High Activity of Hemichannels Permeable to Calcium Ions Leads to ROS Generation and Reduced Cell Viability

Connexins (Cxs) and pannexin1 (Panx1) form hemichannels (HCs) that enable the exchange of ions and small molecules between the intracellular and extracellular compartments. Since an elevated cytoplasmic Ca²⁺ concentration promotes cell death and elevated HC activity has been implicated in pathological conditions, we investigated whether high HC activity contributes to Ca²⁺ influx and cell death. HeLa parental cells and HeLa cells expressing Cx39, Cx43, Cx45, or Panx1 were exposed to an alkaline extracellular solution (pH 8.5) to increase HC activity. Under these conditions, dye uptake assays revealed high HC activity in all transfected cells but not in parental control cells. Previous studies have shown that Cx43 HCs, but not Cx39 and Panx1 HCs, allow the influx of extracellular Ca²⁺. Here, we also found that exposure of Cx45 transfectants to pH 8.5 activated HCs and allowed the influx of extracellular Ca²⁺. Only in cells expressing functional HCs permeable to Ca²⁺ did the elevated HC activity heighten the cytosolic Ca²⁺ concentration, which promoted lipid peroxidation and reduced cell viability. The effects were also abolished by the removal of extracellular divalent cations, suggesting that a Ca²⁺ influx that triggers downstream deleterious effects is required. Our findings identify Cx45 as a novel Ca²⁺-permeable HC, and they reveal that alkaline stress promotes Ca²⁺ entry via Cx43 and Cx45 HCs, which in turn leads to oxidative stress and cell death.