Regulation of connexin43 protein complexes by intracellular acidification.
Ischemia-induced acidification of astrocytes or cardiac myocytes reduces intercellular communication by closing gap junction channels and subsequently internalizing gap junction proteins. To determine whether such coupling changes might be attributable to altered interactions between connexin43 (Cx43) and other proteins, we applied the nigericin/high K+ method to vary intracellular pH (pHi) in cultured cortical astrocytes. Intracellular acidification was accompanied by internalization of Cx43 with retention of Cx43 scaffolding protein Zonula Occludens-1 (ZO-1) at cell surfaces, suggesting that ZO-1 and Cx43 dissociate at low pHi. Coimmunoprecipitation studies revealed decreased binding of ZO-1 and increased binding of c-Src to Cx43 at low pHi. Resonant mirror spectroscopy was used to quantify binding of the SH3 domain of c-Src and the PDZ domains of ZO-1 to the carboxyl terminal domain of Cx43 (Cx43CT). Data indicate that the c-Src/Cx43CT interaction is highly pH dependent whereas the ZO-1/Cx43CT interaction is not. Moreover, binding of c-Src to Cx43CT prevented and reversed ZO-1/Cx43CT binding. We hypothesize that increased affinity of c-Src for Cx43 at low pHi aids in separation of Cx43 from ZO-1 and that this may facilitate internalization of Cx43. These data suggest that protracted acidification may remodel protein-protein interactions involving Cx43 and thus provide an important protective mechanism to limit lesion spread after ischemic injury.
Duffy
Heather S
HS
Department of Neuroscience, Albert Einstein College of Medicine, 1410 Pelham Pkwy S, Bronx, NY 10464, USA. hduffy@aecom.yu.edu
Ashton
Anthony W
AW
O'Donnell
Phyllis
P
Coombs
Wanda
W
Taffet
Steve M
SM
Delmar
Mario
M
Spray
David C
DC
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D018031:Connexin 43 / Q000737:chemistry / Q000378:metabolism*; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000737:chemistry; D007511:Ischemia / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009550:Nigericin / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D062826:Zonula Occludens-1 Protein; D018909:src Homology Domains
D018031:Connexin 43; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C569043:Tjp1 protein, mouse; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; D016392:Proto-Oncogene Proteins pp60(c-src); D009550:Nigericin; D011188:Potassium