) K. Funakoshi, H. Suzuki, S. Takeuchi, Lipid bilayer formation by contacting monolayers in a microfluidic device for membrane protein analysis. Anal. Chem. 78(24), 8169–8174 (2006) D.J. Gillie, S.J. Novick et al., Development of a high-throughput electrophysiological assay for the human ether-à-go-go related potassium channel hERG. J. Pharmacol. Toxicol. Methods 67(1), 33–44 (2013) J.C. Hancox, M.J. McPate, A. El Harchi, The hERG potassium channel and hERG screening for drug-induced torsades de pointes. Pharmacol. Ther. 119(2), 118–132 (2008) A. Hirano-Iwata, A. Oshima et al., Stable Lipid Bilayers Based on Microand Nano-Fabrication as a Platform for Recording Ion-Channel Activities. Anal. Sci. 28(11), 1049–1057 (2012) M.A. Holden, D. Needham, H. Bayley, Functional bionetworks from nanoliter water droplets. J. Am. Chem. Soc. 129(27), 8650–8655 (2007) X.-P. Huang, T. Mangano et al., Identification of human Ether-a-go-go related gene modulators by three screening platforms in an academic drug-discovery setting. Assay. Drug Dev. Tech. 8(6), 727–742 (2010) J. Jiménez-Vargas, R. Restano-Cassulini, L. Possani, Toxin modulators and blockers of hERG K+ channels. Toxicon 60(4), 492–501 (2012) K. Kamiya, R. Niwa et al., Molecular determinants of HERG channel block. Mol. Pharmacol. 69(5), 1709–1716 (2006) R. Kawano, Y. Tsuji, et al. BAutomated Parallel Recordings of Topologically Identified Single Ion Channels.^ Sci. Rep. 3 (2013) J. Kiehn, A.E. Lacerda et al., Molecular physiology and pharmacology of HERG: single-channel currents and block by dofetilide. Circulation 94(10), 2572–2579 (1996) J. Kiehn, A.E. Lacerda, A.M. Brown, Pathways of HERG inactivation. Am. J. Physiol-Heart Cir. Physiol. 277(1), H199–H210 (1999) S. Leptihn, J.R. Thompson et al., In vitro reconstitution of eukaryotic ion channels using droplet interface bilayers. J. Am. Chem. Soc. 133(24), 9370–9375 (2011) G. Liu, J. Zhou et al., Single channel recordings of a rapid delayed rectifier current in adult mouse ventricular myocytes: Basic properties and effects of divalent cations. J. Physiol. 15(556), 401–413 (2004) Y. Long, Z. Lin et al., Mechanism of HERG potassium channel inhibition by tetra-n-octylammonium bromide and benzethonium chloride. Toxicol. Appl. Pharmacol. 267(2), 155–166 (2013) B. Lu, G. Kocharyan, J.J. Schmidt, Lipid bilayer arrays: Cyclically formed and measured. Biotechnol. J. 9(3), 446–451 (2014) A. Oshima, A. Hirano-Iwata et al., Reconstitution of Human Ether-a-gogo-Related Gene Channels in Microfabricated Silicon Chips. Anal. Chem. 85(9), 4363–4369 (2013) S.A. Portonovo, J. Schmidt, Masking apertures enabling automation and solution exchange in sessile droplet lipid bilayers. Biomed. Microdevices 14(1), 187–191 (2012) S.A. Portonovo, C.S. Salazar, J.J. Schmidt, hERG drug response measured in droplet bilayers. Biomed. Microdevices 15(2), 255–259 (2013) J.L. Poulos, S.A. Portonovo et al., Automatable lipid bilayer formation and ion channel measurement using sessile droplets. J. Phys. Condens. Matter 22, 454105 (2010)
Biomed Microdevices (2015) 17:12 M.C. Sanguinetti, C. Jiang et al., A mechanistic link between an inherited and an acquired cardiac arrthytmia: HERG encodes the IKr potassium channel. Cell 81(2), 299–307 (1995) D. Schmidt, S.R. Cross, R. Mackinnon, A Gating model for the Archeal voltage- dependant K channel KvAP in DPhPC and POPE :POPG decane lipid bilayers. J. Mol. Biol. 390(5), 902–912 (2009) P.L. Smith, T. Baukrowitz, G. Yellen, The inward rectification mechanism of the HERG cardiac potassium channel. Nature 379(6568), 833– 836 (1996) X. Tao, R. MacKinnon, Functional analysis of Kv1. 2 and paddle chimera Kv channels in planar lipid bilayers. J. Mol. Biol. 382(1), 24–33 (2008)
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Single channel and ensemble hERG conductance measured in droplet bilayers Viksita Vijayvergiya & Shiv Acharya & Jason Poulos & Jacob Schmidt
