N-terminal integrity governs heptamer assembly and maturation in a mosquito innexin hemichannel

Nonchordate gap junction channels are formed by innexins, yet the structures and subunit assembly mechanisms of insect innexins remain unknown. We determined cryoelectron microscopy structures of AGAP001487, an innexin from the malaria mosquito Anopheles gambiae , reconstituted in lipid nanodiscs. The wild-type protein forms a rotationally symmetric heptameric hemichannel with an ordered N-terminal ring stabilized by intersubunit hydrogen bonds. Whereas deletion of a single N-terminal residue preserves channel activity, removal of two or more residues markedly impairs function. Consistent with these functional defects, the structure lacking two N-terminal residues exhibits disrupted rotational symmetry, extensive N-terminal disorder, pore collapse, and altered pore–lipid interactions. In addition, two-dimensional class averages revealed a minor population of hexameric particles, consistent with a potential assembly intermediate. Together, these structures support a stepwise assembly pathway in which intersubunit hydrogen bonding stabilizes N-terminal ring formation as a maturation step leading to a fully symmetric heptameric channel. Our findings identify N-terminal integrity as a critical determinant of oligomer maturation and channel function and provide mechanistic insight into how defined oligomeric assemblies are established in large-pore channel families.