Whole‐Cell Configuration of the Patch‐Clamp Technique in the hERG Channel Assay to Predict the Ability of a Compound to Prolong QT Interval

Sonia Goineau1, Christophe Legrand1, Guillaume Froget1

1 Porsolt Research Center, Le Genest‐Saint‐Isle, France
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 10.15
DOI:  10.1002/0471141755.ph1015s57
Online Posting Date:  June, 2012
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Abstract

In vitro electrophysiological safety studies have become an integral part of the drug development process since, in many instances, compound‐induced QT prolongation has been associated with a direct block of human ether‐a‐go‐go‐related gene (hERG) potassium channels or its native current, the rapidly activating delayed rectifier potassium current (IKr). Therefore, the in vitro hERG channel patch‐clamp assay is commonly used as an early screen to predict the ability of a compound to prolong QT interval. The protocol described in this unit is designed to assess the effects of new chemical entities after acute or long‐term exposure on the amplitude of IKr in human embryonic kidney 293 (HEK293) cells stably transfected with the hERG channel (whole‐cell configuration of the patch‐clamp technique). Examples of results obtained with terfenadine, arsenic, pentamidine, erythromycin, and sotalol are provided for illustrative purposes. Curr. Protoc. Pharmacol. 57:10.15.1‐10.15.14. © 2012 by John Wiley & Sons, Inc.

Keywords: hERG; patch‐clamp; whole‐cell configuration; QT prolongation; trafficking

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of the Acute Effects of Test Substances in the hERG Channel Test
  • Alternate Protocol 1: Measurement of the Long‐Term Effects of Test Substances in the hERG Channel Test
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of the Acute Effects of Test Substances in the hERG Channel Test

  Materials
  • Human embryonic kidney (HEK) 293 cells stably transfected with the hERG channel supplied by Creacell (the cells can also be obtained from other companies, e.g., ChanTest, Millipore)
  • Cellular culture medium (see recipe)
  • Tyrode's solution (see recipe)
  • Extracellular solution (see recipe)
  • Intra‐pipet solution (see recipe)
  • Test substances (e.g., terfenadine, sotalol or erythromycin) and appropriate vehicle [e.g., if the test substance is soluble, use extracellular solution (see recipe); if the substance is not soluble in extracellular solution, a solvent such as dimethyl sulfoxide (DMSO) can be used]
  • 35‐mm cell culture dishes (Greiner bio‐one, cat. no. 627160)
  • 25‐mm glass coverslip (Warner Instruments, cat. no. CS‐25R)
  • 37°C, 5% CO 2/95% cell culture incubator
  • 1‐liter volumetric flasks
  • 1‐liter glass bottle (Tyrode's solution reservoir)
  • Superfusion system
  • Glass capillary tubes 1.5 mm OD × 1.17 mm ID (GC150TF, Harvard) and 1.0 mm OD × 0.78 mm ID (GC100T, Harvard)
  • C‐Flex and polyethylene tubing for connecting to syringe
  • 1‐ and 10‐ml syringes (Terumo type BS‐01T and SS‐10ES, respectively)
  • Micromanipulator (MP‐285, Sutter Instruments) equipped with pressure port attached to the head stage of electrophysiological amplifier
  • Recording chamber (RC‐40LP, Warner Instruments) mounted in a QE‐1 heating platform coupled with the TC‐344B temperature controller
    • Inflow port connected to Tyrode's solution reservoir previously heated (SH‐27B, Warner Instruments)
    • Outflow port connected to peristalic pump to hold Tyrode's solution effluent
  • Inverted microscope (DMI3000 B, Leica or IX51, Olympus)
  • Antivibration table (Newport)
  • Gassing system (compressed air)
  • Faraday cage
  • Thermostatic device (TC‐344B, Warner Instruments) with a probe placed near the patched cell
  • Reference Ag/AgCl electrode (Phymep, cat. no. E206)
  • Patch pipets [1.5‐mm OD × 1.17‐mm ID glass capillary tubes previously prepared using a microelectrode vertical puller (P30, Sutter Instruments)] filled with intra‐pipet solution (1.5 to 3.5 MΩ tip resistance)
  • Amplifier (e.g., MultiClamp 700B) coupled to Digidata 1322A (or 1440A) digital‐to‐analog converter (Axon Instruments)
  • Micromanipulator (Narishige, cat. no. UM‐3C) for local superfusion of formulations
  • Oscilloscope (Hameg Instruments, cat. no. HM400 or HM407)
  • Appropriate software programs to generate pulse commands, store digital signals (pClamp software, Axon Instruments) and analyze experimental data (pClamp software, Axon Instruments and Microsoft Excel)

Alternate Protocol 1: Measurement of the Long‐Term Effects of Test Substances in the hERG Channel Test

  • Test substances (e.g., pentamidine or arsenic)
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Figures

