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

• 成果类型: Article
• 署名作者: Salgado, Magdiel; Marquez-Miranda, Valeria; Ferrada, Luciano; Rojas, Maximiliano; Poblete-Flores, Gonzalo; Gonzalez-Nilo, Fernando D.; Ardiles, Alvaro O.; Saez, Juan C.
• 署名单位: Universidad de Valparaiso; Universidad Andres Bello; Universidad de Concepcion; Universidad de Valparaiso
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-13258
• DOI: 10.1073/pnas.2405468121
• 发表日期: 2024-06-18
• 关键词: 1 channels; cortical astrocytes; molecular-dynamics; nitric-oxide; calcium; atp; activation; permeability; release; amber

摘要

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 (Delta 371hPanx1) were exposed to alkaline extracellular saline solution, increasing the activity of Panx1 HCs. The Delta 371hPanx1 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 Delta 371hPanx1 cells, which was supported by bioinformatics approaches. The influx of Ca2+ through Delta 371hPanx1 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 (10)Panx1 in Delta 371hPanx1 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 pan-caspase inhibitor, reduced death by similar to 60%, suggesting the involvement of apoptosis. Therefore, alkaline pH increases the activity of Delta 371hPanx1HCs, leading to a critical intracellular free-Ca2+ overload that promotes cell death.