A major interspecies difference in the ionic selectivity of megakaryocyte Ca2+-activated channels sensitive to the TMEM16F inhibitor CaCCinh-A01

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 CaCC_inh-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.

Keywords: Calcium; Scott syndrome; TMEM16F; megakaryocyte; phospholipid scrambling; platelet.

Figure 1.

Detection of Ca²⁺-activated and A01-sensitive TMEM16F-like currents in HEL cells and rat and mouse MKs. A) TMEM16F expression by permeabilised HEL cells (left), primary rat (centre) and mouse (right) MKs assessed by immunocytochemistry with a primary antibody raised against an intracellular epitope of TMEM16F. Strong fluorescence was observed at the periphery of cells treated with primary (αTMEM16F) and secondary (AF647) antibodies, whilst no fluorescence was detected in secondary antibody only controls. B-D) whole-cell patch clamp recordings of Ca²⁺-activated currents from HEL cells (B), rat (C) and mouse (D) MKs. Intracellular and bath solutions contained 150mM NaCl and were K⁺-free. [Ca²⁺]_i was set at either ≈5nM (1mM EGTA) or 100µM as indicated. After 10 minutes in the whole-cell mode, currents were recorded in response to voltage steps of 1s duration (−120 to +120mV, 20mV increments) in the presence of vehicle control (0.04% DMSO) or the TMEM16F antagonist CaCCinh-A01 (A01; 20µM). Representative traces are shown for control or A01-treated MKs in the presence of intracellular EGTA or 100µM [Ca²⁺]_i. Summary current density-voltage relationship data are shown in the right hand panels for EGTA-subtracted currents under control (solid line) or A01-treated (dashed line) conditions. For immunocytochemistry experiments, scale bars represent 10µm. Data are representative of a minimum of three independent experiments for each condition. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively.

Figure 2.

Comparison of ionic selectivity between human, rat and mouse megakaryocytic Ca²⁺-activated, A01 sensitive TMEM16F-like channels. Whole-cell patch clamp recordings of Ca²⁺-activated currents from HEL cells (left), rat (centre) and mouse (right) MKs under conditions designed to isolate TMEM16F channel activity. Intracellular solutions were K⁺-free (150mM NaCl), and membrane currents were assessed after 10 mins in the whole cell configuration with [Ca²⁺]_i set at either ≈5nM (1mM EGTA, not shown) or 100µM. Currents were recorded in response to voltage steps of 1s duration (−120 to +120mV, 20mV increments) in the presence of vehicle control (0.04% DMSO) or the TMEM16F antagonist CaCCinh-A01 (A01; 20µM). For experiments in panels A-B extracellular Cl⁻ was substituted by equimolar gluconate⁻ and in panels C-D, extracellular Na⁺ and Cl⁻ were substituted by NMDG⁺ and gluconate, respectively. B,D) Summary current density-voltage relationship data for Ca²⁺-activated currents under vehicle control (solid line) or A01-treated (dashed line) conditions. Data are representative of a minimum of three independent experiments for each condition. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively.