Cryo-EM structures of human Cx36/GJD2 neuronal gap junction channel

Abstract

Connexin 36 (Cx36) is responsible for signal transmission in electrical synapses by forming interneuronal gap junctions. Despite the critical role of Cx36 in normal brain function, the molecular architecture of the Cx36 gap junction channel (GJC) is unknown. Here, we determine cryo-electron microscopy structures of Cx36 GJC at 2.2–3.6 Å resolutions, revealing a dynamic equilibrium between its closed and open states. In the closed state, channel pores are obstructed by lipids, while N-terminal helices (NTHs) are excluded from the pore. In the open state with pore-lining NTHs, the pore is more acidic than those in Cx26 and Cx46/50 GJCs, explaining its strong cation selectivity. The conformational change during channel opening also includes the α-to-π-helix transition of the first transmembrane helix, which weakens the protomer-protomer interaction. Our structural analyses provide high resolution information on the conformational flexibility of Cx36 GJC and suggest a potential role of lipids in the channel gating.

Structures of wild-type and selected CMT1X mutant connexin 32 gap junction channels and hemichannels

“…S13A). The α-π helix transition, seen previously in the TM1 of Cx36 (35), is not observed in the corresponding regions of the Cx32 structures. The main distinction between these channel structures is the NTH region.…”

“…To date, the structures of four connexin complexes have been experimentally determined: Cx26 GJC (28)(29)(30)(31), Cx46/50 GJC (32,33), Cx31.3 HC (34), Cx36 GJC (35), and Cx43 GJC and HC (36,37). These homologous structures have not only provided important clues to the shared architectural features and assembly of connexin-mediated GJCs and HCs but also revealed substantial differences between them.…”

Assembly and Cation-selectivity Mechanisms of Neuronal Gap Junction Channel Connexin 36 Elucidated by Cryo-EM

“…The first structural insight into connexin assembly mechanism was obtained using Cx26 homododecamer [55], which revealed interactions between extracellular loops that governs hemichannel docking, and together with subsequently determined connexin structures, began to uncover docking mechanisms by which homotypical or heterotypical assembly is specified. Interactions between the opposing first extracellular loops (EL1) are primarily hydrogen bonds conserved in known connexin structures including Cx46/50 [56], Cx43 [57], the recently reported Cx36 [58], and Cx36 in this study. In our structure, these hydrogen bonds were formed by Asn56/Leu58 and their opposing partners in a head-to-toe fashion, and between dihedral symmetry-related Gln59 from a different protomer pair (Figs.…”

Mefloquine-induced conformational shift in Cx36 N-terminal helix leading to channel closure mediated by lipid bilayer

“…region contribute to the specificity of MFQ to Cx36, such as the loosely packed NTH conformation due to the insertion of Ala14, exclusively observed in Cx36. As a result, among currently available connexin structures in the PLN state, Cx36 exhibits relatively broader hydrophobic pockets that serve as binding sites for a hydrophobic molecule (Supplementary Fig 5c) 17 . The broader hydrophobic pocket is primarily attributed to the longer NTH-TM1 loop (residues Ala13-Ser19) owing to the insertion of Ala14 (Supplementary Fig.…”

“…To assess the impact of MFQ and AA on the conformational equilibrium of Cx36 GJC, we performed protomer-focused 3D classification following the methodology detailed in our previous work 17 . Strikingly, both inhibitor-bound structures exclusively exhibited FN protomers, with no observation of PLN protomers (Fig.…”