Abstract
All connexins (Cx) proteins contain both highly ordered domains (i.e., 4 transmembrane domains) and primarily unstructured regions (i.e., n- and c-terminal domains). The c-terminal domains vary in length and amino acid composition from the shortest on Cx26 to the longest on Cx43. With the exception of Cx26, the c-terminal domains contain multiple sites for posttranslational modification (PTM) including serines (S), threonines (T), and tyrosines (Y) for phosphorylation or cysteines (C) for S-nitrosylation. These PTMs are critical for regulating cellular localization, protein–protein interactions, and channel functionality. There are several biochemical techniques that allow for the identification of these PTM including Western blotting and the “Biotin Switch” assay for nitrosylation. Quantitative analysis of Western blots can be achieved through use of secondary antibodies in the near infrared scale and high-resolution scanning on a fluorescent scanner.
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References
Johnstone S, Isakson B, Locke D (2009) Biological and biophysical properties of vascular connexin channels. Int Rev Cell Mol Biol 278:69–118
Straub AC, Johnstone SR, Heberlein KR et al (2010) Site-specific connexin phosphorylation is associated with reduced heterocellular communication between smooth muscle and endothelium. J Vasc Res 47:277–286
Johnstone SR, Kroncke BM, Straub AC et al (2012) MAPK phosphorylation of connexin 43 promotes binding of cyclin E and smooth muscle cell proliferation. Circ Res 111:201–211
Looft-Wilson RC, Billaud M, Johnstone SR et al (2012) Interaction between nitric oxide signaling and gap junctions: effects on vascular function. Biochim Biophys Acta 1818:1895–1902
Solan JL, Lampe PD (2007) Key connexin 43 phosphorylation events regulate the gap junction life cycle. J Membr Biol 217:35–41
Solan JL, Lampe PD (2014) Specific Cx43 phosphorylation events regulate gap junction turnover in vivo. FEBS Lett 588:1423–1429
Le Gal L, Alonso F, Mazzolai L et al (2015) Interplay between connexin40 and nitric oxide signaling during hypertension. Hypertension 65:910–915
D'hondt C, Iyyathurai J, Vinken M et al (2013) Regulation of connexin- and pannexin-based channels by post-translational modifications. Biol Cell 105:373–398
Nishi H, Hashimoto K, Panchenko AR (2011) Phosphorylation in protein-protein binding: effect on stability and function. Structure 19:1807–1815
Campbell AS, Johnstone SR, Baillie GS et al (2014) beta-Adrenergic modulation of myocardial conduction velocity: connexins vs. sodium current. J Mol Cell Cardiol 77:147–154
Johnstone SR, Ross J, Rizzo MJ et al (2009) Oxidized phospholipid species promote in vivo differential cx43 phosphorylation and vascular smooth muscle cell proliferation. Am J Pathol 175:916–924
Solan JL, Lampe PD (2009) Connexin43 phosphorylation: structural changes and biological effects. Biochem J 419:261–272
Berg AP, Talley EM, Manger JP et al (2004) Motoneurons express heteromeric TWIK-related acid-sensitive K+ (TASK) channels containing TASK-1 (KCNK3) and TASK-3 (KCNK9) subunits. J Neurosci 24:6693–6702
Straub AC, Billaud M, Johnstone SR et al (2011) Compartmentalized connexin 43 s-nitrosylation/denitrosylation regulates heterocellular communication in the vessel wall. Arterioscler Thromb Vasc Biol 31:399–407
Meens MJ, Alonso F, Le Gal L et al (2015) Endothelial Connexin37 and Connexin40 participate in basal but not agonist-induced NO release. Cell Commun Signal 13:34
Tannenbaum SR, White FM (2006) Regulation and specificity of S-nitrosylation and denitrosylation. ACS Chem Biol 1:615–618
Jaffrey S.R., Snyder S.H. (2001) The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE 2001:pl1
Wang X, Kettenhofen NJ, Shiva S et al (2008) Copper dependence of the biotin switch assay: modified assay for measuring cellular and blood nitrosated proteins. Free Radic Biol Med 44:1362–1372
Acknowledgements
This work was financially supported by the University of Glasgow, Lord Kelvin Adam Smith Fellowship (SRJ), by NIH grants R00 HL11290402, the Institute for Transfusion Medicine and the Hemophilia Center of Western Pennsylvania (ACS) and postdoctoral fellowships from the Canadian Institutes for Health Research and Alberta Innovates - Health Solutions (AWL).
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Lohman, A.W., Straub, A.C., Johnstone, S.R. (2016). Identification of Connexin43 Phosphorylation and S-Nitrosylation in Cultured Primary Vascular Cells. In: Vinken, M., Johnstone, S. (eds) Gap Junction Protocols. Methods in Molecular Biology, vol 1437. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3664-9_7
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DOI: https://doi.org/10.1007/978-1-4939-3664-9_7
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