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 voltage- dependent currents following step changes in applied potential that were similar to previously reported patch clamp measurements.
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References
A. Algra, J. Tijssen et al., QT interval variables from 24 hour electrocardiography and the two year risk of sudden death. Br. Heart J. 70(1), 43–48 (1993)
M. Asayama, J. Kurokawa et al., Effects of an hERG Activator, ICA-105574, on Electrophysiological Properties of Canine Hearts. J. Pharmacol. Sci. 121(1), 1–8 (2013)
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 Micro- and 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. “Automated 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-go-go-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)
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)
T. Voets, G. Droogmans et al., The principle of temperature-dependent gating in cold- and heat-sensitive TRP channels. Nature 430(7001), 748–754 (2004)
Y. Zhang, T. Phung et al., hERG ion channel pharmacology: cell membrane liposomes in porous-supported lipid bilayers compared with whole-cell patch-clamping. Eur. Biophys. J. 41(11), 949–958 (2012)
Z. Zhou, Q. Gong et al., Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature. Biophys. J. 74(1), 230–241 (1998)
A. Zou, M.E. Curran et al., Single HERG delayed rectifier K+ channels expressed in Xenopus oocytes. Am. J. Physiol-Heart Cir. Physiol. 272(3), H1309–H1314 (1997)
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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Vijayvergiya, V., Acharya, S., Poulos, J. et al. Single channel and ensemble hERG conductance measured in droplet bilayers. Biomed Microdevices 17, 12 (2015). https://doi.org/10.1007/s10544-014-9919-4
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DOI: https://doi.org/10.1007/s10544-014-9919-4