# Springer Science+Business Media New York 2015
Abstract The human ether-a-go-go related gene (hERG) encodes the potassium channel Kv11.1, which plays a key role in the cardiac action potential and has been implicated in cardiac disorders as well as a number of off-target pharmaceutical interactions. The electrophysiology of this channel has been predominantly studied using patch clamp, but lipid bilayers have the potential to offer some advantages, including apparatus simplicity, ease of use, and the ability to control the membrane and solution compositions. We made membrane preparations from hERG-expressing cells and measured them using droplet bilayers, allowing measurement of channel ensemble currents and 13.5 pS single channel currents. These currents were ion selective and were blockable by E-4031 and dofetilide in a dose-dependent manner, allowing determination of IC50 values of 17 nM and 9.65 μM for E-4031 and dofetilide, respectively. We also observed time- and voltagedependent currents following step changes in applied potential that were similar to previously reported patch clamp measurements.
Keywords Ion channel conductance . hERG . Lipid bilayer . Single channel
Electronic supplementary material The online version of this article (doi:10.1007/s10544-014-9919-4) contains supplementary material, which is available to authorized users. V. Vijayvergiya : S. Acharya : J. Schmidt (*) Department of Bioengineering, University of California Los Angeles, Los Angeles, CA 90095, USA e-mail: [email protected] J. Poulos Librede Inc, Sherman Oaks, CA, USA
1 Introduction The human ether-a-go-go related gene (hERG) encodes the pore-forming subunit of the rapid component of the delayed rectifier K+ channel Kv11.1, which is expressed in the heart, various brain regions, smooth muscle cells, endocrine cells, and a wide range of tumor cell lines. Kv11.1 is involved in long QT syndrome, an inherited disorder associated with a markedly increased risk of ventricular arrhythmia and sudden cardiac death (Algra et al. 1993). Blockade of Kv11.1 by a wide range of prescription medications can cause drug-induced QT prolongation with an increase in risk of sudden cardiac arrest (Curran et al. 1995; Sanguinetti et al. 1995). Patch clamp has been the ‘gold standard’ for characterization of hERG (Kiehn et al. 1996; Smith et al. 1996; Zou et al. 1997) and is the basis for development of high-throughput electrophysiological assays (Gillie et al. 2013). These measurements show that the channel opens from a closed state at depolarizing potentials followed by rapid inactivation. Single channel measurements in eukaryotic cellular systems, (Zhou et al. 1998) cardiac myocytes, (Liu et al. 2004) and Xenopus oocytes (Zou et al. 1997) show a 10–14 pS conductance. A number of compounds have been found as hERG activators (Asayama et al. 2013) or inhibitors, (Zhou et al. 1998; Huang et al. 2010; Jiménez-Vargas et al. 2012; Long et al. 2013) including E-4031, astemizole, and dofetilide as well as quaternary ammonium compounds. Measurement of drug potency and determination of IC50 values is important for drug discovery and also safety screening, (Hancox et al. 2008) due to hERG’s involvement in QT prolongation. hERG channels have also been studied in various artificial lipid bilayer platforms such as tethered bilayers (Becucci et al. 2008) and droplet interface bilayers, (Leptihn et al. 2011) as well as bilayers on Teflon, (Zhang et al. 2012) alumina, (Hirano-Iwata et al. 2012) and microfabricated silicon chips
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(Oshima et al. 2013). These artificial bilayer platforms are attractive for their ease of use and ability to allow direct control of temperature, pH, and lipid composition, among other parameters. Droplet bilayers, in which the bilayer is formed by contacting lipid monolayers self-assembled at two aqueous/oil interphases, (Funakoshi et al. 2006; Holden et al. 2007) are particularly advantageous in that pure protein or cellular membrane preparations may be used and measurement of single channels or channel ensembles may be controlled simply through dilution (Leptihn et al. 2011; El-Arabi et al. 2012; Portonovo et al. 2013). The small droplet sizes (~μl or smaller) minimize use of protein and pharmaceutical compounds and their measurement can be parallelized and automated (Kawano