Electrophysiological Characterization of Recombinant and Native P2X Receptors

Wende Niforatos1, Michael F. Jarvis1

1 Abbott Laboratories, Abbott Park, Illinois
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 11.9
DOI:  10.1002/0471141755.ph1109s26
Online Posting Date:  October, 2004
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Abstract

ATP acts as a fast neurotransmitter by activating a family of ligand‐gated ion channels, the P2X receptors. Functional homomeric P2X3 and heteromeric P2X2/3 receptors are highly localized on primary sensory afferent neurons that transmit nociceptive sensory information. Activation of these P2X3‐containing channels may provide a specific mechanism whereby ATP, released via synaptic transmission or by cellular injury, elicits pain. The experimental procedures described in this unit are useful for the electorphysiological characterization of P2X receptors. In addition, these protocols provide methods for the evaluation of ligands that interact with P2X receptors that are either natively expressed on excitable cells or cloned and expressed in heterologous cell systems. These methods are derived from standard electrophysiological principles and procedures that are applicable to a wide variety of ligand‐gated ion channels. Specific attention is given here to the reliable electrophysiological measurement of both quickly (P2X3) and more slowly (P2X2 and P2X2/3) desensitizing receptors.

Keywords: P2X3; P2X2/3; oocyte; ATP; receptors; dorsal root ganglia; DRG; voltage clamp

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of ATP‐Evoked Responses from Xenopus Oocytes Using the Two‐Electrode Voltage Clamp Technique
  • Support Protocol 1: Oocyte Harvesting, Defolliculation, and Injection
  • Support Protocol 2: Construction of a Push/Pull Applicator
  • Basic Protocol 2: Measurement of ATP‐Evoked Responses from Acutely Dissociated Dorsal Root Ganglion Neurons Using Whole‐Cell Patch Clamp
  • Support Protocol 3: Preparation of Acutely Dissociated Dorsal Root Ganglion Neurons from the Rat
  • Alternate Protocol 1: Measurement of ATP‐Evoked Responses from Stably Transfected Cell Lines Expressing P2X Receptors
  • Support Protocol 4: Piezo‐Driven Rapid Application System for Recording Whole‐Cell Currents from Rat Drg Neurons or Stably Transfected Clonal Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of ATP‐Evoked Responses from Xenopus Oocytes Using the Two‐Electrode Voltage Clamp Technique

  Materials
  • 120 mM KCl
  • Bleach (e.g., Clorox)
  • Recording solution (see recipe)
  • Xenopus oocytes expressing P2X receptor of interest ( protocol 2)
  • Test compounds (i.e., agonists and antagonists; Table 11.9.1)
  • Oocyte chamber (Warner Instruments)
  • Antivibration table (Technical Manufacturer) with Faraday cage
  • Stereomicroscope
  • Headstages (Axon Instruments)
  • Recording microelectrodes: 1.5‐mm‐o.d. × 1.1‐mm‐i.d. borosilicate glass with filament (Sutter Instruments, cat. no. BF150‐110‐10)
  • Pipet puller (i.e., model P‐80/PC, Sutter Instruments)
  • Electrode holders (Axon Instruments)
  • Micromanipulators (i.e., Narishige)
  • Amplifier (e.g., Axoclamp 2A, Axon Instruments)
  • Fine forceps
  • Peristaltic pump (e.g., Master Flex Model 7523‐30, Cole‐Palmer Instrument)
  • Vacuum source (e.g., house vacuum)
  • Push/pull applicator (see protocol 3)
  • pCLAMP software (Axon Instruments)
  • Graphics software (e.g., Prism, Graphpad Software)
  • Additional reagents and equipment for placement of a push/pull applicator (Briggs et al., )

Support Protocol 1: Oocyte Harvesting, Defolliculation, and Injection

  Materials
  • 2.5‐year‐old female Xenopus laevis frogs (e.g., Xenopus 1, NASCO)
  • 0.28% (w/v) 3‐aminobenzoic acid ethyl ester methanesulfonate salt (tricaine): dissolve 1.4 g tricane in 500 ml H 2O (prepare fresh)
  • Low‐Ca2+ Barth's solution (see recipe)
  • 2 mg/ml collagenase: 20 mg collagenase type 1A (Sigma) in 10 ml low‐Ca2+ Barth's solution (prepare fresh)
  • Normal Barth's solution (see recipe), 16° to 19°C
  • 1 µg/µl P2X receptor cDNA or mRNA
  • Mineral oil
  • 100 × 20–mm glass petri dishes
  • Surgical instruments (e.g., scissors, forceps, 18‐G needle)
  • Synthetic, absorbable, 4‐0 suture
  • Nonabsorbable, surgical, 4‐0 suture (i.e., black monofilament nylon)
  • 50‐ml conical tubes
  • Dissecting microscope (e.g., Nikon SMZ‐2B stereomicroscope with 0.5× objective and 20× eyepiece)
  • 16° to 19°C incubator (e.g., Sheldon Manufacturing)
  • Micropipet puller (e.g., Model P‐80/PC Flaming/Brown, Sutter Instruments)
  • Injection pipet: 0.5‐mm‐i.d. × 1.0‐mm‐o.d. borosilicate glass (Sutter Instruments, cat. no. B100‐50‐10)
  • Three‐dimensional micromanipulator (Narishige)
  • Fine forceps
  • 60 × 15–mm plastic petri dish
  • 100 × 20–mm glass petri dish with polypropylene mesh glued to the bottom (Warner Instruments)
  • PV830 Pneumatic PicoPump (World Precision Instruments) with either a standard floor vacuum pump or house vacuum attached, and compressed N 2
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.NOTE: All chemicals are purchased from Sigma unless otherwise noted. All laboratory supplies are purchased from VWR unless otherwise noted.

