Abstract
Connexin proteins are the building blocks of gap junctions and connexin hemichannels. Both provide a pathway for cellular communication. Gap junctions support intercellular communication mechanisms and regulate homeostasis. In contrast, open connexin hemichannels connect the intracellular compartment and the extracellular environment, and their activation fuels inflammation and cell death. The development of clinically applicable connexin hemichannel blockers for therapeutic purposes is therefore gaining momentum. This chapter describes a well-established protocol optimized for assessing connexin hemichannel activity by using the reporter dye Yo-Pro1.
Graphical Abstract
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Lukowicz-Bedford R, Farnsworth DR, Miller AC (2021) Connexinplexity: the spatial and temporal expression of connexin genes during vertebrate organogenesis. bioRxiv preprint
Oyamada M, Oyamada Y, Takamatsu T (2005) Regulation of connexin expression. Biochim Biophys Acta Biomembr 1719:6–23
Beyer EC, Paul DL, Goodenough DA (1987) Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol 105:2621–2629
Totland MZ, Rasmussen NL, Knudsen LM et al (2020) Regulation of gap junction intercellular communication by connexin ubiquitination: physiological and pathophysiological implications. Cell Mol Life Sci 77:573–591
Zhu Y (2022) Gap junction-dependent and -independent functions of Connexin43 in biology. Biology (Basel) 11:1–19
Liu W, Cui Y, Wei J et al (2020) Gap junction-mediated cell-to-cell communication in oral development and oral diseases: a concise review of research progress. Int J Oral Sci 12:1–9
Van Campenhout R, Gomes AR, De Groof TWM et al (2021) Mechanisms underlying connexin hemichannel activation in disease. Int J Mol Sci 22:1–14
Peng B, Xu C, Wang S et al (2022) The role of Connexin Hemichannels in inflammatory diseases. Biology (Basel) 11:1–20
Cooreman A, Van Campenhout R, Ballet S et al (2019) Connexin and Pannexin (hemi)channels: emerging targets in the treatment of liver disease. Hepatology 69:1317–1323
Delvaeye T, De Smet MAJ, Verwaerde S et al (2019) Blocking connexin43 hemichannels protects mice against tumour necrosis factor-induced inflammatory shock. Sci Rep 9:1–12
Mugisho OO, Green CR, Zhang J et al (2019) Connexin43 hemichannels: a potential drug target for the treatment of diabetic retinopathy. Drug Discov Today 24:1627–1636
Retamal MA, Fernandez-Olivares A, Stehberg J (2021) Over-activated hemichannels: a possible therapeutic target for human diseases. Biochim Biophys Acta Mol basis Dis 1867:1–15
Caufriez A, Böck D, Martin C et al (2020) Peptide-based targeting of connexins and pannexins for therapeutic purposes. Expert Opin Drug Discov 15:1213–1222
Willebrords J, Maes M, Crespo Yanguas S et al (2017) Inhibitors of connexin and pannexin channels as potential therapeutics. Pharmacol Ther 180:144–160
Goyvaerts C, De Groeve K, Dingemans J et al (2012) Development of the Nanobody display technology to target lentiviral vectors to antigen-presenting cells. Gene Ther 19:1133–1140
Breckpot K, Dullaers M, Bonehill A et al (2003) Lentivirally transduced dendritic cells as a tool for cancer immunotherapy. J Gene Med 5:654–667
Lopez W, Ramachandran J, Alsamarah A et al (2016) Mechanism of gating by calcium in connexin hemichannels. Proc Natl Acad Sci USA 113:7986–7995
Thimm J, Mechler A, Lin H et al (2005) Calcium-dependent open/closed conformations and interfacial energy maps of reconstituted hemichannels. J Biol Chem 280:10646–10654
Harris AL, Contreras JE (2014) Motifs in the permeation pathway of connexin channels mediate voltage and Ca2+ sensing. Front Physiol 5:1–10
Maes M, Crespo Yanguas S, Willebrords J et al (2017) Connexin hemichannel inhibition reduces acetaminophen-induced liver injury in mice. Toxicol Lett 278:30–37
Crespo Yanguas S, da Silva TC, Pereira IVA et al (2018) TAT-Gap19 and Carbenoxolone alleviate liver fibrosis in mice. Int J Mol Sci 19:1–17
Walrave L, Pierre A, Albertini G et al (2018) Inhibition of astroglial connexin43 hemichannels with TAT-Gap19 exerts anticonvulsant effects in rodents. Glia 66:1788–1804
Walrave L, Vinken M, Albertini G et al (2016) Inhibition of connexin43 hemichannels impairs spatial short-term memory without affecting spatial working memory. Front Cell Neurosci 10:1–10
Willebrords J, Cogliati B, Pereira IVA et al (2017) Inhibition of connexin hemichannels alleviates non-alcoholic steatohepatitis in mice. Sci Rep 7:1–11
Wang N, De Vuyst E, Ponsaerts R et al (2013) Selective inhibition of Cx43 hemichannels by Gap19 and its impact on myocardial ischemia/reperfusion injury. Basic Res Cardiol 108:1–16
Acknowledgments
This work was financially supported by the European Research Council (Proof-of-Concept grant 861913), the European Future and Emerging Technologies program (grant 858014), the Research Foundation Flanders-Belgium (grants G012318N, G020018N and G0F7219N), the Methusalem program of the Flemish Government and the University Hospital of the Vrije Universiteit Brussel-Belgium (Scientific Fund Willy Gepts). The authors wish to thank Fien Haenen and Arne Loosen for their dedicated technical assistance.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Van Campenhout, R. et al. (2024). Assessment of Connexin43 Hemichannel Functionality Based on Cytosolic Uptake of Yo-Pro1. In: Mammano, F., Retamal, M. (eds) Connexin Hemichannels. Methods in Molecular Biology, vol 2801. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3842-2_6
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3842-2_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3841-5
Online ISBN: 978-1-0716-3842-2
eBook Packages: Springer Protocols