et al. 2013). Using droplet bilayers we previously measured concentration-dependent changes in conductance of TRPM8 (purified from engineered E. coli and reconstituted into liposomes) and hERG channels (commercial membrane preparations) with several pharmaceutical compounds (El-Arabi et al. 2012; Portonovo et al. 2013). The TRPM8 measurements were not able to resolve the sub-10 ms temporal changes in channel conductance following voltage steps observed in patch clamp studies (Voets et al. 2004). It is unclear whether these rapid changes were unresolvable as a result of the large transient capacitive charging currents arising from step changes in voltage that are characteristic of the ~100– 400 pF capacitance of large area lipid bilayer membranes. In contrast to the rapid kinetics of the TRPM8 channel, hERG channels have longer time constants associated with channel activation (~1 s), which should be observable even after the lipid bilayer membrane’s capacitive charging or discharging is complete. However, our previous measurements of hERG did not result in any observations of the expected temporal or voltage dependent conductance of ion channel ensembles,{Portonovo et al. 2013 #34} similar to others’ measurements, {Zhang et al. 2012 #38} although Oshima and coworkers reported voltage dependent single channel conductance.{Oshima et al. 2013 #33} Temporal and voltage dependence of conductance for purified ion channels reconstituted in lipid bilayers has been previously reported by Tao and MacKinnon (Tao and MacKinnon 2008) and Schmidt and MacKinnon (Schmidt et al. 2009) for K v1.2 and K vAP channels, respectively. In the work described here, we used droplet bilayers to measure ion channel currents from membrane preparations of hERG-expressing cells at the single channel and ensemble levels, demonstrating ion selectivity and dose-dependent inhibition by E-4031 and dofetilide, with IC50 values of 17 nM and 9.65 μM. Time and voltage dependent currents were also observed similar to previous reported patch clamp measurements.
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2 Materials and methods 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was obtained from Avanti Polar Lipids. E-4031, dofetilide, and all commonly used chemicals were purchased from SigmaAldrich. Human embryonic kidney (HEK) cells expressing hERG were obtained from Invitrogen, and mouse L cells expressing hERG were a gift (Dr. Eckhard Ficker, Rammelkamp Center, Cleveland OH). Aqueous solutions used were buffer K130 (130 mM KCl, 1 mM MgCl2, 5 mM EGTA and 10 mM HEPES (pH 7.4)) and buffer K4 (4 mM KCl, 137 mM NaCl 1.8 mM CaCl2 1 mM MgCl2, and 10 mM HEPES (pH 7.4)) similar to those described previously (Zhou et al. 1998). 2.1 Formation of lipid bilayers and conductance measurements Droplet bilayer formation used the same apparatus and methods as previously described (Fig. 1) (El-Arabi et al. 2012; Portonovo and Schmidt 2012; Portonovo et al. 2013). Briefly, the lower chamber was filled with aqueous solution, followed by addition of 80 μl of DPhPC in hexadecane (10 mg/ml) to the middle chamber. A droplet of solution containing diluted membrane vesicles (described below) was placed on the end of a Ag/AgCl electrode mounted on a micromanipulator and lowered into the hexadecane solution in the center well. A waiting time of 30 min allowed for formation of lipid monolayers on the hexadecane/aqueous interfaces of the lower aqueous solution and the droplet. The droplet was lowered further to bring the two monolayers into contact, with the contact area bounded by a 100 μm diameter circular aperture in a 75 μm thick Delrin sheet (McMaster-Carr). Experiments were performed at room temperature (23 °C). Currents were recorded with an Axopatch 200B patch clamp amplifier (Axon Instruments). Signals were filtered with a 1 kHz low pass Bessel filter and digitized at 5 kHz with Digidata 1322A (Axon Instruments). pClamp9 software (Axon Instruments) was used to generate voltage clamp commands, acquire membrane currents, and analyze digitized data. The currents were recorded without leak subtraction or capacitance compensation and low-pass filtered in software with a cut-off frequency of 60–90 Hz after data acquisition. Measurement of hERG inhibition by E-4031 and dofetilide was achieved by addition of aliquots of concentrated E-4031 and dofetilide to the lower chamber in the same buffer. 2.2 Preparation of hERG-enriched membrane vesicles Two million HEK or mouse L cells were washed with phosphate buffered saline (PBS) and resuspended in 5 ml of 10 mM HEPES buffer (pH 7.4) containing 250 mM sucrose and protease inhibitors (Halt Protease inhibitor cocktail, Thermo Scientific), sonicated in a bath sonicator (VWR) at
