Connexin43 phosphorylation in brain, cardiac, endothelial and epithelial tissues

Abstract

Gap junctions, composed of proteins from the connexin family, allow for intercellular communication between cells in essentially all tissues. There are 21 connexin genes in the human genome and different tissues express different connexin genes. Most connexins are known to be phosphoproteins. Phosphorylation can regulate connexin assembly into gap junctions, gap junction turnover and channel gating. Given the importance of gap junctions in development, proliferation and carcinogenesis, regulation of gap junction phosphorylation in response to wounding, hypoxia and other tissue insults is proving to be critical for cellular response and return to homeostasis. Connexin43 (Cx43) is the most widely and highly expressed gap junction protein, both in cell culture models and in humans, thus more research has been done on it and more reagents to it are available. In particular, antibodies that can report Cx43 phosphorylation status have been created allowing temporal examination of specific phosphorylation events in vivo. This review is focused on the use of these antibodies in tissue in situ, predominantly looking at Cx43 phosphorylation in brain, heart, endothelium and epithelium with reference to other connexins where data is available. These data allow us to begin to correlate specific phosphorylation events with changes in cell and tissue function. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Fig. 1. Brain shows phosphorylation at S365, S325/328/330 and S368

(A) For SDS-PAGE and Western blot, two mouse brains were quickly removed and frozen in liquid nitrogen. The tissue was pulverized in the presence of sample buffer containing protease and phosphatase inhibitors to prevent dephosphorylation during tissue homogenization. Samples from brain 1 and 2 (one in each lane) were run on 10% PAGE, blotted and probed with NT1 antibody to total Cx43 or antibodies to Cx43 phosphorylated at S368 (pS368), S365 (pS365) and S325/328/330 (pS330) [73]. (B) Immunofluorescence of OCT embedded frozen sections of brain using total Cx43 (Cx43IF1 [72] – in green) and S325/328/330 ([78] - in red) antibodies. Immunohistochemistry of formalin-fixed, paraffin-embedded tissue sections stained with Hematoxylin/Eosin showing cerebellar cells staining at opposed plasma membranes (brown) for total Cx43 (C) and phospho-S368 (D). Phospho-S368 levels appeared to be highest in the cerebellar regions of the brain. Bars are 20 μm.

Fig. 2. Gap junction remodeling in heart occurs in response to ischemia

Mouse hearts were excised and immediately fixed in formalin (CON) or first incubated without perfusion in non-oxygenated PBS for 30 min (ISCHEMIA). Paraffin embedded sections were co-labeled with a rabbit antibody to Total Cx43 (Sigma-Aldrich, St. Louis, MO) and a mouse antibody, CT1, which recognizes Cx43 unphosphorylated on S364/365 (FHCRC, Seattle, WA). Overlay panel shows the Total Cx43 antibody labeled with Alexa 546-conjugated secondary antibody in red and CT1 labeled with Alexa 488-conjugated secondary antibody in green.