Two-Dimensional Kinetics of Inter-Connexin Interactions from Single-Molecule Force Spectroscopy

Gap junction channels are intercellular channels that form by docking the extracellular loops of connexin protein subunits. While the structure and function of gap junctions as intercellular channels have been characterized using different techniques, the physics of the inter-connexin interaction remain unknown. Moreover, as far as we know, the capacity of gap junction channels to work as adhesion complexes supporting pulling forces has not yet been quantitatively addressed. We report the first quantitative characterization of the kinetics and binding strength of the interaction of a short peptide mimicking extracellular loop 2 of Cx26 with membrane-reconstituted Cx26, combining the imaging and force spectroscopy capabilities of atomic force microscopy. The fast dissociation rate inferred a dynamic bond, while the slow association rate reflected the reduced flexibility and small size of extracellular loops. Our results propose the gap junction channel as an adhesion complex that associates slowly and dissociates fast at low force but is able to support important pulling forces in its native, hexameric form.

► We used atomic force microscopy to probe the two-dimensional kinetics and binding strength of inter-Cx26 interactions.
► We mimicked inter-Cx26 interactions with a short peptide and reconstituted Cx26.
► We found a fast dissociation rate at zero force and slow association rate.
► The binding strength suggests gap junctions as force-bearing adhesion complexes.