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Fig. 1 Bilayer chip schematic. (a) Side wells are loaded with lower aqueous solution and the central well is loaded with hexadecane containing 10 mg/ml DPhPC. The lower aqueous solution and center well are separated by a 75 μm thick Delrin film containing a 100 μm aperture. (b) A Ag/AgCl electrode with sessile aqueous droplet
containing hERG membrane preparation is lowered into the center well from above. Lipid monolayers spontaneously form at the aqueous/ hexadecane interfaces (insets). A lipid bilayer is formed by lowering the sessile droplet into contact with the lower aqueous solution, bounded by the Delrin aperture
100 % power for 30 s, and centrifuged at 1000 g at 4 °C. The pellet was removed and the supernatant was centrifuged again at 7000 g at 4 °C to remove cell debris. The resulting supernatant was centrifuged at 22,000 g for 30 min at 4 °C to collect the membrane vesicles. The membrane vesicles were resuspended in buffer K130. The total protein concentration in the membrane preparation was determined by the Bradford method (Bio-Rad 500–0002). The total protein in the droplet as described above varied from 100 to 1000 ng in the experiments described here. Membrane vesicles were stored at −80 °C and used within one week of preparation by diluting them into buffer K130 in ratios of 1:100 to 1:10000 before use. The membrane samples containing protein were further characterized by Western blot. The membrane samples were boiled in sample buffer (0.12 M Tris–HCl pH 6.8, 2 % SDS, 2 % mercaptoethanol, 20 % glycerol, and 0.001 % bromophenol blue) and electrophoresed on a 4–15 % MiniProtean TGX pre-cast polyacrylamide gel (Bio-Rad 456– 1083). The membrane proteins were then transferred into nitrocellulose filter using a Mini Trans-Blot Module (Bio-Rad 170–3935). After transfer, the filters were blocked with 5 % nonfat dry milk and 0.2 % Tween-20 in PBS for 1 hr. The filters were washed with PBS and mixed with hERG C-20 (Santa Cruz Biotech SC-15968) and incubated for 1 hr. The filters were incubated for 30 min with purified rabbit anti-goat IgG-HRP Antibody (Santa Cruz Biotech, SC-2768) at a 1:1000 dilution at room temperature, washed, and developed. The Western blot showed one primary band near 150 kD (Fig. S1, Supplementary Material), expected for the presence of hERG (Zhou et al. 1998).
measurement of a small constant current (~pA) with 100 mV applied trans-membrane potential, corresponding to a stable low conductance bilayer. However, when the hERGcontaining membrane vesicles were present in the hanging droplet, currents ranging from tens to thousands of pA were observed 30–40 % of the time, with pA currents similar to the control observed the remainder of the time. At a 1:1000– 1:10000 dilution of the membrane preparation, small ~ pA fluctuating currents corresponding to hERG single channels could be observed. At smaller dilutions (1:100–1:1000), we observed larger currents of magnitude hundreds of pA to nA, of two types. The first type consisted of currents for which the measured conductance was constant and independent of applied voltage and independent of time following step changes in voltage. The second type was currents for which the conductance did show voltage dependence and temporal variation following step changes in the applied voltage. Both of these types of macroscopic currents were blockable by E-4031, a hERG-specific ion channel blocker (Kamiya et al. 2006). Below we discuss both types of current measurement in more detail.
