The relationship between Connexin 43 (Cx43) and partner protein, human discs large homologue-1 (Dlg1) during wound closure in keratinocytes

Plasma membrane gap junctions, formed by connexin proteins, are responsible for direct intercellular communication between adjacent cells by allowing the exchange of ions, metabolites, secondary messengers and microRNAs. Connexin 43 (Cx43) is a widely expressed gap junction protein. Cx43 mis-expression is associated with various disease states, including chronic non-healing wounds. Loss of Cx43 from the plasma membrane is important to allow wound resolution. Cx43 expression increases, and gap junctions reform, upon wound closure. Cx43 directly interacts with Dlg1, a membrane-associated guanylate kinase (MAGUK) protein, which controls cell shape and polarity. In this study, AlphaFold3 modelling predicted an interaction between amino acids 263-269 and 302-320 of Cx43 and the SH3, HOOK and GUK domains of Dlg1. Since Dlg1 is required for maintaining Cx43 protein levels at the plasma membrane, we hypothesised that Dlg1 may regulate wound healing via its functional interaction with Cx43. Cx43 and Dlg1 relocated to the cytoplasm of cells at the wound edge in scrape wound assays and co-located in pseudopodia of cells migrating towards the wound edge in live cell imaging experiments. Scratch wound assays carried out in HaCaT cells treated with siRNA targeting Dlg1 showed that Dlg1 depletion resulted in reduced wound closure rates. This was not due to changes in cell migration, suggesting that Dlg1 controlled cell proliferation. The data demonstrate that Cx43 and Dlg1 both contribute to wound closure, but that Dlg1 has other crucial functions in migrating cells separate from its role in maintaining Cx43 at the plasma membrane.