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Electrophysiological and Fluorescence Microscopy Studies with HERG Channel/EGFP Fusion Proteins

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Abstract

HERG (human ether-a-go-go-related gene) encodes the Kv11.1 protein α-subunit that underlies the rapidly activating delayed rectifier K+ current (I Kr) in the heart. Alterations in the functional properties or membrane incorporation of HERG channels, either by genetic mutations or by administration of drugs, play major roles in the development of life-threatening torsades de pointes cardiac arrhythmias. Visualization of ion channel localization is facilitated by enhanced green fluorescent protein (EGFP) tagging, but this process can alter their properties. The aim of the present study was to characterize the electrophysiological properties and the cellular localization of HERG channels in which EGFP was tagged either to the C terminus (HERG/EGFP) or to the N terminus (EGFP/HERG). These fusion constructs were transiently expressed in human embryonic kidney (HEK) 293 cells, and the whole-cell patch-clamp configuration and a confocal laser scanning microscope with primary anti-HERG antibodies and fluorescently labeled secondary antibodies were used. For EGFP/HERG channels the deactivation kinetics were faster and the peak tail current density was reduced when compared to both wild-type HERG channels and HERG/EGFP channels. Laser scanning microscopic studies showed that both fusion proteins were localized in the cytoplasm and on discrete microdomains in the plasma membrane. The extent of labeling with anti-HERG antibodies of HEK 293 cells expressing EGFP/HERG channels was less when compared to HERG/EGFP channels. In conclusion, both electrophysiological and immunocytochemical studies showed that EGFP/HERG channels themselves have a protein trafficking defect. HERG/EGFP channels have similar properties as untagged HERG channels and, thus, might be especially useful for fluorescence microscopy studies.

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References

  • Anderson CL, Delisle BP, Anson BD, Kilby JA, Will ML, Tester DJ, Gong Q, Zhou Z, Ackerman MJ, January CT (2006) Most LQT2 mutations reduce Kv11.1 (hERG) current by a class 2 (trafficking-deficient) mechanism. Circulation 113:365–373

    Article  PubMed  CAS  Google Scholar 

  • Claassen S, Zünkler BJ (2005) Comparison of the effects of metoclopramide and domperidone on HERG channels. Pharmacology 74:31–36

    Article  PubMed  CAS  Google Scholar 

  • Cordes JS, Sun Z, Lloyd DB, Bradley JA, Opsahl AC, Tengowski MW, Chen X, Zhou J (2005) Pentamidine reduces hERG expression to prolong the QT interval. Br J Pharmacol 145:15–23

    Article  PubMed  CAS  Google Scholar 

  • Curran ME, Splawski I, Timothy KW, Vincent GM, Green ED, Keating MT (1995) A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 80:795–803

    Article  PubMed  CAS  Google Scholar 

  • Drolet B, Rousseau G, Daleau P, Cardinal R, Turgeon J (2000) Domperidone should not be considered a no-risk alternative to cisapride in the treatment of gastrointestinal motility disorders. Circulation 102:1883–1885

    PubMed  CAS  Google Scholar 

  • Ficker E, Kuryshev YA, Dennis AT, Obejero-Paz C, Wang L, Hawryluk P, Wible BA, Brown AM (2004) Mechanisms of arsenic-induced prolongation of cardiac repolarization. Mol Pharmacol 66:33–44

    Article  PubMed  CAS  Google Scholar 

  • Furutani M, Trudeau MC, Hagiwara N, Seki A, Gong Q, Zhou Z, Imamura S, Nagashima H, Kasanuki H, Takao A, Momma K, January CT, Robertson GA, Matsuoka R (1999) Novel mechanism associated with an inherited cardiac arrhythmia: defective protein trafficking by the mutant HERG (G601S) potassium channel. Circulation 99:2290–2294

    PubMed  CAS  Google Scholar 

  • Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recordings from cells and cell-free membrane patches. Pfluegers Arch 391:85–100

    Article  CAS  Google Scholar 

  • Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59

    Article  PubMed  CAS  Google Scholar 

  • Jenke M, Sanchez A, Monje F, Stühmer W, Weseloh RM, Pardo LA (2003) C-terminal domains implicated in the functional surface expression of potassium channels. EMBO J 22:395–403

    Article  PubMed  CAS  Google Scholar 

  • Kupershmidt S, Yang T, Chanthaphaychith S, Wang Z, Towbin JA, Roden DM (2002) Defective human ether-a-go-go-related gene trafficking linked to an endoplasmic retention signal in the C-terminus. J Biol Chem 277:27442–27448

    Article  PubMed  CAS  Google Scholar 

  • Kuryshev YA, Ficker E, Wang L, Hawryluk P, Dennis AT, Wible BA, Brown AM, Kang J, Chen X-L, Sawamura K, Reynolds W, Rampe D (2005) Pentamidine-induced long QT syndrome and block of hERG trafficking. J Pharmacol Exp Ther 312:316–323

    Article  PubMed  CAS  Google Scholar 

  • Limon A, Reyes-Ruiz JM, Eusebi F, Miledi R (2007) Properties of GluR3 receptors tagged with GFP at the amino or carboxyl terminus. Proc Natl Acad Sci USA 104:15526–15530

    Article  PubMed  CAS  Google Scholar 

  • Maguy A, Hebert TE, Nattel S (2006) Involvement of lipid rafts and caveolae in cardiac ion channel function. Cardiovasc Res 69:798–807

    Article  PubMed  CAS  Google Scholar 

  • O′Connell KMS, Tamkun MM (2005) Targeting of voltage-gated potassium channel isoforms to distinct cell surface microdomains. J Cell Sci 118:2155–2166

