Department of Molecular and Cell Biology, University of Leicester , Leicester , UK.
National Heart and Lung Institute, Cardio-respiratory Section, Imperial College London , London , UK.
Platelets. 2019;30(8):962-966. doi: 10.1080/09537104.2019.1595560. Epub 2019 Apr 22.
TMEM16F is a surface membrane protein critical for platelet procoagulant activity, which exhibits both phospholipid scramblase and ion channel activities following sustained elevation of cytosolic Ca. The extent to which the ionic permeability of TMEM16F is important for platelet scramblase responses remains controversial. To date, only one study has reported the electrophysiological properties of TMEM16F in cells of platelet/megakaryocyte lineage, which observed cation-selectivity within excised patch recordings from murine marrow-derived megakaryocytes. This contrasts with reports using whole-cell recordings that describe this channel as displaying either selectivity for anions or being relatively non-selective amongst the major physiological monovalent ions. We have studied TMEM16F expression and channel activity in primary rat and mouse megakaryocytes and the human erythroleukemic (HEL) cell line that exhibits megakaryocytic surface markers. Immunocytochemical analysis was consistent with surface TMEM16F expression in cells from all three species. Whole-cell recordings in the absence of K-selective currents revealed an outwardly rectifying conductance activated by a high intracellular Ca concentration in all three species. These currents appeared after 5-6 minutes and were blocked by CaCC-A01, properties typical of TMEM16F. Ion substitution experiments showed that the underlying conductance was predominantly Cl-permeable in rat megakaryocytes and HEL cells, yet non-selective between monovalent anions and cations in mouse megakaryocytes. In conclusion, the present study further highlights the difference in ionic selectivity of TMEM16F in platelet lineage cells of the mouse compared to other mammalian species. This provides additional support for the ionic "leak" hypothesis that the scramblase activity of TMEM16F does not rely upon its ability to conduct ions of a specific type.
TMEM16F 是一种表面膜蛋白，对血小板促凝活性至关重要，在细胞浆 Ca 持续升高后，它表现出磷脂翻转酶和离子通道活性。TMEM16F 的离子通透性对血小板翻转酶反应的重要程度仍存在争议。迄今为止，只有一项研究报告了 TMEM16F 在血小板/巨核细胞谱系细胞中的电生理特性，该研究观察到从鼠骨髓衍生的巨核细胞的分离膜片记录中的阳离子选择性。这与使用全细胞记录的报告形成对比，后者描述该通道对阴离子具有选择性，或者对主要生理单价离子具有相对非选择性。我们研究了原代大鼠和小鼠巨核细胞以及表达巨核细胞表面标志物的人红白血病（HEL）细胞系中 TMEM16F 的表达和通道活性。免疫细胞化学分析与来自所有三种物种的细胞的表面 TMEM16F 表达一致。在不存在 K 选择性电流的情况下进行全细胞记录，揭示了所有三种物种的高细胞内 Ca 浓度激活的外向整流电导。这些电流在 5-6 分钟后出现，并被 CaCC-A01 阻断，这是 TMEM16F 的典型特性。离子取代实验表明，在大鼠巨核细胞和 HEL 细胞中，基础电导主要是 Cl 可渗透的，但在小鼠巨核细胞中，对单价阴离子和阳离子没有选择性。总之，本研究进一步强调了 TMEM16F 在与其他哺乳动物物种相比，在小鼠血小板谱系细胞中离子选择性的差异。这为 TMEM16F 的翻转酶活性不依赖于其对特定类型离子的传导能力的离子“泄漏”假说提供了额外的支持。
