Ca²⁺ permeation through C-terminal cleaved, but not full-length human Pannexin1 hemichannels, mediates cell death

Pannexin1 hemichannels (Panx1 HCs) are found in the membrane of most mammalian cells and communicate the intracellular and extracellular spaces, enabling the passive transfer of ions and small molecules. They are involved in physiological and pathophysiological conditions. During apoptosis, the C-terminal tail of Panx1 is proteolytically cleaved, but the permeability features of hemichannels and their role in cell death remain elusive. To address these topics, HeLa cells transfected with full-length human Panx1 (fl-hPanx1) or C-terminal truncated hPanx1 (Δ371hPanx1) were exposed to alkaline extracellular saline solution, increasing the activity of Panx1 HCs. The Δ371hPanx1 HC was permeable to DAPI and Etd⁺, but not to propidium iodide, whereas fl-hPanx1 HC was only permeable to DAPI. Furthermore, the cytoplasmic Ca²⁺ signal increased only in Δ371hPanx1 cells, which was supported by bioinformatics approaches. The influx of Ca²⁺ through Δ371hPanx1 HCs was necessary to promote cell death up to about 95% of cells, whereas the exposure to alkaline saline solution without Ca²⁺ failed to induce cell death, and the Ca²⁺ ionophore A23187 promoted more than 80% cell death even in fl-hPanx1 transfectants. Moreover, cell death was prevented with carbenoxolone or ¹⁰Panx1 in Δ371hPanx1 cells, whereas it was undetectable in HeLa Panx1^-/- cells. Pretreatment with Ferrostatin-1 and necrostatin-1 did not prevent cell death, suggesting that ferroptosis or necroptosis was not involved. In comparison, zVAD-FMK, a pancaspase inhibitor, reduced death by ~60%, suggesting the involvement of apoptosis. Therefore, alkaline pH increases the activity of Δ371hPanx1HCs, leading to a critical intracellular free-Ca²⁺ overload that promotes cell death.