Visualization and functional blocking of gap junction hemichannels (connexons) with antibodies against external loop domains in astrocytes

Abstract

Astrocytes constitute a metabolically and electrically coupled syncytium which is essential for the regulation of ionic homeostasis of the interstitial brain fluid and for coordinating responses to neuronal activities. The structural avenues that couple individual astrocytes are provided by gap junctions which consist of transmembraneous channels that bridge the extracellular space. Each gap junction channel is composed of two hemichannels (connexons) that dock to each other via their extracellular loops. Here we report on the characterization of external loop antibodies that visualize hemichannels in specific plasma membranes of vital astrocytes. Hemichannels were found to be concentrated on cytoplasmic processes and filopodia of subconfluent cells. Specificity of hemichannel‐binding was evaluated by blockage of Lucifer Yellow (LY) dye‐transfer and Ca2+ transmission, as well as LY uptake under calcium‐deficient conditions. Our data indicate that hemichannels are accessible from the extracytoplasmic side either for direct visualization or functional manipulation. The availability of such a probe will allow in vivo experiments which require selective and/or temporal blocking of gap junctions in animal models. GLIA 24:141–154, 1998. © 1998 Wiley‐Liss, Inc.

ATP release from non-excitable cells

“…Thus, travelling [Ca 2+ ] i waves can also be caused by release of nucleotides, stimulation of P2 receptors and generation of further nucleotide release to feed wave propagation. Travelling [Ca 2+ ] i waves appear to be dependent on the expression of gap junction proteins, raising the question of whether expression of connexins may modulate nucleotide release [90,91]. Forced expression of different connexins (Cx43, Cx32, Cx26) caused a greatly increased UTP-stimulated ATP release in different cell types [90].…”

“…This requires that connexin molecules, in addition to building gap junctions between cells, also localise in the plasma membrane as hemichannels. Using well-characterised antibodies against the extracellular loops of connexins, strong evidence for hemichannel localisation in cellular processes and filipodia of astrocytes has been provided [91]. Lowering extracellular Ca 2+ triggered the uptake of small (>1 kDa) fluid phase fluorophores, and this could be blocked with antibodies against connexins [91].…”

“…Using well-characterised antibodies against the extracellular loops of connexins, strong evidence for hemichannel localisation in cellular processes and filipodia of astrocytes has been provided [91]. Lowering extracellular Ca 2+ triggered the uptake of small (>1 kDa) fluid phase fluorophores, and this could be blocked with antibodies against connexins [91]. Thus, hemichannels have been shown to be functionally present in the plasma membrane and as a consequence of their biophysical structure could allow the exit of ATP.…”

New roles for astrocytes: Gap junction hemichannels have something to communicate

“…Although there is no direct measure of the number of hemichannels inserted in the membrane using connexins labeled with enhanced green-fluorescent protein (eGFP) [22], reaction with antibodies to the extracellular loop indicates substantial and readily visualized accessibility of extracellular epitopes of Cx43 hemichannels in astrocytes [52]. Thus, the observations that only a few hemichannels open in Cx43-expressing cells indicate that open probability, P o , is very low.…”

Lack of “Hemichannel” Activity in Insulin-Producing Cells

“…Consistent with this hypothesis, biochemical, immunolabeling, and expression studies revealed that connexons may be inserted in nonjunctional regions of the cell membrane (Musil et al 1991;Paul et al 1991;De Vries et al 1992). Other studies showed that leakage of cytosolic molecules and uptake of extracellular, membrane-impermeant tracers may be abolished by drugs or conditions blocking gap junction channels (Liu et al 1998;Hofer and Dermietzel 1998;Kondo et al 2000;Quist et al 2000). Finally, electrophysiology of single cells identified currents with voltage sensitivity and unitary conductances approximatively as expected for half junctional channels, made of unpaired connexons (Harris 2001;Stout et al 2004;Saez et al 2005;Spray et al 2006).…”