3 Results and discussion Control experiments, in which the droplet hanging from the electrode contained only buffer K130 or K130 and membrane preparations of non-transfected HEK cells, resulted in
3.1 Single channel measurements Using membrane preparation dilution ratios of 1:1000 and 1:10000, ion selective single channel currents were observed (Fig. 2). With buffer K4 in the lower solution and the droplet containing K130 with hERG membrane preparations from mouse L cells, potassium selective single channel currents were observed while applying −80 mV for 1 s followed by +60 mV for 2 s and steps to −100 to 40 mV in 10 mV increments for 4 s (Fig. 2a). The single channel conductance was obtained from histogram analysis of step changes in the measured currents (for example Fig. S2, Supplementary Material) plotted against the applied potential (Fig. 2b). A linear regression of the single channel steps in Fig. 2b yielded a single channel conductance of 12 pS, consistent with previous reports in bilayers (Leptihn et al. 2011; Oshima et al. 2013; Portonovo et al. 2013) and patch clamp (Zhou et al. 1998;
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Fig. 2 (a) Measured currents from membrane preparations of hERGexpressing mouse L cells in asymmetric buffers K130 and K4 using applied voltages: −80 mV for 1 s, +60 mV for 2 and 4 s steps from +40 to −100 mV in 10 mV increments. Histograms of the currents measured during the step voltages were fit to a double Gaussian function to find the
single channel current as a function of voltage (B). The average current measured during each step voltage was also plotted as a function of voltage (B). The traces shown were electronically filtered at 1 kHz, acquired at 5 kHz, and notch-filtered in software at 60 Hz
Kiehn et al. 1999). The single channel currents indicated potassium selectivity and were blocked following addition of E4031 (Fig. S3, Supplementary Material).
At dilution ratios between 1:100 and 1:1000, larger currents ranging from tens of pA to tens of nA were measured (Fig. 3). These currents were blockable by 10 μM E-4031. These currents were predominantly representative of a voltage- and time-independent constant conductance similar to previously reported bilayer measurements of hERG (Zhang et al. 2012; Portonovo et al. 2013). These voltage- and time-independent constant conductance currents were also observed to be potassium selective, with Erev =−84 mV for the trace average currents in Fig. 2b (also Fig. S4, Supplementary Material).
Measurement of these currents as the concentration of E4031 was increased in the surrounding solution showed a concentration-dependent reduction in amplitude, similar to our previous measurements (Portonovo et al. 2013). Sometimes these currents were not completely blocked following application of 10 μM E-4031, possibly as a result of the presence of other channels in the membrane preparation or bilayer leak currents. This is commonly seen in patch clamp experiments on over-expressing cells which have some endogenous channels in the membrane that are not responsive to hERG specific blockers (Kiehn et al. 1996). Likewise with those experiments we have subtracted these unblockable currents from the total measured current when determining the concentration-dependent drug inhibition. These currents, normalized to the zero concentration value, were plotted versus E-4031 concentration and fit to the Hill equation with an
Fig. 3 Measurement of macroscopic currents from HEK cell membrane preparations using a dilution ratio of 1:100. Measurement solutions were K130 in both the droplet and lower solution. Voltage protocol, Top: The applied voltage was held at −80 mV for 1 s followed by a constant voltage
step progressively increasing from −60 to +60 mV in steps of 20 mV for 1 s, followed by −50 mV for 1 s. Middle: The resultant measured current. Bottom: The measured current measured from the same sample with the same applied potentials but following the addition of 10 μM E-4031
3.2 Macroscopic current measurements
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Fig. 4 Concentration dependent inhibition of measured currents in symmetric K130 solutions. (Left) I/I max plotted versus E-4031 concentration (points) with a fit of this data to the Hill equation (lines) for two experiments, shown in red and blue. IC50 values from each fit
were 14.5±1.1 and 19.6±1.5 nM. (Right) I/Imax plotted versus dofetilide concentration (points) with a fit of this data to the Hill equation (lines) for two experiments. IC50 values from each fit were 9.4±0.5 and 9.9± 0.3 μM
