Proceedings of The Physiological Society

Kings College London (2005) J Physiol 565P, PC161


On the mechanism that opens Connexin 43 hemichannels in metabolically inhibited rat astrocytes.

Retamal, Mauricio A.; Cortes, Constanza J.; Bukauskas, Feliksas F.; Bennett, Michael V.L.; Saez, Juan C.;

1. Physiology, P. Universidad Catolica de Chile, Santiago, Chile. 2. Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA.

Gap junction hemichannels (HCs) are hexamers of protein subunits named connexins (Cxs). Unapposed HCs can open to connect intra- and extra-cellular space and allow permeation by ions and small molecules. In astrocytes subject to metabolic inhibition (MI) increased opening of connexin43 HCs (Cx43-HCs) contributes to cell death (Contreras et al, 2002). Desphosphorylation and/or oxidation of Cx43-HCs have been proposed as gating mechanisms that open Cx43-HCs during MI or ischemia. Our objectives were to determine the effects of MI on the phosphorylation state and function of Cx43-HCs and to study the effect of reducing agents on Cx43-HCs during MI. Primary cultures of cortical astrocytes obtained from humanely killed neonatal rats were MI with 100 μM iodoacetic acid and 5 ng/ml antimycin A for different periods up to 75 min at 37°C. Membrane proteins of the extracellular surface were labeled with sulfo-NHS-SS-biotin and isolated with neutravidin beads. The phosphorylation state of Cx43 was analyzed by immunoblot determination of mobility using an antibody to the C-terminal. Cx43 at the cell surface of unpermeabilized cells were visualized by immunofluorescence using an antibody to the first extracellular loop. Opening of Cx43-HCs was evaluated through uptake of ethidium bromide (100 μM) in cells exposed to the dye for 5 min. Under basal conditions, 8.8±2.6‰ (mean±SD, n=4) of total Cx43 was at the cell surface and was mainly phosphorylated. After 15 min MI, surface Cx43 was 1.8±0.5 (mean±SD, n=4) times that in control cells and remained high for at least 60 min. In agreement, surface Cx43 immunoreactivity was greatly increased by MI. After 15-30 min MI levels of unphosphorylated Cx43 increased at least up to 75 min. Dye uptake after 50 min MI was decreased by 10 mM DTT or 100 μM trolox added 30 min before the uptake assay. DTT did not prevent the increase in unphosphorylated Cx43 levels after MI. We propose that the massive Cx43-HC opening observed in metabolic inhibited astrocytes results from a combination of HC recruitment to the cell surface and oxidation, presumably of Cx43.

Where applicable, experiments conform with Society ethical requirements