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

Electrical synapses are essential components of neural circuits. Dysfunction of electrical synapses can lead to obstacles in learning and memory. Neural signal transduction across electrical synapses is primarily mediated by a gap junction channel, Connexin 36 (Cx36), the lack of which causes impaired electrical couplings in cortical interneurons and thalamic reticular nucleus (TRN) neurons. Unique characteristics of Cx36 gap junction channel include its insensitivity in transjunctional membrane potential, as well as its preference for homomeric assembly, prone to exclude other paralogous connexins from co-assembly. However, the structural basis underlying Cx36 function and assembly remains elusive. Here, we report the cryo-EM structure of human Cx36 at 2.67 Å resolution and identified critical residues underpinning its obligatory homomeric assembly. In particular, we found non-canonical electrostatic interactions between protomers from opposing hemichannels and a steric complementary site between adjacent protomers within a hemichannel, which together offer a structural explanation for the assembly specificity in homomeric and heteromeric gap junction channels. Moreover, the narrowest restriction along the channel axis overlaps with an acidic patch, where Glu43, Asp47 and Asp48 may contribute to cation-selectivity. Also, the amino-terminal helix reported to be responsible for sensing membrane potential in other connexins was disordered in our Cx36 structure, in line with its channel activity independent of membrane potential. Together, this work elucidated the assembly mechanisms of the electrical synaptic gap junction channel, and offered possible explanations for sustained Cx36 activity upon membrane depolarization, which allows efficient action potential propagation across electrical synapses.