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.
Bibliographical noteFunding Information:
We gratefully acknowledge the generosity of Drs. Alan Lau and Bonnie Cramer, Klaus Willecke and Claudia Elfgang, as well as Christian Naus, for providing cell lines used in some of these studies. We also thank Drs. Jim Nagy and Gerhard Dahl for providing pannexin 1 antibodies. This work was supported by NIH Grant GM-46277 to RGJ and JDS. RGJ initiated these studies, developed the basic protocols for both mechanical stimulation and dye uptake, performed some of the early experiments, and supervised the work with the connexin antibodies. JDS developed and modeled the theoretical background for this work, including the explicit inclusion of membrane potential effects on uptake and leakage, and created the basic macros and protocols for imaging cells and analyzing fluorescence intensities. He also supervised the work on fluorescence intensities. The work described in Online Resource 7 was carried out by BAC in the JCS laboratory, with RGJ?s involvement in the initial studies. LTF performed dye leakage experiments that informed similar experiments reported here. DWL developed the peptide-specific antibodies for Cx43 used here. All other co-authors were students or technicians in the Johnson-Sheridan lab who performed experiments that led to the development of the uptake/leakage methods or provided the findings described here. RGJ and JDS also wrote this manuscript.
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- Dye permeability