Whole‐Cell Patch‐Clamp Electrophysiology of Voltage‐Sensitive Channels

Timothy J. Shafer1

1 U.S. Environmental Protection Agency, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 11.12
DOI:  10.1002/0471140856.tx1112s17
Online Posting Date:  November, 2003
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Abstract

Ion channels are critical modulators of electrical excitability in neurons and are targets of a wide variety of neurotoxicants, including metals and insecticides. The only way to directly examine effects of toxicants on ion channel function in an individual cell on a physiologically relevant time scale is to utilize patch‐clamp electrophysiological techniques. This unit presents the basics of utilizing whole‐cell patch‐clamp techniques to study effects of toxicants on voltage‐sensitive Ca2+ channels in neurons grown in culture.

Keywords: Patch clamp; electrophysiology; ion channels; neurotoxicants; pesticides; metals

     
 
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Table of Contents

  • Unit Introduction
  • Strategic Planning
  • Basic Protocol: Examination of Voltage‐Sensitive Ca2+ Currents in Neurons
  • Support Protocol: Tissue Culture for PC12 Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol: Examination of Voltage‐Sensitive Ca2+ Currents in Neurons

 Materials
  • Extracellular bath solution (see recipe)
  • Cells plated on glass coverslips (see Support Protocol)
  • Intracellular (patch pipet) solution (see recipe)
  • Tetrodotoxin stock solution (see recipe), optional
  • QX‐314 solution (see recipe), optional
  • Glass blanks for pipets
  • Multistage pipet puller
  • Microforge/heated wire polisher
  • Chamber to hold cells
  • Inverted microscope with phase‐contrast, DIC, or Hoffman modulation optics
  • Faraday cage (can be constructed inexpensively)
  • Hydrolic, motorized, or piezoelectric micromanipulators
  • Computer with monitor
  • Patch‐clamp amplifier
  • Analog to digital (A/D) conversion board
  • Software
  • Vibration isolation table
  • Drug delivery device (optional, but recommended)
  • Oscilloscope

Support Protocol: Tissue Culture for PC12 Cells

 Materials
  • PC12 cells
  • Dulbecco's modified Eagle medium (DMEM), supplemented with 44 mM NaHCO3, 2 mM HEPES, 7.5% (v/v) fetal bovine serum (FBS), and 7.5% (v/v) horse serum
  • 50 µg/ml poly‐l‐lysine
  • 50 ng/ml human recombinant nerve growth factor (NGF) or 100 ng/ml mouse 7s NGF
  • 25‐cm2 polystyrene culture flasks
  • 25‐mm glass coverslips, sterile
  • 6‐ or 12‐well polystyrene multiwell plates
  • 15‐ml conical centrifuge tubes

NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.
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Figures

  •  FigureFigure 11.12.1 Diagram of patch clamp set‐up and information flow. This schematic depicts the major electronic components of a patch‐clamp electrophysiology setup, as well as the general functions of each component and the flow of information between them. Not depicted are other important items such as a Faraday cage, vibration‐isolation table, drug delivery systems, and an oscilloscope. Many software programs have components that carry out the function of an oscilloscope. However, an oscilloscope can be useful in assessing noise and noise reduction.
    View Image
  •  FigureFigure 11.12.2 Current density–voltage relationship for Ca2+ current in PC12 cells following treatment with nerve growth factor (NGF). PC12 cells were plated onto glass coverslips and treated with NGF. At the indicated time intervals, coverslips were placed into extracellular buffer solution and whole‐cell recordings were made. The current‐voltage protocol described in the Basic Protocol, step , was applied to cells, and then the peak amplitudes obtained at each test potential were divided by the capacitance of each cell to obtain the current density, which was plotted as a function of the test potential (in mV).
    View Image
  •  FigureFigure 11.12.3 Reversible inhibition of Ca2+ currents in PC12 cells. The data presented in this figure were obtained using the protocol described in the Basic Protocol, step . A test potential to +20 mV was delivered every 10 sec from a holding potential of −70 mV. When an inhibitor of Ca2+ channel function was applied to the bath solution (solid bar), peak current amplitude decreased rapidly. Stopping perfusion of the inhibitor (by washing) resulted in a rapid recovery of the current amplitude. The traces labeled A through E on the inset (trace C is repeated to facilitate comparisons) correspond to the points labeled A through E on the plot of the current amplitude versus time. In the inset, the scale bars correspond to 200 pA and 25 msec.
    View Image
  •  FigureFigure 11.12.4 Example of a current trace from a cell where space clamp is poor. In this example trace, both the activation (left arrow) and inactivation (right arrow) of the Ca2+ current contains pronounced shoulders that indicate that the cell is inadequately space‐clamped. Compare the shape of this current trace to the examples in the inset of Figure 11.12.3.
    View Image

