Distinct modes of N-terminal remodeling through a conserved inhibitor pocket in connexin-46/50

Selective pharmacological control of gap junction communication remains challenging because existing small-molecule connexin inhibitors act across multiple isoforms. The structural basis of this broad activity remains incompletely understood. Here, we used single-particle cryo-electron microscopy to examine native connexin-46/50 gap junction channels treated with two commonly used and chemically distinct inhibitors, 2-aminoethoxydiphenyl borate (2-APB) and carbenoxolone (CBX). Both treatments perturb the hydrophobic N-terminal (NT)-docking groove, but produce distinct conformational ensembles. 2-APB yields ordered destabilized-open and gated NT states resembling conformations observed during physiological pH- and Ca2+-dependent gating, with ligand- and lipid-associated densities accompanying the gated state. In contrast, CBX-compatible density occupies the same groove and is associated with loss of an ordered NT conformation, consistent with steric competition and altered physicochemical complementarity at the NT-docking site. Comparison with inhibitor-treated connexin-32 and connexin-36 structures identifies this region as a structurally conserved, pharmacologically sensitive pocket. These findings reveal how chemically diverse inhibitors perturb a conserved element of the native gating machinery while producing divergent NT-gating ensembles, providing a structural rationale for broad cross-isoform activity and identifying sequence-variable pocket margins that may support more selective modulation.