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

• Published in: Biochemical and biophysical research communications 2013-03, Vol.432 (4), p.707-712
• Main Authors: D’hondt, Catheleyne, Iyyathurai, Jegan, Wang, Nan, Gourdie, Robert G., Himpens, Bernard, Leybaert, Luc, Bultynck, Geert
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Animals; Asparagine - chemistry; Asparagine - genetics; ATP release; Biotin - chemistry; Ca2+-wave propagation; Calcium - metabolism; Cattle; Connexin; Connexin 43 - chemistry; Connexin 43 - genetics; HeLa Cells; Hemichannels; Humans; Peptides; Proline - chemistry; Proline - genetics; Protein Structure, Secondary; Protein Structure, Tertiary; Thrombin - chemistry
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2013.01.066

In 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. [Display omitted] ► 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. 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.