Calcium-dependent phospholipid scrambling by TMEM16F

TLDR: It is shown that TMEM16F (transmembrane protein 16F) is an essential component for the Ca2+-dependent exposure of PtdSer on the cell surface, which results from a defect in phospholipid scrambling activity and is found to carry a mutation at a splice-acceptor site of the gene encoding TMEM 16F, causing the premature termination of the protein.

Abstract: In all animal cells, phospholipids are asymmetrically distributed between the outer and inner leaflets of the plasma membrane. This asymmetrical phospholipid distribution is disrupted in various biological systems. For example, when blood platelets are activated, they expose phosphatidylserine (PtdSer) to trigger the clotting system. The PtdSer exposure is believed to be mediated by Ca(2+)-dependent phospholipid scramblases that transport phospholipids bidirectionally, but its molecular mechanism is still unknown. Here we show that TMEM16F (transmembrane protein 16F) is an essential component for the Ca(2+)-dependent exposure of PtdSer on the cell surface. When a mouse B-cell line, Ba/F3, was treated with a Ca(2+) ionophore under low-Ca(2+) conditions, it reversibly exposed PtdSer. Using this property, we established a Ba/F3 subline that strongly exposed PtdSer by repetitive fluorescence-activated cell sorting. A complementary DNA library was constructed from the subline, and a cDNA that caused Ba/F3 to expose PtdSer spontaneously was identified by expression cloning. The cDNA encoded a constitutively active mutant of TMEM16F, a protein with eight transmembrane segments. Wild-type TMEM16F was localized on the plasma membrane and conferred Ca(2+)-dependent scrambling of phospholipids. A patient with Scott syndrome, which results from a defect in phospholipid scrambling activity, was found to carry a mutation at a splice-acceptor site of the gene encoding TMEM16F, causing the premature termination of the protein.

Figures

Figure 3. Effect of TMEM16F on the exposure of phosphatidylethanolamine. a, Ba/F3 cells transformed with vector-, wild-type-, or D409G-mutant TMEM16F were incubated with biotin-labeled Ro09-0198 with or without BAPTA-AM pretreatment, followed by staining with APC-streptavidin. b, Ba/F3 cells transformed with vector or the wild-type TMEM16F were preincubated with biotin-Ro09-0198 and APC-streptavidin. A23187 was added, and the fluorescence was monitored by flow cytometry. The y-axis is fluorescence intensity observed by FACS analysis, and shown in arbitrary units.

Figure 2. No effect of TMEM16F on the Ca2+ influx. a, The vector-, wild-type-, or D409G mutant TMEM16F-transformed Ba/F3 cells were loaded with Fluo-4AM, and analyzed by FACS. Open area represents the profile without Fluo-4AM. b, The vector- or wild-type TMEM16F-transformed Ba/F3 were preincubated with Fluo-4-AM. A23187 was added at the point indicated by an arrow, and the fluorescence was monitored.

Figure 7. The effect of knock-down of TMEM16F on the Ca2+-dependent PS exposure. The pRS shRNA vector for TMEM16F was introduced into Ba/F3 cells. Among the Ba/F3 stable transformant clones in which the TMEM16F mRNA level is reduced, 5 clones (sh16F #1-5) were picked and

Figure 6. No inhibition of the phospholipid-internalization by an inhibitor of the endocytosis. The Ba/F3 cells transformed with the vector or D409 mutant TMEM16F were preincubated at 37°C for 30 min with or without 20 µM

Figure 1. Constitutive binding of MFG-E8 to BaF/3 cells expressing the D409G mutant TMEM16F. Ba/F3 cells were infected with the empty, wild-type TMEM16F- or D409G mutant TMEM16F-carrying retrovirus vector. The cells were incubated with 0.4 µg/ml of mouse MFG-E8, followed by successive

Figure 5. No breakdown of NBD-PC and NBD-SM incorporated in the cells. NBD-PC (a) or NBD-SM (b) was added to Ba/F3 expressing the D409G mutant of TMEM16F, incubated at room temperature for 6 or 8 min, and treated with fatty-acid-free BSA. The phospholipids incorporated into the cells were extracted from the cells, and analyzed by thin-layer chromatography together with the input NBD-PC and NBD-SM. As reference, NBD-derivatives of phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and sphingomyelin (SM) were chromatographed in parallel, and their positions on the chromatogram are indicated on right.

