Electrophysiological Analysis of Tetrodotoxin‐Resistant Sodium Channel Pharmacology

Xiao‐Wei Dong1, Tony Priestley1

1 Schering‐Plough Research Institute, Kenilworth, New Jersey
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 11.8
DOI:  10.1002/0471141755.ph1108s23
Online Posting Date:  February, 2004
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes essential procedures to record the activity of and the effects of pharmacological manipulation upon tetrodotoxin‐resistant (TTXr) sodium channels. Readers are guided through the process of assembling a functional |patch‐clamp| electrophysiology apparatus and construction of software‐controlled voltage protocols that address various aspects of channel gating. Guidelines are also presented for analysis and display of the data typically generated by these experiments. For investigators wishing to study native channels in their appropriate cellular environment, protocols describing the isolation of viable cells from the main tissues that commonly express TTXr channels are also included.

Keywords: sodium channel; TTX‐resistant; activation; inactivation; state‐dependent; use‐dependent; voltage‐dependent; open channel; inactivated channel; tonic block

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Pharmacological Modulation of TTXr Sodium Channels Assessed Using the Whole‐Cell Voltage Clamp Technique
  • Support Protocol 1: Preparation of Acutely Dissociated Rat Dorsal Root Ganglia (DRG) Neurons for Patch‐Clamp Recordings
  • Support Protocol 2: Preparation of Acutely Dissociated Cardiac Myocytes from Adult Rats For Patch‐Clamp Recordings
  • Support Protocol 3: Fabrication of Laminin‐Coated Glass Cover Slips for Cell Attachment
  • Support Protocol 4: Voltage Protocols, Data Acquisition and Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Pharmacological Modulation of TTXr Sodium Channels Assessed Using the Whole‐Cell Voltage Clamp Technique

  Materials
  • Modified aCSF (see recipe)
  • Stock solution(s) of test compound(s): e.g., 10 mM lidocaine (Sigma) in H 2O or 10 mM cypermethrin (Alexis Biochemicals) in ethanol
  • Acutely dissociated cells (see protocol 2 and protocol 32) or cultured clonal cells (see unit 6.3) attached to coverslips
  • Internal pipet solution (see recipe)
  • Electrophysiological setup (see ) including:
    • Patch pipets (also see unit 6.3 and Rae and Levis, )
    • Electronic components
    • Perfusion control system
    • Optical system
    • Mechanics
    • Incubator for cell cultures and acutely dissociated cells
  • Additional reagents and equipment for positioning recording electrodes and obtaining whole‐cell configuration (unit 11.2, protocol 4, steps 1 to 7 and 10 to 12)

Support Protocol 1: Preparation of Acutely Dissociated Rat Dorsal Root Ganglia (DRG) Neurons for Patch‐Clamp Recordings

  Materials
  • 6‐ to 8‐week old Sprague‐Dawley rats (Charles River Laboratories)
  • Sodium pentobarbital
  • Dulbecco's Modified Eagle's Medium (DMEM, Sigma) with and without 1000 U/ml penicillin and 1000 U/ml streptomycin
  • 5% CO 2/95% O 2
  • Trypsin (Type III, Sigma)
  • Collagenase (Type IA, Sigma)
  • Deoxyribonuclease II (Type IV, Sigma)
  • Soybean trypsin inhibitor (Type I‐S, Sigma)
  • Animal hair clippers
  • Dissecting instruments including:
    • Scalpel
    • Hemostat
    • Bone rongeur
    • Small stainless steel spring scissors
    • Small fine tip forceps
  • Small petri dish
  • 15‐ml polypropylene conical tubes
  • 34°C shaking water bath
  • Fire‐polished Pasteur pipets of progressively smaller bore (optional)
  • Poly‐L‐lysine‐coated ( appendix 3D) 12‐mm glass coverslips
  • 12‐well tissue culture plates
NOTE: All solutions and equipment coming into contact with live cells must be sterile, and proper aseptic technique should be used accordingly.

Support Protocol 2: Preparation of Acutely Dissociated Cardiac Myocytes from Adult Rats For Patch‐Clamp Recordings

  Materials
  • 250 to 300 g Sprague‐Dawley adult rats (Charles River Laboratories)
  • Sodium pentobarbital
  • 500 U/ml heparin
  • Krebs‐Henseleit buffer (KHB; see recipe), with and without 2.5 mM Ca2+
  • 5% CO 2/95% O 2
  • Collagenase (Type IA, Sigma)
  • Hyaluronidase (Type I‐S; Sigma)
  • Bovine serum albumin (BSA)
  • 50 mM CaCl 2 stock solution
  • Bovine serum albumin fraction V (BSA; sigma)
  • Hyaluronidase (Type I‐S) (Sigma)
  • Hair clippers
  • Dissecting instruments including:
    • Scalpel
    • Scissors
    • Hemostat
    • Forceps
    • Small stainless steel spring scissors
  • Retractors
  • Polythene tubing (PE 240, Becton Dickinson)
  • Rodent ventilator (Model 683, Harvard Apparatus)
  • Retrograde Langendorff apparatus for heart perfusion (Model ML176, ADInstruments)
  • Petri dish
  • 25‐ml Erlenmeyer flask
  • 200‐µm nylon mesh
  • 12‐mm glass cover slips coated with laminin (see protocol 4)
  • Additional reagents and equipment for determining cell viability via trypan blue exclusion (unit 12.3)
NOTE: All solutions and equipment coming into contact with live cells must be sterile, and proper aseptic technique should be used accordingly.

