Molecular determinants of low affinity complexes formed by the electrical synapse proteins Connexin 36 and ZO-1

Although chemical and electrical synapses function fundamentally differently, they evidently share common design principles. Like neurotransmitter receptors, Connexin 36 (Cx36) containing gap junction channels, key constituents of electrical synapses, are anchored to scaffolding proteins that stabilize the connexin at the synapse. One of the most prominent proteins that has been described in this context is the Zonula occludens protein 1 (ZO-1). ZO-1 interacts with Cx36 via one of its three PDZ domains. This interaction is inherently weak and was suggested to facilitate the dynamic regulation of electrical synapses. In the present study, we have combined Gaussian accelerated molecular dynamics simulations and binding assays to identify the exact residues in the PDZ binding motif of Cx36 that are necessary to sustain these low affinity interactions. Among the different Cx36 mutations we have generated, we discovered a single substitution at position 319 within the PDZ binding motif that massively increases binding in different experimental settings. In addition to this site, we found that acidic residues adjacent to the PDZ binding motif (PBM) in Cx36 and its fish orthologues are evolutionarily tuned to weaken PDZ interactions as well. We were able to enhance PDZ1 binding drastically by substituting these residues with hydrophobic or positively charged amino acids. Finally, we demonstrate that the weak PDZ1/Cx36 interaction is sensitive to CaMKII mediated phosphorylation of Cx36, suggesting that ZO-1 unbinding may be a necessary event to potentiate electrical synapses. In summary, our study provides a detailed analysis of different mechanisms that can be exploited to modify the interaction between two key components of an electrical synapse: Cx36 and ZO-1.