Assessment of Gap Junctional Intercellular Communication

James E. Klaunig1, Yuhui Shi1

1 Indiana University School of Medicine, Indianapolis, Indiana
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.17
DOI:  10.1002/0471140856.tx0217s41
Online Posting Date:  August, 2009
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Gap junctions are important plasma membrane organelles through which most cells in normal tissues communicate with each other. They exist in two neighboring cells and each cell contributes half of the structure. One gap junction consists of two hexameric connexons that dock with each other to create a channel. Six of the basic subunits referred to as connexins form a connexon. Less than one hundred to several thousand gap junction channels cluster together in the plane of the membrane. The gap junction channels serve as a regulated conduit for the intercellular exchange of small molecules. Maintenance of the integrity of gap junctional intercellular communication (GJIC) is important and required for normal electrical coupling, homeostasis, and embryogenesis. Aberrations of gap junctions have been related to human diseases such as cancer, cardiac arrhythmia, Charcot‐Marie‐tooth disease, and visceroatrial heterotaxia syndrome. This unit describes methods for measuring gap junctional intercellular communication using primary mouse hepatocytes as a model. Focus is only on functional evaluation based on dye coupling. Other methods, such as intracellular calcium waves and dual patch clamp, have been used to measure gap junctional communication, but these are not described in this unit. Curr. Protoc. Toxicol. 41:2.17.1‐2.17.10. © 2009 by John Wiley & Sons, Inc.

Keywords: gap junction; intercellular communication; dye coupling

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Table of Contents

  • Introduction
  • Basic Protocol 1: Evaluation of the Molecular Permeability of Gap Junctional Intercellular Communication in Primary Mouse Hepatocytes
  • Alternate Protocol 1: Scrape Loading Dye Transfer
  • Alternate Protocol 2: Fluorescence Recovery After Photobleaching (FRAP)
  • Support Protocol 1: Primary Mouse Hepatocyte Isolation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1: Evaluation of the Molecular Permeability of Gap Junctional Intercellular Communication in Primary Mouse Hepatocytes

  • Primary hepatocytes on 60‐mm culture dishes (see protocol 4)
  • Chemicals to alter GJIC
  • 5% (w/v) Lucifer yellow CH (Sigma‐Aldrich) in 0.1 M LiCl
  • 0.1 M lithium chlorine (LiCl) solution
  • Capillary glass tubing (1.5‐mm diameter with filament; World Precision Instruments)
  • PuL‐1 pipet puller (World Precision Instruments)
  • Nikon Diaphot‐TMB inverted phase‐contrast microscope with epifluorescence equipment (Fryer)
  • Hamilton syringe (Hamilton Company)
  • MicroFil needle to fill micropipet (World Precision Instruments)
  • Micropipet storage jar (World Precision Instruments)
  • Narashige MO‐388 joystick hydraulic micromanipulator (Fryer)
  • Narashige MM‐188 three‐dimensional motor drive course manipulator (Fryer)
  • Power source (6 to 9 Valkaline battery with variable reostat for iontophoresis)

Alternate Protocol 1: Scrape Loading Dye Transfer

  • Cells cultured in 60‐cm tissue culture dishes
  • Ca2+/Mg2+‐PBS buffer (see recipe)
  • Fluorescence dye(s):
    • 0.5 mg/ml Lucifer yellow CH lithium salt (Sigma‐Aldrich) in Ca2+/Mg2+‐PBS (store in dark at −20°C)
    • 0.5 mg/ml Lucifer yellow and 0.5 mg/ml tetramethylrhodamine‐dextran (Molecular Probes) in Ca2+/Mg2+‐PBS (store in dark at −20°C)
    • 0.5 mg/ml tetramethylrhodamine‐dextran in Ca2+/Mg2+‐PBS (store in dark at −20°C)
  • 4% (w/v) formalin in Ca2+/Mg2+‐PBS
  • Scalpel blades
  • Ultima interactive laser cytometer (Meridian Instrument)

Alternate Protocol 2: Fluorescence Recovery After Photobleaching (FRAP)

  • 6 mg/ml stock solution of calboxyfluorescein diacetate (CFDA; Molecular Probes) in DMSO
  • DMEM/F12 complete medium (see recipe)
  • Ultima interactive laser cytometer (Meridian Instruments)

Support Protocol 1: Primary Mouse Hepatocyte Isolation

  • Perfusion buffer 1 (see recipe)
  • Perfusion buffer 2 (see recipe)
  • Liberase Blendzyme 3 (Roche)
  • Mouse (B6C3F1 or C57B, male or female, 18 to 30 g body weight)
  • 10 mg/ml pentobarbital solution in PBS (100 mg/kg body weight)
  • 70% ethanol
  • DMEM/F12 complete medium (see recipe)
  • 41°C water bath
  • Dissection tools: foam board, curved forceps, dissecting scissors, hemostat, T‐pins, angiocath with cannula, needle and syringe, gauze and cotton swabs, 3‐0 silk ligatures
  • Warming lamp
  • Peristaltic perfusion pump, tubing
  • 60‐ and 100‐mm tissue culture dishes
  • Wide‐bore pipets
  • 100‐µm cell strainer (BD Biosciences)
  • 50‐ml centrifuge tubes
  • Additional reagents and equipment for cell counting ( appendix 3B)
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Literature Cited

Literature Cited
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   el‐Fouly, M.H., Trosko, J.E., and Chang, C.C. 1987. Scrape‐loading and dye transfer. A rapid and simple technique to study gap junctional intercellular communication. Exp. Cell Res. 168:422‐430.
   Falk, M.M. 2000. Biosynthesis and structural composition of gap junction intercellular membrane channels. Eur. J. Cell Biol. 79:564‐574.
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