Controllable gap junctions by vitamin B ₁₂ and light

Gap junctions mediate rapid signal transduction between contiguous cells, which are indispensable for multicellular organisms to coordinate cellular activities across numerous physiological processes. However, precise control of gap junctions remains elusive. Herein, we present CarGAP, a single-component chemo-optogenetic tool that utilizes the C-terminal adenosylcobalamin (AdoB₁₂) binding domain of a photoreceptor protein (i.e., CarH_C) to achieve reversible control over both vertebrate and invertebrate gap junctions with spatiotemporal precision. The vertebrate CarGAP (i.e., Cx-CarGAP), created by genetically fusing connexins with CarH_C in mammalian cells, can efficiently block the gap junction channels through AdoB₁₂-induced protein oligomerization and subsequently reinstate them via green light-induced protein disassembly. We further introduced the CarGAP system (i.e., Inx-CarGAP) to the <i>Drosophila</i> ovary, enabling reversible control over the heterotypic gap junctions formed by innexin2 (Inx2) and innexin4 (Inx4, also known as zero population growth, Zpg), thereby uncovering the roles of gap junctions in stem cell-niche interactions. This study illustrates CarGAP as a generalizable chemo-optogenetic tool for interrogating the functions of gap junctions in various biological contexts.