Support Protocol 3: Fabrication of Laminin‐Coated Glass Cover Slips for Cell Attachment

  Materials
  • 50 µg/ml laminin stock (Becton Dickinson) in sterile, serum‐free medium (e.g., PBS)
  • Medium for rinsing (e.g., PBS)
  • 12‐mm glass cover slips (Warner Instruments)
  • 12‐well tissue culture plate
  • UV light source

Support Protocol 4: Voltage Protocols, Data Acquisition and Analysis

  Materials
  • Personal computer
  • Analog‐to digital (A/D) board (Digidata 1200A, 1200B, 1320A, Axon Instruments)
  • Patch‐clamp amplifier (Axopatch‐1D or Axopatch 200B, Axon Instruments)
  • Data acquisition software (pClamp8, Axon Instruments)
  • Data analysis software (pClamp8, Axon Instruments; Excel, Microsoft; Prism, GraphPad; Origin, Microcal Software)
  • Additional reagents and equipment for whole‐cell voltage clamp analysis of TTXr sodium channel pharmacology (see protocol 1)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Axon Instruments. 1993. The Axon Guide. Axon Instruments, Foster City, Calif.
   Bezanilla, F. and Armstrong, C.M. 1977. Inactivation of the sodium channel. I. Sodium current experiments. J. Gen. Physiol. 70:549–566.
   Boulton, A.A., Baker, G.B., and Walz, W. 1995. Neuromethods 26: Patch‐Clamp Applications and Protocols. Humana Press, Totowa, N.J.
   Cahalan, M.D. and Almers, W. 1979. Interactions between quaternary lidocaine, the sodium channel gates, and tetrodotoxin. Biophys. J. 27:39–55.
   Catterall, W.A. 1992. Cellular and molecular biology of voltage‐gated sodium channels. Physiol. Rev. 72:15–48.
   Farmer, B.B., Mancina, M., Williams, E.S., and Watanabe, A.M. 1983. Isolation of calcium tolerant myocytes from adult rat hearts: Review of the literature and description of a method. Life Sci. 33:1–18.
   Hille, B. 2001. Ionic Channels of Excitable Membranes. Sinauer Associates, Sunderland, Mass.
   Hondeghem, L.M. and Katzung, B.G. 1984. Antiarrhythmic agents: The modulated receptor mechanism of action of sodium and calcium channel‐blocking compounds. Annu. Rev. Pharmacol. Toxicol. 24:387–423.
   Kettenmann, H. and Grantyn, R. 1992. Practical Electrophysiological Methods, Wiley‐Liss, N.Y.
   Lai, J., Gold, M.S., Kim, C.S., Bian, D., Ossipov, M.H., Hunter, J.C., and Porreca, F. 2002. Inhibition of neuropathic pain by decreased expression of the tetrodotoxin‐resistant sodium channel, Nav 1.8. Pain 95:143–152.
   Novakovic, S.D., Tzoumaka, E., McGivern, J.G., Haraguchi, M., Sangameswaran, L., Gogas, K.R., Eglen, R.M., and Hunter, J.C. 1998. Distribution of the tetrodotoxin‐resistant sodium channel PN3 in rat sensory neurons in normal and neuropathic conditions. J. Neurosci. 18:2174–2187.
   Ogden, D. 1994. Microelectrode Techniques. The Company of Biologists Ltd., Cambridge, U.K.
   Rae, J.L. and Levis, R.A. 1997. Fabrication of patch pipets. In Current Protocols in Neuroscience (J.N. Crawley, C.R. Gerfen, M.A. Rogawski, D.R. Sibley. P. Skolnick,, and, S. Wray, eds.) pp.6.3.1–6.3.31. John Wiley & Sons, New York.
   Roza, C., Laird, J.M., Souslova, V., Wood, J.N., and Cervero, F. 2003. The tetrodotoxin‐resistant Na+ channel Nav1.8 is essential for the expression of spontaneous activity in damaged sensory axons of mice. J. Physiol. 550:921–926.
   Rush, A.M., Brau, M.E., Elliott, A.A., and Elliott, J.R. 1998. Electrophysiological properties of sodium current subtypes in small cells from adult rat dorsal root ganglia. J. Physiol. 511:771–789.
   Sakmann, B. and Neher, E. 1995. Single‐Channel Recording. Plenum Press, New York.
   Shapiro, B.I. 1977. Effects of strychnine on the sodium conductance of the frog node of Ranvier. J. Gen. Physiol. 69:915–926.
   Strichartz, G., Rando, T., and Wang, G.K. 1987. An integrated view of the molecular toxinology of sodium channel gating in excitable cells. Annu. Rev. Neurosci. 10:237–267.
   Ten Eick, R.E. 1990. Biophysical techniques for the study of cardiac tissue. In Cardiac Electrophysiology: A Textbook (M.R. Rosen, M.J. Janse,, and, A.L. Wit, eds.). Futura, Mount Kisco, N.Y.
   Trezise, D.J., John, V.H., and Xie, X.M. 1998. Voltage‐ and use‐dependent inhibition of Na+ channels in rat sensory neurones by 4030W92, a new antihyperalgesic agent. Br. J. Pharmacol. 124:953–963.
Key References
  Axon Instruments. 1993. See above.
  This is an excellent handbook, available at no cost from Axon Instruments, covering most aspects of in vitro electrophysiological recording techniques.
  Boulton et al., 1995. See above.
  This book provides concise survey of patch‐clamp technique and its advantages and limitations.
  Hille, 2001 See above.
  An excellent ion channel reference book
  Kettenmann and Grantyn, 1992. See above.
  A good guide for in vitro electophysiological methods.
  Ogden, 1994. See above.
  The book provides detailed description of both practical and theoretical aspects of electrodes.
  Sakmann and Neher, 1995. See above.
  A very detailed practical guide to patch‐clamp recording, data acquisition, and analysis techniques.
Internet Resources
  http://www.axon.com/MR_Axon_Guide.html
  URL for the Axon Guide (Axon Instruments, ) on the Web.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library