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.

Graphical abstractIn this study, we have identified Asp378 & Asp379 and Pro375 & Pro377 as critical residues within the last amino acids of the C-terminal tail of Cx43, provided as a TAT-fused peptide, to restore the activity of non-functional C-terminally truncated Cx43M239-based hemichannels. Arg374 and Arg376 were dispensable for TAT-Cx43CT activity. Similar results were obtained using full-length Cx43 hemichannels under conditions that inhibit Cx43-hemichannel opening due to loss of endogenous loop/tail interactions. Our functional observations were underpinned by surface plasmon resonance measurements, showing that Asp378 & Asp379 are essential for binding the cytoplasmic loop domain.Figure optionsDownload full-size imageDownload as PowerPoint slideHighlights► Intramolecular loop/tail interactions are essential for Cx43-hemichannel activity. ► The last 10 aa in the C-terminal tail of Cx43 (Cx43CT) are critical for this. ► We determined the aa in Cx43CT critical for loop binding in Cx43 hemichannels. ► Two Asp residues in Cx43CT mediate loop binding in Cx43 hemichannels. ► Two Pro residues in Cx43CT may help in the proper coordination of the Asp residues.