Multi-ion permeation and dynamic conductance modulation in connexin gap junction channels.

Gap junction channels formed by connexins mediate direct intercellular communication and are essential for electrical signaling and tissue homeostasis. Despite their large, solvent-accessible pores, connexin channels exhibit distinct conductance, selectivity, and rectification properties, but the molecular mechanisms underlying these behaviors remain incompletely understood. Here, we performed ∼67 μs of all-atom computational electrophysiology simulations of connexin-46 (Cx46), connexin-50 (Cx50), and heterotypic Cx46/50 gap junction channels based on high-resolution open-state structures, enabling characterization of both ion permeation and long-timescale channel dynamics. Simulations reveal a multi-ion, multi-pathway permeation mechanism governed by isoform-specific energetic barriers and transient ion coordination sites that shape conductance and selectivity. In heterotypic Cx46/50 channels, asymmetric energetic landscapes establish a mechanistic basis for rectification. Unexpectedly, the microsecond-timescale simulations further revealed dynamic interactions between the intracellular loop (ICL) region and N-terminal domain (NT) that transiently constrict the pore and attenuate ionic currents. These findings suggest that the open-state comprises an ensemble of rapidly interconverting conductance microstates rather than a single static conformation, providing structural information of potential mechanistic importance beyond what has been learned from cryo-EM studies. Together, our results provide a mechanistic basis for ion permeation and current modulation in gap junction channels and highlight the importance of long-timescale protein dynamics in shaping intercellular communication.