Abstract
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 Ca2+. 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 Ca2+ concentration in all three species. These currents appeared after 5–6 minutes and were blocked by CaCCinh-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.
Highlights
Procoagulant activity resulting from exposure of anionic membrane phospholipids is critical for thrombosis and haemostasis
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
We compared the ionic selectivity of the Ca2+-activated conductances of primary megakaryocytes from rat and mouse, and a human megakaryocyte cell line, under conditions previously used to characterise TMEM16F
Summary
Procoagulant activity resulting from exposure of anionic membrane phospholipids is critical for thrombosis and haemostasis. The underlying mechanisms of this lipid redistribution process are incompletely understood, a Ca2+-activated phospholipid scramblase is known to be important [1,2,3]. TMEM16F is a ubiquitously expressed Ca2+-dependent ion channel and phospholipid scramblase [2,4]. Missense mutations of the TMEM16F gene occur in patients with Scott syndrome, a rare bleeding diathesis characterised by defective Ca2+-dependent phospholipid scrambling [5,6,7]. This disease is phenocopied in TMEM16F−/− mice [3,8].
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