Connexin 46 and connexin 50 gap junction channel open stability and unitary conductance are shaped by structural and dynamic features of their N-terminal domains

Abstract

<jats:title>Abstract</jats:title><jats:p>The connexins form intercellular communication channels, known as gap junctions (GJs), that facilitate diverse physiological roles in vertebrate species, ranging from electrical coupling and long-range chemical signaling, to coordinating development and nutrient exchange. GJs formed by different connexins are expressed throughout the body and harbor unique channel properties that have not been fully defined mechanistically. Recent structural studies have implicated the amino-terminal (NT) domain as contributing to isoform-specific functional differences that exist between the lens connexins, Cx50 and Cx46. To better understand the structural and functional differences in the two closely related, yet functionally distinct GJs, we constructed models corresponding to CryoEM-based structures of the wildtype Cx50 and Cx46 GJs, NT domain swapped chimeras (Cx46-50NT and Cx50-46NT), and point variants at the 9<jats:sup>th</jats:sup> residue (Cx46-R9N and Cx50-N9R) for comparative MD simulation and electrophysiology studies. All of these constructs formed functional GJ channels, except Cx46-50NT, which correlated with increased dynamical behavior (instability) of the NT domain observed by MD simulation. Single channel conductance (γ<jats:sub>j</jats:sub>) also correlated well with free-energy landscapes predicted by MD, where γ<jats:sub>j</jats:sub> of Cx46-R9N was increased from Cx46 and the γ<jats:sub>j</jats:sub>s of Cx50-46NT and Cx50-N9R was decreased from Cx50, but to a surprisingly greater degree. Additionally, we observed significant effects on transjunctional voltage-dependent gating (V<jats:sub>j</jats:sub>-gating) and open-state dwell times induced by the designed NT domain variants. Together, these studies indicate that the NT domains of Cx46 and Cx50 play an important role in defining channel properties related to open-state stability and single channel conductance.</jats:p>