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

Connexins (Cxs) and pannexin1 (Panx1) form hemichannels (HCs) that allow the ex-change of ions and small molecules between the intracellular and extracellular com-partments. Since elevated cytoplasmic Ca2+ concentration promotes cell death and el-evated 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 influx of extracellular Ca2+. Here, we also found that exposure of Cx45 transfectants to pH 8.5 activated HCs and allowed influx of extracellular Ca2+. Only in cells expressing functional HCs permeable to Ca2+, the elevated HC activity heightened the cytosolic Ca²⁺ concentration, which promoted lipid peroxidation and reduced cell viability. The effects were also abolished by removal of extracellular divalent cations, suggesting the requirement of Ca²⁺ influx that triggers downstream deleterious effects. 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.