Mercè Cases, Jonatan Dorca-Arévalo, Marta Blanch, Sergi Rodil, Beatrice Terni, Mireia Martín-Satué, Artur Llobet, Juan Blasi, Carles Solsona
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Activation of calcium-activated chloride channels (CaCCs) and a decrease in the PM surface were recorded using the two-electrode voltage-clamp technique. To evaluate intracellular Ca<sup>2+</sup> levels and scramblase activity, fluorescent dyes were employed. Extracellular vesicles were imaged using light and electron microscopy, while toxin oligomers were identified through western blots. Etx triggered intracellular Ca<sup>2+</sup> mobilization in the Xenopus oocytes expressing hMAL, leading to the activation of CaCCs, ATP release, and a reduction in PM capacitance. The toxin induced the activation of scramblase and, thus, translocated phospholipids from the inner to the outer leaflet of the PM, exposing phosphatidylserine outside in Xenopus oocytes and in an Etx-sensitive cell line. Moreover, Etx caused the formation of extracellular vesicles, not derived from apoptotic bodies, through PM fission. These vesicles carried toxin heptamers and doughnut-like structures in the nanometer size range. In conclusion, ATP release was not directly attributed to the formation of pores in the PM, but to scramblase activity and the formation of extracellular vesicles.</p>","PeriodicalId":19948,"journal":{"name":"Pharmacology Research & Perspectives","volume":null,"pages":null},"PeriodicalIF":2.9000,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11423345/pdf/","citationCount":"0","resultStr":"{\"title\":\"The epsilon toxin from Clostridium perfringens stimulates calcium-activated chloride channels, generating extracellular vesicles in Xenopus oocytes.\",\"authors\":\"Mercè Cases, Jonatan Dorca-Arévalo, Marta Blanch, Sergi Rodil, Beatrice Terni, Mireia Martín-Satué, Artur Llobet, Juan Blasi, Carles Solsona\",\"doi\":\"10.1002/prp2.70005\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The epsilon toxin (Etx) from Clostridium perfringens has been identified as a potential trigger of multiple sclerosis, functioning as a pore-forming toxin that selectively targets cells expressing the plasma membrane (PM) myelin and lymphocyte protein (MAL). Previously, we observed that Etx induces the release of intracellular ATP in sensitive cell lines. Here, we aimed to re-examine the mechanism of action of the toxin and investigate the connection between pore formation and ATP release. We examined the impact of Etx on Xenopus laevis oocytes expressing human MAL. Extracellular ATP was assessed using the luciferin-luciferase reaction. Activation of calcium-activated chloride channels (CaCCs) and a decrease in the PM surface were recorded using the two-electrode voltage-clamp technique. To evaluate intracellular Ca<sup>2+</sup> levels and scramblase activity, fluorescent dyes were employed. Extracellular vesicles were imaged using light and electron microscopy, while toxin oligomers were identified through western blots. Etx triggered intracellular Ca<sup>2+</sup> mobilization in the Xenopus oocytes expressing hMAL, leading to the activation of CaCCs, ATP release, and a reduction in PM capacitance. 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引用次数: 0
摘要
来自产气荚膜梭菌的epsilon毒素(Etx)已被确定为多发性硬化症的潜在诱因,它是一种孔形成毒素,可选择性地靶向表达质膜(PM)髓鞘和淋巴细胞蛋白(MAL)的细胞。此前,我们观察到 Etx 可诱导敏感细胞株释放细胞内 ATP。在此,我们旨在重新研究该毒素的作用机制,并调查孔形成与 ATP 释放之间的联系。我们研究了 Etx 对表达人类 MAL 的爪蟾卵母细胞的影响。使用荧光素-荧光素酶反应评估了细胞外 ATP。使用双电极电压钳技术记录了钙激活氯通道(CaCCs)的激活和 PM 表面的下降。为了评估细胞内 Ca2+ 水平和加扰酶活性,使用了荧光染料。使用光镜和电子显微镜对细胞外囊泡进行成像,并通过 Western 印迹鉴定毒素寡聚体。在表达hMAL的爪蟾卵母细胞中,Etx引发细胞内Ca2+动员,导致CaCCs活化、ATP释放和PM电容降低。在爪蟾卵母细胞和对Etx敏感的细胞系中,毒素诱导了scramblase的活化,从而将磷脂从PM的内叶转移到外叶,使磷脂酰丝氨酸暴露在外面。此外,Etx 还能通过 PM 裂变形成细胞外囊泡,而这些囊泡并非来自凋亡体。这些囊泡携带毒素七聚体和纳米级的甜甜圈状结构。总之,ATP的释放并不直接归因于PM中孔隙的形成,而是归因于scramblase活性和细胞外囊泡的形成。
The epsilon toxin from Clostridium perfringens stimulates calcium-activated chloride channels, generating extracellular vesicles in Xenopus oocytes.
The epsilon toxin (Etx) from Clostridium perfringens has been identified as a potential trigger of multiple sclerosis, functioning as a pore-forming toxin that selectively targets cells expressing the plasma membrane (PM) myelin and lymphocyte protein (MAL). Previously, we observed that Etx induces the release of intracellular ATP in sensitive cell lines. Here, we aimed to re-examine the mechanism of action of the toxin and investigate the connection between pore formation and ATP release. We examined the impact of Etx on Xenopus laevis oocytes expressing human MAL. Extracellular ATP was assessed using the luciferin-luciferase reaction. Activation of calcium-activated chloride channels (CaCCs) and a decrease in the PM surface were recorded using the two-electrode voltage-clamp technique. To evaluate intracellular Ca2+ levels and scramblase activity, fluorescent dyes were employed. Extracellular vesicles were imaged using light and electron microscopy, while toxin oligomers were identified through western blots. Etx triggered intracellular Ca2+ mobilization in the Xenopus oocytes expressing hMAL, leading to the activation of CaCCs, ATP release, and a reduction in PM capacitance. The toxin induced the activation of scramblase and, thus, translocated phospholipids from the inner to the outer leaflet of the PM, exposing phosphatidylserine outside in Xenopus oocytes and in an Etx-sensitive cell line. Moreover, Etx caused the formation of extracellular vesicles, not derived from apoptotic bodies, through PM fission. These vesicles carried toxin heptamers and doughnut-like structures in the nanometer size range. In conclusion, ATP release was not directly attributed to the formation of pores in the PM, but to scramblase activity and the formation of extracellular vesicles.
期刊介绍:
PR&P is jointly published by the American Society for Pharmacology and Experimental Therapeutics (ASPET), the British Pharmacological Society (BPS), and Wiley. PR&P is a bi-monthly open access journal that publishes a range of article types, including: target validation (preclinical papers that show a hypothesis is incorrect or papers on drugs that have failed in early clinical development); drug discovery reviews (strategy, hypotheses, and data resulting in a successful therapeutic drug); frontiers in translational medicine (drug and target validation for an unmet therapeutic need); pharmacological hypotheses (reviews that are oriented to inform a novel hypothesis); and replication studies (work that refutes key findings [failed replication] and work that validates key findings). PR&P publishes papers submitted directly to the journal and those referred from the journals of ASPET and the BPS