Long-term editing of brain circuits using an engineered electrical synapse

Design of the Cx34.7 and Cx35 mutant library A semi-rational design approach was used to design the mutant library. Sequence alignments between the M. americana connexins and the connexins for which the most structure–function data existed (Cx26, Cx32, Cx36, Cx40 and Cx43) were performed in ClustalW. Sites identified by previous studies as conferring specificity for docking were used, as well as those identified by homology modelling from the structures of Cx26 (ref. 56 ). Specifically, we primarily focused on residues in the extracellular loops: four residues at the interface in EL2, KEVE/KDVE ( M. americana Cx34.7 and Cx35), and one residue in EL1. The homologous residues in other connexins had been demonstrated to be highly tolerant to mutations and critical for docking specificity 57 . Mutations were modelled in Swiss PDB Viewer using homology models of Cx34.7 and Cx35 from a Cx26 and Cx32 interface structure so as not to create mutations with obvious steric hindrance. A wide range of substitutions were made for these five residues of interest, including those intended to introduce compatible electrostatic interactions, as well as less likely candidates. Mutations were also created that targeted other residues nearby and/or adjacent to these five for which there was some published evidence that they contributed to docking specificity. …