A protein kinase A-ezrin complex regulates connexin 43 gap junction communication in liver epithelial cells

Communication between adjacent cells can occur via gap junctions (GJ) composed of connexin (Cx) hexamers that allow passage of small molecules. One of the most widely and highly expressed Cxs in human tissues is Cx43, shown to be regulated through phosphorylation by several kinases including PKA. Ezrin is a membrane associated protein that can serve as an A-kinase anchoring protein (AKAP) and hold an anchored pool of PKA. Here, we used the liver epithelial cell line IAR20, which expresses Cx43 as the predominant GJ protein, to test the hypothesis that Ezrin may associate with Cx43 in cell types that form stable GJs and serve as an AKAP. Our biochemical and proteomics data indicate that Ezrin associates with Cx43 in epithelial cells. Analyses by confocal immunofluorescence microscopy and proximity ligation assays demonstrate that Ezrin and Cx43 co-localize, together with zonula occludens-1 (ZO-1) and PKA RIα and RIIα, at the cell membrane. Quantitative gap-FRAP experiments show increased GJ intercellular communication after cAMP stimulation. Moreover, loading of cells with the Ht31 peptide that displaces both PKA RIα and RIIα from the AKAP or a peptide that disrupts the Cx43-Ezrin interaction reverts the effect and reduces the level of communication, supporting the hypothesis that in IAR20 cells Ezrin associates with Cx43 (in complex with ZO-1) which places PKA in proximity to Cx43, enabling its phosphorylation and GJ opening.