Use of Electrophysiological Methods in the Study of Recombinant and Native Neuronal Ligand‐Gated Ion Channels

Philip Thomas1, Trevor G. Smart1

1 Department of Neuroscience, Physiology and Pharmacology, Division of Biosciences, University College London, London, United Kingdom
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 11.4
DOI:  10.1002/0471141755.ph1104s59
Online Posting Date:  December, 2012
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Abstract

Detailed in this unit are protocols for studying the effects of externally and internally applied agents on the behavior of ligand‐gated ion channels (LGICs), specifically the GABAA receptor. These assays include a number of electrophysiological techniques applied to whole‐cell and excised patch recordings of recombinant and native GABAA receptor subtypes used in the generation and analysis of a pharmacological data. Although applied to GABAA receptors, these techniques are equally applicable to other LGICs. The analysis is extended to incorporate consideration of post‐synaptic inhibitory events. In addition, complementary descriptions of how tissues for such studies are prepared for studying recombinant and native receptors are included. Curr. Protoc. Pharmacol. 59:11.4.1‐11.4.37. © 2012 by John Wiley & Sons, Inc.

Keywords: ligand‐gated ion channels; electrophysiology; transfection; tissue culture

     
 
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Table of Contents

  • Introduction
  • Acquisition and Analysis of Pharmacological Data Using Electrophysiological Techniques
  • Desensitization and Deactivation of Receptor Responses
  • Current‐Voltage Relationships
  • Synaptic Current Data Analysis
  • Basic Protocol 1: Data Acquisition Procedures for Whole‐Cell Currents from HEK A293 Cells Expressing GABAA Receptors
  • Support Protocol 1: Transient Expression of Recombinant GABAA Receptors in HEK A293 Cells by Calcium Phosphate Precipitation
  • Support Protocol 2: Production and Maintenance of Primary Hippocampal Neuronal Cultures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Data Acquisition Procedures for Whole‐Cell Currents from HEK A293 Cells Expressing GABAA Receptors

  Materials
  • HEK (human embryonic kidney)
  • A293 cells (ATCC #CRL‐1573)
  • γ‐Aminobutyric acid (GABA; or other appropriate agonist)
  • Krebs perfusion medium (see recipe)
  • Test compounds
  • Patch‐pipet recording electrolytes (see recipe)
  • Patch electrodes (borosilicate, 3‐5 MOhm; World precision Instruments, cat. no. TW150‐4); stored in a clean container
  • Patch‐clamp equipment (e.g., AxoPatch 200B, Axon Instruments)
  • U‐tube (see Rapid Application of Compounds: Fabrication of a U‐Tube)
  • Amplifier (Axopatch amplifiers)
  • Computer software (e.g., pClamp, Molecular Devices)

Support Protocol 1: Transient Expression of Recombinant GABAA Receptors in HEK A293 Cells by Calcium Phosphate Precipitation

  Materials
  • Cultured HEK A293 cells (density ∼70% in 100‐mm petri dishes), room temperature
  • CMF‐HBSS [Ca2+‐ and Mg2+‐free (CMF); e.g., Life Technologies], room temperature
  • 0.25% (w/v) trypsin/1 mM EDTA, room temperature
  • HEK A293 DMEM‐based growth medium (see recipe), room temperature
  • cDNA (or mRNA) receptor subunit constructs and appropriate reporter (e.g., eGFP)
  • Calcium chloride solution (340 mM in distilled water): Divide into 500‐µl aliquots and store up to 6 months at −20°C (keep at 4°C when in use)
  • 2× HEPES buffered saline (HBS: 50 mM HEPES; 280 mM NaCl; 2.8 mM Na 2HPO 4, pH 7.2)
  • 10‐ml and 50‐ml conical centrifuge tubes
  • Centrifuge
  • Sterilized Pasteur pipet, fire‐polished to half‐bore diameter
  • Poly‐L‐lysine‐coated, 22‐mm coverslips (see recipe)
  • Microscope (e.g., Nikon E600FN or equivalent) with epifluorescence facility (mercury or LED lamp) and appropriate bandwidth filters for the visualization of reporter products (eGFP) or tracers (e.g., biocytin or lucifer yellow)
  • Hemacytometer (to count cell density), optional

Support Protocol 2: Production and Maintenance of Primary Hippocampal Neuronal Cultures

  Materials
  • Pregnant rodent [embryonic day (E) 17 to 19]
  • HBSS (with Ca2+ and Mg2+; e.g., Life Technologies)
  • Ice
  • Trypsin solution (see recipe), 37°C
  • Hippocampal plating medium (see recipe), 37°C
  • Hippocampal trypsin inhibition medium (see recipe), 37°C
  • Hippocampal maintenance medium (see recipe), 37°C
  • 100‐ml beaker 2/3 full of 70% ethanol
  • 100‐ml beaker of sterile water, slightly fuller than the 70% ethanol beaker
  • 100‐ml beaker of HBSS, slightly fuller again than the beaker containing sterile water
  • 250‐ml beaker containing 50 ml HBSS, 4°C
  • CO 2 chamber or other means of euthanasia approved by regulatory authorities
  • Stainless steel scissors
  • Sterile hypodermic needle (no.16; Becton Dickinson) or equivalent, for use as a dissection pin
  • Sterilized 100‐mm dissecting dish half‐filled with cured Sylgard (Dow Corning)
  • Watchmaker's forceps (straight no.5; John Weiss and Sons)
  • Micro‐spatula
  • Large forceps
  • Sterile 35‐ and 60‐mm plastic petri dish (Nunc) containing HBSS (4°C)
  • Upright microscope (up to 50× magnification)
  • Hemostats fitted with a 5‐ to 20‐mm section of cut razor (blade down): a scalpel and blade may suffice
  • Sterile 5‐ and 10‐ml syringes
  • Sterile 100‐mm plastic Kwill filling tubes (Scientific Laboratory Supplies) or equivalent
  • Fire‐polished, sterile Pasteur pipets with decreasing diameters of 1, 1/2, and 1/4 of the original bore
  • Sterile 10‐ and 50‐ml conical centrifuge tubes (Falcon; Dow Corning)
  • Poly‐D‐lysine‐coated glass coverslips (22‐mml; see recipe)
  • 37°C incubator
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to governmental regulations regarding the care and use of laboratory animals.
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Figures

Videos

Literature Cited

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Internet Resources
  http://www.synaptosoft.com
  Analysis software, MiniAnalysis.
  www.strath.ac.uk/physpharm/staff/
  Analysis software, EDR, Dr John Dempster, University of Strathclyde.
  www.axon.com
  Analysis software, pClamp.
  www.atcc.org
  Cell line depository.
  www.novagen.com
  Supplier of GeneJuice transfection reagent.
  www.promega.com
  Supplier of FuGENE transfection reagent.
  www.invitrogen.com
  Supplier of lipofectamine and related transfection reagents.
  www.ozbiosciences.com
  Supplier of MagnetoFection transfection reagent.
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