Mechanism Of Cell Toxicity Of Connexin31EKV-associated Mutant

Connexins form gap junctions between adjacent cells to mediate cell-cell communication by allowing small molecules less than1KD to pass through gap junctional channels. In addition, Connexins can also form hemichannels on plasma membrane to mediate exchanges of small molecules between cell and extracellular environment. In general, open probability of Connexin hemichannels is very low. However, Connexin hemichannels may open under certain conditions, for example, decreased extracellular calcium, membrane depolarization, oxidation or reduction. Some pathogenic mutations of Connexin result in excessive hemichannel opening, leading to cell death.Mutations in Connexin31gene (GJB3) were identified to cause hereditary diseases, including hearing loss, erythrokeratodermia variabilis (EKV) and peripheral neuropathy. Expression of EKV-associated Cx31mutations can cause ER stress and cell death. However, the mechanism of the cell death induced by these mutations remains largely unknown. Our results show that expression of EKV pathogenic mutant Cx31R42P induces cell death with necrotic characteristics. Inhibition of hemichannel activity by a Connexin hemichannel inhibitor or high extracellular calcium suppresses Cx31R42P-induced cell death. Expression of Cx31R42P induces ER stress resulting in reactive oxygen species (ROS) production, in turn, to regulate gating of Cx31R42P hemichannels and Cx31R42P induced cell death. Moreover, Cx31R42P hemichannels play an important role in mediating ATP release from the cell. In contrast, no hemichannel activity was detected with cells expressing wildtype Cx31. Together, the results suggest that Cx31R42P forms constitutively active hemichannels to promote necrotic cell death. The Cx31R42P active hemichannels are likely resulted by an ER stress mediated ROS overproduction.Moreover, we identified two small molecules that significantly rescue Cx31R42P-induced cell death, including WIN55,212-2and Rimonabant, using high throughput screening,. Both molecules inhibit Cx31R42P hemichannel activity. However, the mechanisms are likely distinct. The study opens a new avenue to develop effective treatment of Cx31diseases.