Metabolic inhibition increases activity of connexin-32 hemichannels permeable to Ca2+ in transfected HeLa cells.
Numerous cell types express functional connexin (Cx) hemichannels (HCs), and membrane depolarization and/or exposure to a divalent cation-free bathing solution (DCFS) have been shown to promote HC opening. However, little is known about conditions that can promote HC opening in the absence of strong depolarization and when extracellular divalent cation concentrations remain at physiological levels. Here the effects of metabolic inhibition (MI), an in vitro model of ischemia, on the activity of mouse Cx32 HCs were examined. In HeLa cells stably transfected with mouse Cx32 (HeLa-Cx32), MI induced an increase in cellular permeability to ethidium (Etd). The increase in Etd uptake was directly related to an increase in levels of Cx32 HCs present at the cell surface. Moreover, MI increased membrane currents in HeLa-Cx32 cells. Underlying these currents were channels exhibiting a unitary conductance of approximately 90 pS, consistent with Cx32 HCs. These currents and Etd uptake were blocked by HC inhibitors. The increase in Cx32 HC activity was preceded by a rapid reduction in mitochondrial membrane potential and a rise in free intracellular Ca(2+) concentration ([Ca(2+)](i)). The increase in free [Ca(2+)](i) was prevented by HC blockade or exposure to extracellular DCFS and was virtually absent in parental HeLa cells. Moreover, inhibition of Cx32 HCs expressed by HeLa cells in low-confluence cultures drastically reduced cell death induced by oxygen-glucose deprivation, which is a more physiological model of ischemia-reperfusion. Thus HC blockade could reduce the increase in free [Ca(2+)](i) and cell death induced by ischemia-like conditions in cells expressing Cx32 HCs.
Sánchez
Helmuth A
HA
Departamento de Ciencias Fisiológicas, Pontificia Universidad Católica de Chile, Alameda 340, Santiago, Chile.
Orellana
Juan A
JA
Verselis
Vytas K
VK
Sáez
Juan C
JC
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D010100:Oxygen / Q000378:metabolism*; D014162:Transfection; D000097002:Gap Junction beta-1 Protein
D002118:Calcium; D017630:Connexins; D010100:Oxygen; D000097002:Gap Junction beta-1 Protein