Characterizing the mode of action of extracellular Connexin43 channel blocking mimetic peptides in an in vitro ischemia injury model.
Non-selective Connexin43 hemichannels contribute to secondary lesion spread. The hemichannel blocking peptidomimetic Peptide5, derived from the second extracellular loop of the human Connexin43 protein, prevents lesion spread and reduces vascular permeability in preclinical models of central nervous system injury. The molecular mode of action of Peptide5, however, was unknown and is described here.



Human cerebral microvascular endothelial cells and APRE-19 cells were used. Scrape loading was used to assess gap junction function and hypoxic, acidic ion-shifted Ringer solution induced ATP release used to assess hemichannel function. Peptide modifications, including amino acid substitutions and truncations, and competition assays were used to demonstrate Peptide5 functional specificity and site of action respectively.



Peptide5 inhibits Connexin43 hemichannel-mediated ATP release by acting on extracellular loop two of Connexin43, adjacent to its matching sequence within the protein. Precise sequence specificity is important for hemichannel block, but less so for uncoupling of gap junction channels (seen only at high concentrations). The SRPTEKT motif is central to Peptide5 function but on its own is not sufficient to inhibit hemichannels. Both the SRPTEKT motif and Peptide5 reduce gap junction communication, but neither uncoupling below 50%.



Reduced gap junction coupling at high peptide concentrations appears to be relatively non-specific. However, Peptide5 at low concentrations acts upon extracellular loop two of Connexin43 to block hemichannels in a precise, sequence specific manner.



The concentration dependent and sequence specific action of Peptide5 supports its development for the treatment of retinal injury and chronic disease, as well as other central nervous system injury and disease conditions.
Department of Ophthalmology, New Zealand National Eye Centre, University of Auckland, Private Bag 92019, Auckland, New Zealand.
Yeri
Y
Kim
Centre for Brain Research, Department of Anatomy Medical Imaging, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.
Jarred M
JM
Griffin
School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Private Bag 92019, Auckland, New Zealand; School of Biological Sciences, New Zealand.
Paul W R
PW
Harris
School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.
Sin Hang Crystal
SH
Chan
Centre for Brain Research, Department of Anatomy Medical Imaging, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Private Bag 92019, Auckland, New Zealand.
Louise F B
LF
Nicholson
School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Private Bag 92019, Auckland, New Zealand; School of Biological Sciences, New Zealand.
Margaret A
MA
Brimble
Centre for Brain Research, Department of Anatomy Medical Imaging, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.
Simon J
SJ
O'Carroll
Department of Ophthalmology, New Zealand National Eye Centre, University of Auckland, Private Bag 92019, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Private Bag 92019, Auckland, New Zealand. Electronic address: c.green@auckland.ac.nz.
Colin R
CR
Green
D000255:Adenosine Triphosphate / Q000378:metabolism; D002460:Cell Line; D002493:Central Nervous System Diseases / Q000188:drug therapy / Q000378:metabolism; D054022:Cerebrum / Q000187:drug effects* / Q000378:metabolism; D018031:Connexin 43 / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007473:Ion Channels / Q000378:metabolism; D007511:Ischemia / Q000188:drug therapy* / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology*; D015427:Reperfusion Injury / Q000188:drug therapy* / Q000378:metabolism; D012164:Retinal Diseases / Q000188:drug therapy / Q000378:metabolism
D018031:Connexin 43; D007473:Ion Channels; D010455:Peptides; D000255:Adenosine Triphosphate
Connexin43; Gap junctions; Hemichannel; Ischemia; Peptide5; Peptidomimetics