Development of a nanobody tool kit to study connexin channels

There are 21 different connexin isoforms in human that play crucial roles in intercellular communication during development and physiology. At molecular level, six connexin proteins assemble as hexamers to form an hemichannel that is inserted in the plasma membrane. The docking of two hemichannels from adjacent cells form a gap junction channel (GJC); both hemichannels and GJCs are permeable to ions and small metabolites (<1 kDa). Several human connexin mutations and multiple pathological conditions exacerbate the activity of undocked hemichannel causing cellular dysfunction. A major limitation in the field is the lack of adequate pharmacological and genetic tools to dissect out the role of undocked connexin hemichannels versus their functions as GJCs. The objective of this proposal is to create recombinant nanobodies targeting Cx43 protein that differentially recognize hemichannels from GJCs to overcome this technical barrier. These nanobodies have reduced size (15 kDa) allowing higher tissue penetration, reduced immunogenicity and easy genetic manipulation and engineering. To achieve this goal, we have selected peptides corresponding to extracellular regions, N-terminal helix and intracellular loop of Cx43 protomer for llama immunization. To identify and generate recombinant Cx43 nanobodies present in this serum, we will use the immunized llama’s B-cells as source of nanobodies-coding RNA to produce a nanobodies-phage display library. The selected nanobodies will be screening to recognize Cx43 via ELISA, Western blot and immunofluorescence. In addition, we will assess the nanobodies capabilities to block Cx43 hemichannel activity in vitro and their wide applicability using heterologous expression system and disease models. Considering the compelling evidence for a pathological role of Cx43 hemichannels in various diseases and our willingness to share the resulting antibodies with the whole scientific community, the present project is highly innovative and significant.