Sah-Teli, S. K.; Eshriew, E.; Abettan, A.; Kedari, A.; Kumpula, E.-P.; Saffitz, J.; Sharma, V.; Huiskonen, J. T.

Gap junction channels formed by the 21-member human connexin family enable direct intercellular exchange of ions and small signaling metabolites, coordinating electrical coupling across cardiac, neural and epithelial tissues. Connexin 45 (Cx45), encoded by GJC1, mediates impulse conduction in the atrioventricular node, His bundle, and Purkinje fibers, where disease-linked mutations cause progressive atrioventricular block and familial atrial fibrillation, yet no experimental structure has been reported, and its regulatory mechanism remains undefined. Here, we determine the structural basis of Cx45 gating and calcium regulation using cryo-electron microscopy, mutational analysis, and molecular dynamics simulations. Cryo-EM structures of the apo (2.76 Angstrom), calcium-bound (2.65 Angstrom), and E41A mutant (3.55 Angstrom) channel reveal a neck constriction formed by Y45, establishing a steric gate distinct from other connexins. Calcium associates with E41, stabilizing the neck via electrostatic remodeling without global conformational change. Together, these data define a dual steric-electrostatic mechanism for Cx45 regulation and provide a structural framework for isoform-specific connexin gating relevant to cardiac physiology and conduction disease.