Patch‐Clamp Studies of Human Cardiac Ion Channels in the Evaluation of Cardiac Electrophysiological Effects of Compounds

William J. Crumb1, Icilio Cavero2

1 Zenas Technologies, New Orleans, Louisiana, 2 Bonneuil sur Marne, France
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 10.8
DOI:  10.1002/0471141755.ph1008s20
Online Posting Date:  May, 2003
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Abstract

Drugs prolonging the QT interval appear to consistently inhibit the outward, rapid delayed rectifier K+ current (IKr) conveyed by the HERG channel. Hence, for determining whether a new drug candidate blocks the latter channel, this unit presents a basic electrophysiology protocol to conduct patch clamp studies in single cell preparations expressing heterologously cloned HERG channels. An additional protocol details the isolation of myocytes from specimens of human atria which are used in the study of native cardiac ion currents (INa, ICa, Ito, Isus, IK1). The results of these tests are useful for determining whether drug candidates have the desired cardiac safety profile for human use.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of the Effects of Compounds on Current Responses Generated by Activation of Cloned hERG K+ Channels
  • Alternate Protocol 1: Study of the Effects of Drugs on Current Responses Conveyed by Na+, Ca2+ and K+Channels Other than hERG in Human Atrial Myocytes
  • Alternate Protocol 2: Stimulation‐Voltage Protocols
  • Support Protocol 1: Fabrication of Patch‐Clamp Pipet Electrodes
  • Support Protocol 2: Isolation of Myocytes from Human Atrial Specimens
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of the Effects of Compounds on Current Responses Generated by Activation of Cloned hERG K+ Channels

  Materials
  • Test compound(s) and appropriate vehicle
  • External K+ solution (see recipe), 37°C
  • K+ internal pipet electrode solution (see recipe)
  • HEK‐203 cells stably expressing hERG channels
  • 0.25% trypsin/1 mM EDTA (Life Technologies)
  • MEM (unit 5.2)
  • Reference compound—e.g., 0.1 µM E‐4031 (RBI) or 0.01 to 0.1 µM dofetilide (Pfizer) in recipeexternal solution
  • Electrophysiological setup for whole‐cell voltage clamp configuration (Fig. ):
  •  Amplifier (e.g., AxoClamp‐1B or ‐2B, Axopatch‐1D; Axon Instruments)
  •  Monitor to visualize seal formation—i.e., oscilloscope (e.g., Tektronix digital 2‐channel) or software—e.g., Clampex, Axoscope, and/or AxoTape (Axon Instruments), or NeuroPro (R.C. Electronics)
  •  Computer with analog‐to‐digital (A/D) boards (Digidata 1200 or 1320, Axon Instruments; ISC‐6 or ‐16, R.C. Electronics)
  •  Appropriate software programs to generate pulse commands and store digital signals (pClamp software; Axon Instruments) and analyze experimental data (Axon Instruments pClamp, Microsoft Excel, or Microcal Origin).
  •  Thermostatic device (Cambion/Midland Ross model 806‐7243‐01) with coupled thermistor placed in proximity to the patched cell so that the local temperature is maintained at 37° ± 1°C.
  •  Plastic (polyethylene or equivalent) tubing
  • Antivibration table (Technical Manufacturing or Newport)
  • Chamber superfusion system: ∼1‐ml chamber with transparent bottom
  • Recording patch‐clamp pipet electrodes with ∼2.0‐ to 4.0‐MΩ tip resistance when filled with K+ internal solution (see protocol 4)
  • Micromanipulator (Soma, Sutter, Narishige, Burleigh) equipped with pressure port attached to head stage of electrophysiological amplifier (Fig. )
  • P‐160 polyethylene tubing (Intramedic/Clay Adams, Becton Dickinson)
  • 10‐ml syringe or mouthpiece (e.g., 100‐µl pipet tip)
  • Inverted microscope (Nikon or Zeiss)

Alternate Protocol 1: Study of the Effects of Drugs on Current Responses Conveyed by Na+, Ca2+ and K+Channels Other than hERG in Human Atrial Myocytes

  • Na+ and Ca2+ external solutions (see recipe)
  • Na+ and Ca2+ internal solutions (see recipe)

