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

## TL;DR

High activity of certain cell channels lets calcium in, causing cell damage and death through increased oxidative stress.

## Contribution

Identifies Cx45 as a new calcium-permeable hemichannel and links its activity to oxidative stress and cell death.

## Key findings

- Cx45 hemichannels allow calcium influx, leading to increased cytosolic calcium levels.
- Elevated hemichannel activity under alkaline conditions causes lipid peroxidation and reduced cell viability.
- Calcium influx through Cx43 and Cx45 hemichannels triggers oxidative stress and cell death.

## Abstract

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.