    Article  PubMed  CAS  Google Scholar 

  • O′Connell KMS, Martens JR, Tamkun MM (2004) Localization of ion channels to lipid raft domains within the cardiovascular system. Trends Cardiovasc Med 14:37–42

    Article  PubMed  CAS  Google Scholar 

  • Paulussen A, Raes A, Matthijs G, Snyders DJ, Cohen N, Aerssens J (2002) A novel mutation (T65P) in the PAS domain of the human potassium channel HERG results in the long QT syndrome by trafficking deficiency. J Biol Chem 277:48610–48616

    Article  PubMed  CAS  Google Scholar 

  • Paulussen ADC, Raes A, Jongbloed RJ, Gilissen RAHJ, Wilde AAM, Snyders DJ, Smeets HJM, Aerssens J (2005) HERG mutation predicts short QT based on channel kinetics but causes long QT by heterotetrameric trafficking deficiency. Cardiovasc Res 67:467–475

    Article  PubMed  CAS  Google Scholar 

  • Petrecca K, Atanasiu R, Akhavan A, Shrier A (1999) N-Linked glycosylation sites determine HERG channel surface membrane expression. J Physiol 515:41–48

    Article  PubMed  CAS  Google Scholar 

  • Rossenbacker T, Mubagwa K, Jongbloed RJ, Vereecke J, Devriendt K, Gewillig M, Carmeliet E, Collen D, Heidbüchel H, Carmeliet P (2005) Novel mutation in the Per-Arnt-Sim domain of KCNH2 causes a malignant form of long-QT syndrome. Circulation 111:961–968

    Article  PubMed  CAS  Google Scholar 

  • Sanguinetti MC, Mitcheson JS (2005) Predicting drug–hERG channel interactions that cause acquired long QT syndrome. Trends Pharmacol Sci 26:119–124

    Article  PubMed  CAS  Google Scholar 

  • Sanguinetti MC, Jiang C, Curran ME, Keating MT (1995) A mechanistic link between an inherited and an acquired cardiac arrhythmia. Cell 81:299–307

    Article  PubMed  CAS  Google Scholar 

  • Sasano T, Ueda K, Orikabe M, Hirano Y, Kawano S, Yasunami M, Isobe M, Kimura A, Hiraoka M (2004) Novel C-terminus frameshift mutation, 1122fs/147, of HERG in LQT2: additional amino acids generated by frameshift cause accelerated inactivation. J Mol Cell Cardiol 37:1205–1211

    Article  PubMed  CAS  Google Scholar 

  • Splawski I, Shen J, Timothy KW, Lehmann MH, Priori S, Robinson JL, Moss AJ, Schwartz PJ, Towbin JA, Vincent GM, Keating MT (2000) Spectrum of mutations in long-QT syndrome genes: KVLQT1, HERG, SCN5A, KCNE1, and KCNE2. Circulation 102:1178–1185

    PubMed  CAS  Google Scholar 

  • Sun H, Liu X, Xiong Q, Shikano S, Li M (2006) Chronic inhibition of cardiac Kir2.1 and hERG potassium channels by celastrol with dual effects on both ion conductivity and protein trafficking. J Biol Chem 281:5877–5884

    Article  PubMed  CAS  Google Scholar 

  • Teng S, Ma L, Dong Y, Lin C, Ye J, Bähring R, Vardanyan V, Yang Y, Lin Z, Pongs O, Hui R (2004) Clinical and electrophysiological characterization of a novel mutation R863X in HERG C-terminus associated with long QT syndrome. J Mol Med 82:189–196

    Article  PubMed  CAS  Google Scholar 

  • Trudeau MC, Warmke JW, Ganetzky B, Robertson GA (1995) HERG, a human inward rectifier in the voltage-gated potassium channel family. Science 269:92–95

    Article  PubMed  CAS  Google Scholar 

  • Vandenberg JI, Torres AM, Campbell TJ, Kuchel PW (2004) The HERG K+ channel: progress in understanding the molecular basis of its unusual gating kinetics. Eur Biophys J 33:89–97

    Article  PubMed  CAS  Google Scholar 

  • Wang L, Wible BA, Wan X, Ficker E (2007) Cardiac glycosides as novel inhibitors of human ether-a-go-go-related gene channel trafficking. J Pharmacol Exp Ther 320:525–534

    Article  PubMed  CAS  Google Scholar 

  • Warmke JW, Ganetzky B (1994) A family of potassium channel genes related to eag in Drosophila and mammals. Proc Natl Acad Sci USA 91:3438–3442

    Article  PubMed  CAS  Google Scholar 

  • Zünkler BJ (2006) Human ether-a-go-go-related (HERG) gene and ATP-sensitive potassium channels as targets for adverse drug effects. Pharmacol Ther 112:12–37

    Article  PubMed  Google Scholar 

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Acknowledgement

We thank Dr. A. I. Roth for critically reading the manuscript and Ms. A. Wessel for preparing the figures.

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Authors and Affiliations

  1. Federal Institute for Drugs and Medical Devices, Kurt-Georg-Kiesinger-Allee 3, 53175, Bonn, Germany

    Sonja Claassen & Bernd J. Zünkler

  2. Molecular Bioenergetics, Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, 53115, Bonn, Germany

    Sarah Schwarzer & Jost Ludwig

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  1. Sonja Claassen
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  2. Sarah Schwarzer
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  3. Jost Ludwig
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  4. Bernd J. Zünkler
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Correspondence to Bernd J. Zünkler.

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Claassen, S., Schwarzer, S., Ludwig, J. et al. Electrophysiological and Fluorescence Microscopy Studies with HERG Channel/EGFP Fusion Proteins. J Membrane Biol 222, 31–41 (2008). https://doi.org/10.1007/s00232-008-9101-0

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  • DOI: https://doi.org/10.1007/s00232-008-9101-0

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