title: Connexin Hemichannels: Methods for Dye Uptake and Leakage.
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.
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 55455, USA. gaplab@umn.edu.
authors: Ross G
authors: RG
authors: Johnson
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 55455, USA.
authors: Hung C
authors: HC
authors: Le
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 55455, USA.
authors: Kristen
authors: K
authors: Evenson
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 55455, USA.
authors: Shelby W
authors: SW
authors: Loberg
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 55455, USA.
authors: Tori M
authors: TM
authors: Myslajek
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 55455, USA.
authors: Andrea
authors: A
authors: Prabhu
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 55455, USA.
authors: Ann-Marie
authors: AM
authors: Manley
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 55455, USA.
authors: Colette
authors: C
authors: O'Shea
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 55455, USA.
authors: Haiying
authors: H
authors: Grunenwald
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 55455, USA.
authors: Madelaine
authors: M
authors: Haddican
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 55455, USA.
authors: Patrick M
authors: PM
authors: Fitzgerald
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 55455, USA.
authors: Timothy
authors: T
authors: Robinson
authors: Departmento de Fisiologia, Pontificia Universidad Católica de Chile, Santiago, Chile.
authors: Bruno A
authors: BA
authors: Cisterna
authors: Departmento de Fisiologia, Pontificia Universidad Católica de Chile, Santiago, Chile.
authors: Juan C
authors: JC
authors: Sáez
authors: Department of Physiology and Biophysics, College of Life Sciences, Peking University, Beijing, People's Republic of China.
authors: Tai-Feng
authors: TF
authors: Liu
authors: Department of Anatomy and Cell Biology, University of Western Ontario, London, ON, Canada.
authors: Dale W
authors: DW
authors: Laird
authors: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 55455, USA.
authors: Judson D
authors: JD
authors: Sheridan
mesh_terms: D000465:Algorithms; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004396:Coloring Agents / Q000378:metabolism; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D008962:Models, Theoretical; D010539:Permeability
chemical_list: D004396:Coloring Agents; D018031:Connexin 43; D017630:Connexins; D002118:Calcium
keywords: Connexin; Dye permeability; Hemichannel