Membrane Phospholipid Scrambling by TMEM16F

The plasma membrane of animal cells contains a phospholipid bilayer; the outer leaflet largely contains phosphatidylcholine and sphingomyelin and the inner leaflet is mainly composed of phosphatidylserine (PS) and phosphatidylethanolamine (PE). This lipid asymmetry is maintained by an ATP-dependent enzyme called the aminophospholipid translocase. Scrambling of membrane phospholipids leading to dissipation of lipid asymmetry was originally observed during platelet activation¹  and subsequently has been identified in other important processes, including apoptosis and the release of neurotransmitters and microvesicles. PS is a component of the surface for assembly of the prothrombinase and intrinsic pathway factor X activation complexes, which are necessary for normal blood coagulation. In 1979, a female with a history of excessive surgical bleeding was identified with platelets displaying impaired procoagulant activity.²  This rare bleeding disorder, called Scott syndrome after this patient, is associated with defective phospholipid scrambling in platelets and other blood cells.

Dr. Suzuki et al. in the laboratory of Shigekazu Nakata in Kyoto have identified a protein known as TMEM16F, which participates in the scrambling of PS in plasma membranes. The authors used a mouse B-cell line, Ba/F3, which exposes PS on its cell surface after treatment with a calcium ionophore. A Ba/ F3 subline with considerably increased PS exposure compared to the parental cell line was isolated by repetitive fluorescence-activated cell sorting. A complementary DNA library was then constructed from this subline and was introduced into the parental cell line. After repetitive sorting and expansion, the team isolated a cell line, designated LD-PS4, that constitutively exposed PS in the absence of calcium ionophore. The cDNA of the LD-PS4 was compared to the GenBank database revealing the gene encoding TMEM16F, which has a predicted polypeptide molecular mass of 106 kDa and eight transmembrane segments. Compared to wild-type TMEM16F, LD-PS4 TMEM16F contains an A-to-G mutation at nucleotide 1226 in codon 409, leading to an aspartate to glycine (D409G) replacement. LD-PS4 cells also constitutively expressed PE, consistent with scramblase activity. Inhibiting endogenous TMEM16F expression in Ba/F3 cells with a TMEM16F short hairpin RNA resulted in a significant decrease in the rate of calcium ionophore-induced exposure of PS and PE. RT–PCR analysis of TMEM16F in a B-cell line from a patient with Scott syndrome revealed the absence of exon 13 and a frameshift producing premature termination of exon 14, caused by a homozygous G-to-T mutation at the splice-acceptor site in intron 12. These results strongly indicate that TMEM16F is an essential component of the entire composition of a phospholipid scramblase.

TMEM16F is a member of the TMEM16 family, which consists of 10 members in both humans and mice. The original member of this family, TMEM16A, is a calcium-dependent chloride channel, suggesting that TMEM16F may bind to and be regulated by calcium. The constitutive activity of the D409G mutant may be due to an increased affinity for calcium and binding at endogenous calcium levels.