VISUALIZATION AND FUNCTIONAL BLOCKING OF GAP JUNCTION HEMICHANNELS (CONNEXONS) WITH ANTIBODIES AGAINST EXTERNAL LOOP DOMAINS IN ASTROCYTES

Astrocytes constitute a metabolically and electrically coupled syncyti um which is essential for the regulation of ionic homeostasis of the i nterstitial brain fluid and for coordinating responses to neuronal act ivities. The structural avenues that couple individual astrocytes are provided by gap junctions which consist of transmembraneous channels t hat bridge the extracellular space. Each gap junction channel is compo sed of two hemichannels (connexons) that dock to each other via their extracellular loops. Here we report on the characterization of externa l loop antibodies that visualize hemichannels in specific plasma membr anes of vital astrocytes. Hemichannels were found to be concentrated o n cytoplasmic processes and filopodia of subconfluent cells. Specifici ty of hemichannel-binding was evaluated by blockage of Lucifer Yellow (LY) dye-transfer and Ca2+ transmission, as well as LY uptake under ca lcium-deficient conditions. Our data indicate that hemichannels are ac cessible from the extracytoplasmic side either for direct visualizatio n or functional manipulation. The availability of such a probe will al low in vivo experiments which require selective and/or temporal blocki ng of gap junctions in animal models. GLIA 24:141-254, 1998. (C) 1998 Wiley-Liss, Inc.