average IC50 value of 17 nM (Fig. 4), similar to our previous work (Portonovo et al. 2013) and patch clamp measurements (Zhou et al. 1998). Measurements with dofetilide also showed a concentration-dependent reduction in amplitude with an average IC50 value of 9.65 μM. This is several orders of magnitude larger than previously reported values (Kiehn et al. 1996; Ficker et al. 2001), which may be related to the lack of inactivation observed in the currents (similar to Fig. 3). Previous reports have described that non-inactivating ether a-go-go channels are relatively insensitive to block by dofetilide with IC50 in the μM range (Ficker et al. 2001). In some experiments, the observed currents exhibited timeand voltage-dependence following step changes in applied voltage. For example, in one experiment measuring hERGexpressing HEK membrane preparations with K130 buffer in the droplet and lower solution, a trans-membrane voltage of 0 mV was applied for 4 s, followed by a step to −120, −100, −80, or −60 mV for 4 s, a step to 0 mV for 4 s, and a step to 60, 80, 100, or 120 mV for 4 s, and a final step to 0 mV for 6 s
before repeating the pulse sequence and changing the step voltages (Fig. 5). The measured current showed a large amplitude that decayed in ~1 s when the voltage was stepped from 0 to −120 mV. However, when stepping from 0 mV to positive potentials, such currents were not observed. These currents were blocked by E-4031 (Fig. S5, Supplementary Material). These currents resemble the macro-patch hERG currents described by Kiehn et al. from Xenopus oocytes measured in symmetric 100 mM KCl solutions, where steps from 0 mV to negative voltages showed large transient currents which were absent following steps from 0 mV to positive voltages (Kiehn et al. 1996). Tao and Mackinnon (Tao and MacKinnon 2008) and Schmidt, Cross, and MacKinnon (Schmidt et al. 2009) have also observed time and voltage dependent ion channel conductance in lipid bilayers. In that work, Kv1.2 and KvAP channels were expressed in yeast or E. coli, purified, and reconstituted into liposomes. These vesicles were fused to freestanding lipid bilayer membranes and subsequently
Fig. 5 Measurements of droplet bilayers formed with hERG-expressing HEK cell membrane preparations at a 1:100 dilution with K130 buffer in the droplet and lower solution with the displayed voltage protocol (top) showed resultant currents (bottom) that large transient behavior
immediately following the step from 0 mV to negative voltages (bottom left). The time scale of this behavior varied with the magnitude of negative potential. Such time and voltage dependence was not observed for steps from 0 mV to positive potentials (bottom right)
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measured. Ion channel ensemble currents of several nA were observed that exhibited characteristic voltage-dependent activation and inactivation. However, there were differences in voltage activation and pharmacological inhibition of Kv1.2 measured in bilayers compared to other studies of Kv1.2 in Xenopus oocytes that they hypothesized could be due to differences in protein expression or differences in the bilayer environment compared to a normal cellular system.
4 Conclusions We have measured membrane preparations of hERGcontaining HEK cells and mouse L cells using droplet bilayers, obtaining single channel and macroscopic currents. These currents were blockable with E-4031 and dofetilide in a dose dependent manner with IC50 values close to previously reported results. Macroscopic currents with time- and voltagedependent conductance similar to previously reported patch clamp measurements were also observed. Droplet bilayer reconstitution of ion channels from membrane preparations requires much less labor and material compared to purified ion channels. The ability to use mammalian cells also lifts any restrictions on ion channel expression in yeast or bacteria. We are continuing to work with this system to extend these results to additional measurement conditions and drugs, to determine the system’s compatibility with measurement of other ion channels, and to parallelize the measurement (Lu et al. 2014). The demonstrated compatibility of droplet bilayers with automated measurement (Poulos et al. 2010) raises the possibility of its use for the performance of ion channel drug screening assays. Acknowledgments We thank Carl Salazar and Prof. Takasi Nisisako for technical support. Research reported in this publication was supported by NIGMS of the National Institutes of Health under award number R44GM097763 and R44GM088890. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Conflict of interest statement Librede Inc. has licensed intellectual property invented by Schmidt and Poulos from The Regents of the University of California. Schmidt and Poulos have a financial interest in Librede.
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