Long-term precision editing of neural circuits using engineered gap junction hemichannels

The coordination of activity between brain cells is a key determinant of neural circuit function; nevertheless, approaches that selectively regulate communication between two distinct cellular components of a circuit, while leaving the activity of the presynaptic brain cell undisturbed remain sparce. To address this gap, we developed a novel class of electrical synapses by selectively engineering two connexin proteins found in Morone americana (white perch fish): connexin34.7 (Cx34.7) and connexin35 (Cx35). By iteratively exploiting protein mutagenesis, a novel in vitro assay of connexin docking, and computational modeling of connexin hemichannel interactions, we uncovered the pattern of structural motifs that broadly determine connexin hemichannel docking. We then utilized this knowledge to design Cx34.7 and Cx35 hemichannels that dock with each other, but not with themselves nor other major connexins expressed in the human central nervous system. We validated these hemichannels in vivo, demonstrating that they facilitate communication between two neurons in Caenorhabditis elegans and recode a learned behavioral preference. This system can be applied to edit circuits composed by pairs of genetically defined brain cell types across multiple species. Thus, we establish a potentially translational approach, Long-term integration of Circuits using connexins (LinCx), for context-precise circuit-editing with unprecedented spatiotemporal specificity.