Electrostatic Interaction Determines the Docking Specificity and the Formation of Functional Cx36 Gap Junction Channels

Connexin36 (Cx36) is broadly expressed in neurons and serves as the principal protein to form interneuronal gap junctions (GJs), also known as electrical synapses. Recent high-resolution structures of human Cx36 GJ have revealed crucial electrostatic interactions (ESIs) between two docked Cx36 hemichannels at the second extracellular (E2) loops. Despite their structural importance, the functional roles of these ESIs remain poorly understood. To investigate their significance, we systematically designed and tested a series of missense variants targeting key E2 interface residues, aiming to disrupt or modulate the electrostatic landscape at the docking interface. Based on the ESI pairs defined from the crystal structure, our combined computational calculations and dual patch-clamp experiments in engineered HEK293 cell pairs suggest that at least three ESI residual pairs per E2-E2 interface are required to support functional GJ formation. Furthermore, we found that these unique ESIs of Cx36 ensure its docking specificity to itself, preventing the formation of heterotypic GJs with other brain connexins (Cx26, Cx30, Cx31.3, Cx32, Cx43, Cx45, Cx47). Overall, these findings provide essential molecular and functional insights into the mechanisms governing Cx36 GJ formation and partner specificity, paving the way for future therapeutic approaches targeting connexin dysfunction in human diseases.