Tricellular adherens junctions provide a cell surface delivery platform for connexin 26/30 oligomers in the cochlea

In the cochlea, connexins 26 (Cx26) and 30 (Cx30) largely co-assemble into heteromeric gap junctions, which connect adjacent non-sensory epithelial cells. These channels are believed to ensure the rapid removal of K⁺ away from the base of sensory hair cells, resulting in K⁺ recycling back to the endolymph to maintain cochlear homeostasis. Many of the mutations in GJB2 and GJB6, which encode CX26 and CX30, impair the formation of membrane channels and cause autosomal hearing loss in humans. Although recent advances have been made, several important questions remain about connexin trafficking and gap junction biogenesis. Here we show that tricellular adherens junctions present at the crossroad between adjacent gap junction plaques, provide an unexpected cell surface delivery platform for Cx26/Cx30 oligomers. Using an in situ proximity ligation assay, we detected the presence of non-junctional Cx26/Cx30 oligomers within lipid raft-enriched tricellular junction sites. In addition, we observed that cadherin homophilic interactions are critically involved in microtubule-mediated trafficking of Cx26/Cx30 oligomers to the cell surface. Overall, our results unveil an unexpected role for tricellular junctions in the trafficking and assembly of membrane channels.