Intramolecular loop/tail interactions are essential for connexin 43-hemichannel activity.
Connexin-assembled gap junctions (GJs) and hemichannels coordinate intercellular signaling processes. Although the regulation of connexins in GJs has been well characterized, the molecular determinants controlling connexin-hemichannel activity are unresolved. Here we investigated the regulation of Cx43-hemichannel activity by actomyosin contractility and intracellular [Ca(2+)] ([Ca(2+)](i)) using plasma membrane-permeable TAT peptides (100 μM) designed to interfere with interactions between the cytoplasmic loop (CL) and carboxy-terminal (CT) in primary bovine corneal endothelial cells and HeLa, C6 glioma, and Xenopus oocytes ectopically expressing Cx43. Peptides corresponding to the last 10 CT aa (TAT-Cx43CT) prevented the inhibition of Cx43-hemichannel activity by contractility/high [Ca(2+)](i), whereas a reverse peptide (TAT-Cx43CTrev) did not. These effects were independent of zonula occludens-1, a cytoskeletal-associated Cx43-binding protein. In contrast, peptides corresponding to CL (TAT-L2) inhibited Cx43-hemichannel responses, whereas a mutant peptide (TAT-L2(H126K/I130N)) did not inhibit. In these assays, TAT-Cx43CT acted as a scaffold for TAT-L2 and vice versa, a finding supported by surface plasmon resonance measurements. Loop/tail interactions appeared essential for Cx43-hemichannel activity, because TAT-Cx43CT restored the activity of nonfunctional hemichannels, consisting of either Cx43 lacking the C-terminal tail (Cx43(M239)) or intact Cx43 ectopically expressed in Xenopus oocytes. We conclude that intramolecular loop/tail interactions control Cx43-hemichannel activity, laying the basis for developing hemichannel-specific blockers.
Ponsaerts
Raf
R
Department Molecular Cell Biology, KU Leuven, Leuven, Belgium.
De Vuyst
Elke
E
Retamal
Mauricio
M
D'hondt
Catheleyne
C
Vermeire
Dieter
D
Wang
Nan
N
De Smedt
Humbert
H
Zimmermann
Pascale
P
Himpens
Bernard
B
Vereecke
Johan
J
Leybaert
Luc
L
Bultynck
Geert
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D045744:Cell Line, Tumor; D018031:Connexin 43 / Q000378:metabolism*; D003315:Cornea / Q000166:cytology / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism; D015696:Gene Products, tat / Q000378:metabolism; D006367:HeLa Cells; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D013917:Thrombin / Q000378:metabolism; D014982:Xenopus laevis / Q000378:metabolism
D018031:Connexin 43; D015696:Gene Products, tat; D006576:Heterocyclic Compounds, 4 or More Rings; D007473:Ion Channels; C472645:blebbistatin; D000255:Adenosine Triphosphate; D013917:Thrombin; D002118:Calcium