Negatively charged residues (Asp378 and Asp379) in the last ten amino acids of the C-terminal tail of Cx43 hemichannels are essential for loop/tail interactions.

Connexin 43 (Cx43)-hemichannel activity is controlled by intramolecular interactions between cytoplasmic loop and C-terminal tail. We previously identified the last 10 amino acids of the C-terminal tail of Cx43 as essential for Cx43-hemichannel activity. We developed a cell-permeable peptide covering this sequence (TAT-Cx43CT). In this study, we examined the critical molecular determinants in TAT-Cx43CT to restore Cx43-hemichannel activity. Using amino acid substitutions in TAT-Cx43CT, we identified the two aspartate (Asp378 and Asp379) and two proline (Pro375 and Pro377) residues as critical for TAT-Cx43CT activity, since TAT-Cx43CTDD/AA and TAT-Cx43CTPP/GG did not overcome the inhibition of Cx43-hemichannel activity induced by thrombin, micromolar cytoplasmic Ca2+ concentration or truncation of Cx43 at M239. Consistent with this, we found that biotin-Cx43CTDD/AA was much less efficient than biotin-Cx43CT to bind the purified CL domain of Cx43 in surface plasmon resonance experiments. In conclusion, we postulate that Asp378 and Asp379 in the C-terminal part of Cx43 are essential for loop/tail interactions in Cx43 hemichannels, while Pro375 and Pro377 may help to properly coordinate the critical Asp residues.