Connexin Hemichannels: Methods for Dye Uptake and Leakage

Ross G. Johnson, Hung C. Le, Kristen Evenson, Shelby W. Loberg, Tori M. Myslajek, Andrea Prabhu, Ann Marie Manley, Colette O’Shea, Haiying Grunenwald, Madelaine Haddican, Patrick M. Fitzgerald, Timothy Robinson, Bruno A. Cisterna, Juan C. Sáez, Tai Feng Liu, Dale W. Laird, Judson D. Sheridan

Research output: Contribution to journalReview articlepeer-review

19 Citations (Scopus)

Abstract

It is now clear that connexin-based, gap junction “hemichannels” in an undocked state are capable of opening and connecting cytoplasm to the extracellular milieu. Varied studies also suggest that such channel activity plays a vital role in diverse cell processes and abnormal hemichannel activity contributes to pathogenesis. To pursue fundamental questions in this area, investigators require methods for studying hemichannel permeability and dynamics that are quantitative, sensitive, versatile, and available to most cellular and molecular laboratories. Here we first provide a theoretical background for this work, including the role of cellular membrane potentials. We then describe in detail our computer-assisted methods for both dye uptake and leakage along with illustrative results from different cell systems. A key feature of our protocol is the inclusion of a mechanical stimulation step. We describe dye uptake, interpreted as connexin dependent, that is shown to be enhanced with reduced extracellular Ca2+, mechanically responsive, inhibited by TPA, inhibited by EL186 antibodies for Cx43 and sustained for more than 15 min following mechanical stimulation. We describe dye leakage that displays these same properties, with estimates of hemichannel numbers per cell being derived from leakage rates. We also describe dye uptake that is shown to be unaffected by a reduction in external Ca2+, insensitive to EL186 antibodies and relatively short-lived following mechanical stimulation; this uptake may occur via pannexin 1 channels expressed in the cells studied here. It is unlikely that cell damage plays a significant role in dye uptake following mechanical stimulation, given compelling results from various control experiments.

Original languageEnglish
Pages (from-to)713-741
Number of pages29
JournalJournal of Membrane Biology
Volume249
Issue number6
DOIs
Publication statusPublished - 1 Dec 2016

Keywords

  • Connexin
  • Dye permeability
  • Hemichannel

ASJC Scopus subject areas

  • Biophysics
  • Physiology
  • Cell Biology

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