Alternate Protocol 2: Stimulation‐Voltage Protocols

  Materials
  • Cyanoacrylate glues
  • Internal electrode solution (see recipe)
  • Two‐ or three‐stage electrode/pipet puller (e.g., P‐87; Sutter Instruments)
  • Medium‐wall glass capillary tubing of 0.86 mm internal diameter, 1.5 mm external diameter, 10 cm length, with or without internal filament (Sutter Instruments)
  • Microforge (optional)
  • Inverted microscope with 100× and 600× magnification
  • P‐10 and P‐160 polyethylene tube (Intramedic/Clay Adams, Becton Dickinson)
  • 13‐mm‐diameter 0.2‐µm syringe‐filter unit (Nylon Acrodisk 13; Gelman Sciences) and 1‐ml syringe
  • Micromanipulator (Soma, Sutter, Narishige, Burleigh) equipped with pressure port attached to head sage of electrophysiological amplifier (Fig. 10.8.2)
  • 10‐ml syringe or mouthpiece (e.g., 100‐µl pipet tip)

Support Protocol 1: Fabrication of Patch‐Clamp Pipet Electrodes

  Materials
  • Cardioplegia solution, 0° to 4°C (Table 10.8.1)
  • No‐Ca2+ wash solution, room temperature (Table 10.8.1)
  • 100% O 2
  • Enzyme solution, 37°C (Table 10.8.1)
  • 1 mg/ml collagenase in enzyme solution (Table 10.8.1) containing 100 µM CaCl 2, 37°C
  • Modified Kraftbruhe solution, room temperature (Table 10.8.1)
  • 50‐ml conical tube
CAUTION: Human tissue is a biohazard and should be handled according to the Occupational Safety and Health Administration (OSHA) regulations for blood‐borne pathogens (29CFR1910‐1030). This document is available at http://www.osha‐slc.gov/OshStd_data/1910_1030.html. Institutional guidelines must be strictly followed.
Table 0.8.1   Materials   Composition of Solutions to Prepare Myocytes from Human Atrial Tissue Specimen a, b   Composition of Solutions to Prepare Myocytes from Human Atrial Tissue Specimen,

Chemical Cardioplegic solutions Enzyme solution Modified Kraftbruhe solution No‐Ca2+ solution
NaCl 137 mM 137 mM
KCl 25 mM
KH 2PO 4 50 mM 5 mM 10 mM 5 mM
MgSO 4 8 mM 1 mM 10 mM
NaHCO 3 10 mM
HEPES 10 mM 5 mM 5 mM
Glucose 25 mM 10 mM 22 mM 10 mM
Adenosine 5 mM
Mannitol 100 mM
Taurine 140 mM 10 mM 5 mM 25 mM
EGTA 0.5 mM 100 µM
Collagenase type V 1.0 mg/ml
Protease type XXIV(Sigma) 0.5 mg/ml
Glutamic Acid 55 mM
Bovine albumin 0.1% (w/v) 0.1% (w/v)
NaOH c pH 7.4 pH 7.4
KOH c pH 7.4 pH 7.4

 aAll solutions should have 100% O 2 bubbled through them.
 bAll solutions can be premade and stored at approximately −20°C.
 cUse to adjust pH to 7.4.
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Figures

