Abstract
Connexin 43 (Cx43) hemichannels establish local signaling networks via the release of ATP and other molecules, but their excessive opening may result in cell death. Hence, the activity of Cx43-hemichannels ought to be critically controlled. This involves interactions between the C-terminal tail (CT) and the cytoplasmic loop (CL), more particularly the L2 domain within CL. Previous work revealed an important role for the last nine amino acids of the Cx43 CT by targeting the L2 domain, as these nine amino acids were sufficient to restore the activity of CT-truncated Cx43-hemichannels. However, we discovered that deletion of the last 19 amino acids of the CT only partially lowered the binding to the L2 domain, indicating that a second L2-binding region is present in the CT. We here provide evidence that the SH3-binding domain is another CT region that targets the L2 domain. At the functional level, the SH3-binding domain was able to restore the activity of CT-truncated Cx43-hemichannels and alleviate the inhibition of full-length Cx43-hemichannels by high intracellular Ca2+ concentration ([Ca2+]i) as demonstrated by various approaches including patch clamp studies of unitary Cx43-hemichannel activity. Finally, we show that in full-length Cx43-hemichannels, deletion of either the SH3-binding domain or the CT9 region suppresses the hemichannel activity, while deletion of both domains completely annihilates the hemichannel activity. These results demonstrate that the Cx43 SH3-binding domain, in addition to the CT9 region, critically controls hemichannel activity at high [Ca2+]i, which may be involved in pathological hemichannel opening.
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Acknowledgements
The authors are grateful to Anja Florizoone, Marina Crabbe, Tomas Luyten and Kirsten Welkenhuyzen for technical support. This work was supported by research Grant (G.0298.11 to GB and LL) and krediet aan navorsers (15117.14 N to CDH and GB) from the Research Foundation–Flanders (FWO) and by NIH grant CA196214 and Welch Foundation grant AQ-1507 (to JXJ). GB and LL are part of a Scientific Research Community supported by the FWO. The authors are thankful to Raf Ponsaerts and Elke De Vuyst for preliminary work and previous support and to Joris Vriens for helpful suggestion for displaying the electrophysiological recordings. The authors are also grateful to Mario A Delmar (New York University School of Medicine, NY, USA) and Paul L Sorgen (University of Nebraska Medical Center, NE, USA) for providing Cx43-expression plasmids.
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GB and LL conceived the study, supervised the work and interpreted data. JI, NW and CDH performed experiments and analyzed data. JJX provided critical reagents. JI performed cloning, protein purifications, SPR experiments, ATP release assays. CDH performed Ca2+-wave propagations. NW performed all electrophysiology experiments. GB drafted the manuscript with further input and corrections by LL, JI, NW and CDH. All authors have read and approved the manuscript.
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Iyyathurai, J., Wang, N., D’hondt, C. et al. The SH3-binding domain of Cx43 participates in loop/tail interactions critical for Cx43-hemichannel activity. Cell. Mol. Life Sci. 75, 2059–2073 (2018). https://doi.org/10.1007/s00018-017-2722-7
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DOI: https://doi.org/10.1007/s00018-017-2722-7