Videos

Literature Cited

   The Axon Guide for Electrophysiology and Biophysics Laboratory Techniques. Axon Instruments, Foster City, Calif.
   Crumb, W.J. Jr. 2000. Loratadine blockade of K(+) channels in human heart: comparison with terfenadine under physiological conditions. J. Pharmacol. Exp. Ther. 292:261‐264.
   Davie, C., Pierre‐Valentin, J., Pollard, C., Standen, N., Mitcheson, J., Alexander, P., and Thong, B. 2004. Comparative pharmacology of guinea pig cardiac myocyte and cloned hERG (IKr) channel. J. Cardiovasc. Electrophysiol. 11:1302‐1309.
   Ficker, E., Kuryshev, Y.A., Dennis, A.T., Obejero‐Paz, C., Wang, L., Hawryluk, P., Wible, B.A., and Brown, A.M. 2004. Mechanisms of arsenic‐induced prolongation of cardiac repolarization. Mol. Pharmacol. 66:33‐44.
   Gintant, G.A. 2011. An evaluation of hERG current assay performance: Translating preclinical safety studies to clinical QT prolongation. Pharmacol. Ther. 129:109‐119.
   Gintant, G.A., Su, Z., Martin, R.L., and Cox, B.F. 2006. Utility of hERG assays as surrogate markers of delayed cardiac repolarization and QT safety. Toxicol. Pathol. 34:81‐90.
   Goineau, S., Picard, S., Legrand, C., Froget, G. 2011. Use of excipients/solvents other than dimethylsulfoxide for testing hydrophobic substances in hERG channel assay. J. Pharmacol. Toxicol. Methods 64:e6 (abstract).
   Himmel, H.M. 2007. Suitability of commonly used excipients for electrophysiological in‐vitro safety pharmacology assessment of effects on hERG potassium current and on rabbit Purkinje fiber action potential. J. Pharmacol. Toxicol. Methods 56:145‐158.
   ICH Harmonized Tripartite Guideline (S7A). 2000. Safety Pharmacology Studies for Human Pharmaceuticals.
   ICH Harmonized Tripartite Guideline (S7B). 2005. The Non‐Clinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) by Human Pharmaceuticals.
   Kirsch, G.E., Trepakova, E.S., Brimecombe, J.C., Sidach, S.S., Erickson, H.D., Kochan, M.C., Shyjka, L.M., Lacerda, A.E., and Brown, A.M. 2004. Variability in the measurement of hERG potassium channel inhibition: Effects of temperature and stimulus pattern. J. Pharmacol. Toxicol. Methods 50:93‐101.
   Kuryshev, Y.A., Ficker, E., Wang, L., Hawryluk, P., Dennis, A.T., Wible, B.A., Brown, A.M., Kang, J., Chen, X.L., Sawamura, K., Reynolds, W., and Rampe, D. 2005. Pentamidine‐induced long QT syndrome and block of hERG trafficking. J. Pharmacol. Exp. Ther. 312:316‐323.
   Monahan, B.P., Ferguson, C.L., Killeavy, E.S., Lloyd, B.K., Troy, J., and Cantilena, L.R. Jr. 1990. Torsades de pointes occurring in association with terfenadine use. J. Am. Med. Assoc. 264:2788‐2790.
   Picard, S. and Lacroix, P. 2003. QT interval prolongation and cardiac risk assessment for novel drugs. Curr. Opin. Investig. Drugs 4:303‐308.
   Picard, S., Goineau, S., Guillaume, P., Henry, J., Hanouz, J.L., and Rouet, R. 2011. Supplemental studies for cardiovascular risk assessment in safety pharmacology: A critical overview. Cardiovasc. Toxicol. 11:285‐307.
   Rampe, D., Roy, M.L., Dennis, A., and Brown, A.M. 1997. A mechanism for the proarrhythmic effects of cisapride (Propulsid): High affinity blockade of the human cardiac potassium channel HERG. FEBS Lett. 417:28‐32.
   Vandenberg, J.I., Varghese, A., Lu, Y., Bursill, J.A., Mahaut‐Smith, M.P., and Huang, C.L. 2006. Temperature dependence of human ether‐a‐go‐go‐related gene K+ currents. Am. J. Physiol. 291:C165‐C175.
   Walker, B.D., Valenzuela, S.M., Singleton, C.B., Tie, H., Bursill, J.A., Wyse, K.R., Qiu, M.R., Breit, S.N., and Campbell, T.J. 1999. Inhibition of HERG channels stably expressed in a mammalian cell line by the antianginal agent perhexiline maleate. Br. J. Pharmacol. 127:243‐251.
   West, P.D., Martin, D.K., Bursill, J.A., Wyse, K.R., and Campbell, T.J. 1997. Modulation of the electrophysiologic actions of E‐4031 and dofetilide by hyperkalemia and acidosis in rabbit ventricular myocytes. J. Cardiovasc. Pharmacol. Ther. 2:205‐212.
   Yang, T. and Roden, D.M. 1996. Extracellular potassium modulation of drug block of IKr. Implications for torsade de pointes and reverse use‐dependence. Circulation 93:407‐411.
Internet Resources
   http://www.axon.com
  The Axon Instruments home page.
   http://www.ich.org/products/guidelines/safety/article/safety‐guidelines.html
  The safety guidelines.
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