Structural insights into the opening mechanism of human Cx43/GJA1 gap junction channel

Abstract Gating of the gap junction intercellular channel (GJCh) is tightly regulated by several cellular factors; however, the underlying mechanism is poorly understood. A cryo-EM study of human Cx43 GJCh revealed detailed structural changes induced by PIP₂. Cx43 protomers in a phospholipid environment show dynamic equilibrium among several N-terminal helix (NTH) conformations, including gate-covering NTH (GCN) and pore-lining NTH (PLN). Upon treatment with a water-soluble PIP₂ analog, the conformational equilibrium shifted from GCN to PLN in a dose-dependent manner, resulting in a decrease in the pore-occluding density and an increase in the open probability. The PIP₂ head interacts closely with basic residues in the membrane opening between neighboring protomers and the cytoplasmic loop (CL). These ionic interactions strengthen the binding of CL to a transmembrane helix, which consequently inhibits the GCN conformation through steric hindrance. This study provides structural insights into the mechanisms underlying the opening of Cx43 GJCh.