Videos

Literature Cited

Literature Cited
    Atchison, W.D., Adgate, L., and Beaman, C.M. 1988. Effects of antibiotics on uptake of calcium into isolated nerve terminals. J. Pharmacol. Exp. Ther. 245:394‐401.
    Audesirk, G. 1993. Electrophysiology of lead intoxication: Effects on voltage‐sensitive ion channels. Neurotoxicol. 14:137‐147.
    Catterall, W.A. 1998. Structure and function of neuronal Ca2+ channels and their role in neurotransmitter release. Cell Calcium 24:307‐323.
    Chow, R.H. 1991. Cadmium block of squid calcium currents. Macroscopic data and a kinetic model. J. Gen. Physiol. 98:751‐770.
    Colston, J.T., Valdes, J.J., and Chambers, J.P. 1998. Ca2+ channel alpha 1‐subunit transcripts are differentially expressed in rat pheochromocytoma (PC12) cells following nerve growth factor treatment. Int. J. Dev. Neurosci. 16:379‐389.
    Ffrench‐Constant, R.H., Anthony, N., Aronstein, K., Rocheleau, T., and Stilwell, G. 2000. Cyclodiene insecticide resistance: From molecular to population genetics. Annu. Rev. Entomol. 45:449‐466.
    Hamill, O.P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F.J. 1981. Improved patch‐clamp techniques for high‐resolution current recording from cells and cell‐free membrane patches. Pflügers Arch. 391:85‐100.
    Hille, B. 2001. "Ion channels of excitable membranes". 3rd Edition. Sinauer Associates, Sunderland, MA.
    Marszalec, W. and Narahashi, T. 1993. Use‐dependent pentobarbital block of kainate and quisqualate currents. Brain Res. 608:7‐15.
    Narahashi, T., Ginsburg, K.S., Nagata, K., Song, J.H., and Tatebayashi, H. 1998. Ion channels as targets for insecticides. Neurotoxicol. 19:581‐590.
    Shafer, T.J. 1999. "The role of ion channels in neurotoxicity". In Neurotoxicology. (H.A. Tilson and G.J. Harry, eds.) pp. 99‐137. Taylor & Francis, Philadelphia, PA.
    Shafer, T.J. and Atchison, W.D. 1991. Transmitter, ion channel and receptor properties of pheochromocytoma (PC12) cells: A model for neurotoxicological studies. Neurotoxicol. 12:473‐492.
    Sirois, J.E. and Atchison, W.D. 1996. Effects of mercurials on ligand‐ and voltage‐gated ion channels: A review. Neurotoxicol. 17:63‐84.
 Key References
    Catterall, 1998. See above.

This review provides an excellent overview of the different types of voltage‐sensitive calcium channels and their roles in neurotransmitter release.

    Ffrench‐Constant et al., 2000. See above.

This paper provides a review of work completed by this and other groups demonstrating that insecticide resistance to cyclodienes is the result of specific mutations in the structure of GABAA receptors. It is an excellent example of how the coupling of molecular biology techniques with electrophysiology is increasing the understanding of mechanisms of the effects of neurotoxicants.

    Hamill et al., 1981. See above.

This is the seminal paper describing the use of patch‐clamp techniques. Sakmann and Neher won the 1991 Nobel Prize for their related work utilizing these techniques to describe the function of ion channels at the level of an individual channel.

    Hille, 2001. See above.

Also known as “The gospel according to St. Bertil”—no disrespect intended. This book by Bertil Hille provides in‐depth coverage of the biophysics of ion channels from the basics such as equivalent electrical circuits in cells, Ohm's law, and the Nernst equation to more advanced topics such as the relationship between molecular structure and function, theory of gating, and mechanisms of block. It is one of the most important books for an electrophysiologist to own.

 Internet Resources
    http://www.axon.com/MR_Axon_Guide.html

Axon Instruments is a commercial vendor of amplifiers and other cellular electrophysiology equipment. However, the mention of this Web site is not an endorsement of their equipment. Rather, it is listed because they do make available, free of charge, a publication called The Axon Guide. This publication contains additional practical and theoretical information about path clamp electrophysiology.

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