Support Protocol 2: Construction of a Push/Pull Applicator

  Materials
  • 1.44‐mm‐o.d. × 1.2‐mm‐i.d.glass capillary (Warner Instruments)
  • Silicone tubing, sizes 13 and 14 (Masterflex, Cole Palmer Instrument)
  • T‐fittings that can accommodate size 13 silicone tubing
  • PE50 polyethylene tubing (Becton‐Dickinson)
  • Microscope
  • Peristaltic pump, three‐channel (Masterflex Model 7523‐30, Cole Palmer Instruments)
  • Micromanipulator (i.e., Narishige)
  • Oocyte chamber (Warner Instruments)
  • Computer‐driven solenoid valve with valve driver (Series 1, General Valve)
  • PC computer with analog‐to‐digital (A/D) board (i.e., Digidata 1200) and pCLAMP software (Axon Instruments)
  • Ring stand
  • Antivibration table (Technical Manufacturing)
  • 10‐ml plastic syringes with Luer‐Loks
  • Rubber stoppers, size 1
  • 18‐G × 2‐in. stainless steel Luer‐Lok hypodermic needles
  • Female Luer fittings
NOTE: See Figure for a schematic of this apparatus.

Basic Protocol 2: Measurement of ATP‐Evoked Responses from Acutely Dissociated Dorsal Root Ganglion Neurons Using Whole‐Cell Patch Clamp

  Materials
  • Internal electrode solution (see recipe)
  • Cultured rat DRG neurons (see protocol 5)
  • Extracellular recording solution (see recipe)
  • Test compounds (agonist, antagonist; Table 11.9.1)
  • Micropipet puller (Sutter Instruments)
  • Recording microelectrodes: borosilicate glass with 1.5‐mm filament (World Precision Instruments)
  • Pipet filler: 1‐ml syringe with 34‐G microfiller attached (World Precision Instruments)
  • Electrode holder with pressure port (Axon Instruments)
  • Micromanipulator attached to microscope stage to hold headstage with electrode holder (Burliegh Instruments)
  • PE‐160 tubing
  • 10‐ml plastic syringe
  • Piezo‐driven rapid application system (see protocol 7)
  • Cyberamp (Axon Instruments)
  • pCLAMP software for data acquisition and analysis (Axon Instruments)
  • Graphics software (e.g., Prism, Graphpad Software)
  • Recording chamber (Warner Instruments)

Support Protocol 3: Preparation of Acutely Dissociated Dorsal Root Ganglion Neurons from the Rat

  Materials
  • Dissection solution: Dulbecco's modified Eagle's medium (DMEM) containing 100 U/ml penicillin and 100 mg/ml streptomycin (store up to 1 week at 4°C)
  • Male Sprague‐Dawley rats, 7‐ to 8‐weeks old or 250 to 300 g (Charles River)
  • 80% CO 2/20% O 2
  • Ethanol
  • 0.3% collagenase solution (see recipe)
  • Trypsin solution (see recipe)
  • Dulbecco's modified Eagle's medium (DMEM; Invitrogen)
  • Dulbecco's phosphate‐buffered saline (DPBS) without Ca2+ or Mg2+ (Invitrogen)
  • DRG incubation medium (see recipe)
  • 0.5‐, 0.75‐, and 1.0‐mm‐i.d. Pasteur pipets
  • Surgical instruments (e.g., scissors, fine forceps, microspring scissors; Fine Science Tools)
  • Dissecting microscope
  • 60 × 15–mm plastic petri dishes
  • Rodent nose‐cone
  • Rodent guillotine
  • PEI‐coated coverslips (see recipe)
  • 37°C, 5% CO 2 incubator

Alternate Protocol 1: Measurement of ATP‐Evoked Responses from Stably Transfected Cell Lines Expressing P2X Receptors

  • Cells (e.g., 1321N1 cells; Communi and Boeynaems, ) transfected with receptor of interest (e.g., human astrocytoma cells transfected with P2X receptor)
  • Incubation medium (see recipe)

Support Protocol 4: Piezo‐Driven Rapid Application System for Recording Whole‐Cell Currents from Rat Drg Neurons or Stably Transfected Clonal Cells

  Materials
  • Extracellular recording solution (see recipe)
  • Micropipet puller (Sutter Instruments)
  • Glass theta tube (FHC)
  • Fine forceps
  • Syringe needles
  • Silicone rubber adhesive sealant or equivalent (e.g., epoxy)
  • PE‐tubing connected to multichannel fittings
  • 20‐ml syringe barrels
  • Ring stand
  • Piezo‐electric rapid application system (Burliegh Instruments) attached to microscope stage
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Figures

Videos

Literature Cited

Literature Cited
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