Videos

Literature Cited

Literature Cited
   Axon Instruments, 1993. Instrument Guide. Axon Instruments, Foster City, Calif.
   Bednar, M.M., Harrigan, E.P., Anziano, R.J., Camm, A.J., and Ruskin, J.N. 2001. The QT interval. Prog. Cardiovasc. Dis. 43:1‐45.
   Cavero, I. and Crumb, W. 2001. Native and cloned ion channels from human heart: Laboratory models for evaluating the cardiac safety of new drugs. Eur. Heart J. 3:53K‐63K.
   Cavero, I., Mestre, M., Guillon, J‐M., and Crumb, W. 2000. Drugs that prolong QT as an unwanted effect: Assessing their likelihood of inducing hazardous cardiac dysrhythmias. Exp. Opin. Pharmacother. 2:947‐973.
   Ekins, S., Crumb, W.J., Sarazan, R.D., Wikel, J.H., and Wrighton, S.A. 2002. Three‐dimensional quantitative structure‐activity relationship for inhibition of human ether‐a‐go‐go‐related gene potassium channel. J. Pharmacol. Exp. Ther. 301:427‐434.
   Haverkamp, W., Breithardt, G., Camm, A.J., Janse, M.J., Rosen, M.R., Antzelevitch, C., Escande, D., Franz, M., Malik, M., Moss, A., and Sha, R. 2000. The potential for QT prolongation and proarrhythmia by non‐antiarrhythmic drugs: Clinical and regulatory implications. Report on a policy conference of the European Society of Cardiology. Europ. Heart J. 21:1216‐1231.
   Hamill, O.P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. 1981. Improved patch‐clamp techniques for high‐resolution current recording from cells and cell‐free membrane patches. Pflugers Arch. 391:85‐100.
   Johnson, J.P., Balser, J.R., and Bennett, P.B., Enhancement of hERG K+ currents by Cd2+ destabilization of the inactivated state. 1999. Biophys J. 77:2534‐2541
   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. FEBS Lett. 417:28‐32.
   Saldeen, J., Curiel, D.T., Eizirik, D.L., Andersson, A., Strandell, E., Buschard, K., and Welsh, N. 1996. Efficient gene transfer to dispersed human pancreatic islet cells in vitro using adenovirus‐polylysine/DNA complexes or polycationic liposomes. Diabetes 45:1197‐1203.
   Tie, H., Walker, B.D., Singleton, C.B., Valenzuela, S.M., Bursill, J.A., Wyse, K.R., and Campbell, T.J. 2000. Inhibition of hERG potassium channels by the antimalarial agent. Br. J. Pharmacol. 130:1967‐1975.
   Tseng, G.N. 2001. IKr: The hERG channel. J.Mol. Cell Cardiol. 33:835‐49.
   Vandenberg, J.I., Walker, B.D., and Campbell, T.J. 2001. hERG K+ channels; friend and foe. Trends Pharmacol. Sci. 22:241‐246.
   Walker, B.D., Singleton, C.B., Bursill, J.A., Wyse, K.R., Valenzuela, S.M., Qiu, S.N., and Campbell, T.J. 1999. Inhibition of the human ether‐a‐go‐go‐related gene (hERG) potassium channel by cisapride: Affinity for open and inactivated states. Br. J. Pharmacol 128:444‐450.
   Wang, H.Z., Shi, H., Liao, S.J., and Wang, Z. 1999. Inactivation gating determines nicotine blockade of human hERG channels. Am. J. Physiol. 277:H1081‐H1088.
   Yang, T. and Roden, D.M. 1996. Extracellular potassium modulation of drug block of IKr. Importance for torsade de pointes and reverse use‐dependence. Circulation 93:407‐411.
   Zhou, Z., Gong, Q., Ye, B., Fan, Z., Makielski, J.C., Robertson, G.A., and January, C.T. 1998. Properties of hERG channels stably expressed in HEK 293 cells studied at physiological temperature. Biophys J. 74:230–241.
Internet Resources
  http://www.axon.com
  The Axon Instruments homepage.
  http://www.health.gov.au/tga/docs/pdf/euguide/swp/098696en.pdf
  Points to Consider: The Assessment of the Potential for QT Interval Prolongation by Non‐Cardiovascular Medicinal Products. Issued in 1997 by the EMEA Committee for Proprietary Medicinal Products.
  http://www.hc‐sc.gc.ca/hpb‐dgps/therapeut/htmleng/guidmain.html#QT_Prolong
  The Assessment of the QT prolongation potential on non‐antiarrhythmics drugs from the Therapeutic Products Directorate of Health Canada.
  http://www.emea.eu.int/pdfs/human/ich/042302.pdf
  Guideline on Safety Pharmacology Studies for Assessing the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) by Human Pharmaceuticals (S7B) from the International Conference for Harmonization (ICH).
  http://www.fenichel.net/pages/Professional/subpages/QT/subpages/pqt_prolongation.htm
  Robert R. Fenichel's table of drugs that do and do not affect cardiac ion channels.
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