Frequently asked questions

Q1. What was the effect of the phospholipid scramblase on the PS?

Upon reduction of the intracellular Ca2+ concentration, the phospholipid scramblase lost activity, and flippases returned the PS to the inner leaflet. 

Q2. What is the role of TMEM16F in the release of phospholipids?

The PS exposure or scrambling of phospholipids occurs in other biological processes1,4,26-29, such as the apoptotic cell death, the fusion of muscle, bone or trophoblast cells, and the release of neurotransmitters and microvesicles. 

Q3. What is the mechanism of the PS exposure?

The PS exposure is believed to be mediated by Ca2+-dependent phospholipid scramblases that bidirectionally transport phospholipids1,4, but its molecular mechanism has remained elusive. 

Q4. What was the cause of the strong PS exposure in Ba/F3-PS19?

Onewas the over-expression or over-activation of phospholipid scramblase, and the other was the inactivation of flippase10 that transports PS from the outer to inner leaflet of the plasma membranes. 

Q5. How many cells were exposed to PS?

At the third cycle of sorting and expansion (Library-Derived (LD)-PS3), about 35% of the cells exposed PS without A23187 treatment, and this cell population (LDPS4) was characterized. 

Q6. How many times did the cell line have to be sorted?

The sorting and expansion was repeated another seven times, and the resulting cell line (Ba/F3-PS19) was used for further studies. 

Q7. What is the mRNA sequence of the TMEM16F?

this method yielded TMEM16F carrying a point mutation that rendered the process extremely sensitive to Ca2+, such that in the cells expressing the mutated TMEM16F, the phopholipid scramblase functioned even in resting cells, in which the cytosolic Ca2+ concentration is below 100 nM24. 

Q8. What caused the TMEM16F mRNA to be skipped?

This skipping caused a frame shift, which resulted in the premature termination of the protein in exon 14 (Fig. 4e) at the third transmembrane segment of human TMEM16F (Fig. 4f). 

Q9. What is the role of dynamin in the internalization of phospholipids?

Dynasore that inhibits dynamin-mediated endocytosis18 did slightly or not inhibited the internalization of these phospholipids (Supplementary Fig. 6), suggesting that the contribution of endocytosis in the TMEM16F-mediated phospholipid-internalization may not be great. 

Q10. What is the effect of the treatment of the wild-type TMEM16F?

When the cells expressing the wild-type TMEM16F were treated with A23187, they incorporated NBD-PC faster than the parental cells, and about 40% of the cell-associated NBD-PC was inside of the cells within 4 min (Fig. 2g Similar results, i.e., constitutive internalization by cells expressing the mutant TMEM16F, and the enhanced A23817-induced incorporation by cells expressing the wild-type TMEM16F, were obtained with NBD-sphingomyelin (NBDSM)(Supplementary Fig. 4). 

Q11. What was the cDNA that caused the subline to expose PS?

A cDNA library was constructed from the subline, and a cDNA that caused Ba/F3 to spontaneously expose PS was identified by expression cloning. 

Q12. What is the mRNA of the TMEM16F?

An RTPCR analysis of the TMEM16F mRNA (GenBank NM_001025356) showed that the 5’ part (1320 bp) corresponding to exons 1-12 was identical among the patient and the parents, while its 3’ half corresponding to exons 11-20 was shorter in the patient than that in the parents (Fig. 4b). 

Q13. What is the mRNA level of TMEM16F?

As shown in Fig. 3a and Supplementary Fig. 7, the expression level of TMEM16F mRNA in 5 transformants was reduced to 20-35% of that in the cells expressing the control shRNA.