The C-terminus of connexin43 adopts different conformations in the Golgi and gap junction as detected with structure-specific antibodies

The C-terminus of the most abundant and best-studied gap-junction protein, connexin43, contains multiple phosphorylation sites and protein-binding domains that are involved in regulation of connexin trafficking and channel gating. It is well-documented that SDS/PAGE of NRK (normal rat kidney) cell lysates reveals at least three connexin43-specific bands (PO, PI and P2). P1 and P2 are phosphorylated on multiple, unidentified serine residues and are found primarily in gap-junction plaques. In the present study we prepared monoclonal antibodies against a peptide representing the last 23 residues at the C-terminus of connexin43. Immunofluorescence studies showed that one antibody (designated CT1) bound primarily to connexin43 present in the Golgi apparatus, whereas the other antibody (designated IF1) labelled predominately connexin43 present in gap-junctions. CT1 immunoprecipitates predominantly the PO form whereas IF1 recognized all three bands. Peptide mapping, mutational analysis and protein-protein interaction experiments revealed that unphosphorylated Ser(364) and/or Ser(365) are critical for CT1 binding. The IF1 paratope binds to residues Pro(375)-Asp(379) and requires Pro(375) and Pro(377). These proline residues are also necessary for ZO-1 interaction. These studies indicate that the conformation of Ser(364)/Ser(365) is important for intracellular localization, whereas the tertiary structure of Pro(375)-ASp(379) is essential in targeting and regulation of gap junctional connexin43.