Abstract
TMEM16A and 16B work as Cl(-) channel, whereas 16F works as phospholipid scramblase. The function of other TMEM16 members is unknown. Using TMEM16F(-/-) cells, TMEM16C, 16D, 16F, 16G, and 16J were shown to be lipid scramblases. Some TMEM16 members are divided into two Cl(-) channels and five lipid scramblases. Learning the biochemical function ofTMEM16family members is essential to understand their physiological role. Asymmetrical distribution of phospholipids between the inner and outer plasma membrane leaflets is disrupted in various biological processes. We recently identified TMEM16F, an eight-transmembrane protein, as a Ca(2+)-dependent phospholipid scramblase that exposes phosphatidylserine (PS) to the cell surface. In this study, we established a mouse lymphocyte cell line with a floxed allele in the TMEM16F gene. When TMEM16F was deleted, these cells failed to expose PS in response to Ca(2+) ionophore, but PS exposure was elicited by Fas ligand treatment. We expressed other TMEM16 proteins in the TMEM16F(-/-) cells and found that not only TMEM16F, but also 16C, 16D, 16G, and 16J work as lipid scramblases with different preference to lipid substrates. On the other hand, a patch clamp analysis in 293T cells indicated that TMEM16A and 16B, but not other family members, acted as Ca(2+)-dependent Cl(-) channels. These results indicated that among 10 TMEM16 family members, 7 members could be divided into two subfamilies, Ca(2+)-dependent Cl(-) channels (16A and 16B) and Ca(2+)-dependent lipid scramblases (16C, 16D, 16F, 16G, and 16J).
Highlights
TMEM16A and 16B work as ClϪ channel, whereas 16F works as phospholipid scramblase
To demonstrate TMEM16F involvement in Ca2ϩ-dependent phospholipid scrambling and to determine whether TMEM16F plays a role in exposing PS to the cell surface during apoptotic cell death, we established from fetal thymus tissue a TMEM16F-deficient mouse cell line that expresses a small number of TMEM16 family members, including TMEM16F
Embryos were genotyped at embryonic day 14.5, and fetal TMEM16Fflox/flox thymus cells were infected with retroviruses carrying H-rasV12 and c-myc to establish immortalized fetal thymocyte (IFET) cell lines
Summary
TMEM16A and 16B work as ClϪ channel, whereas 16F works as phospholipid scramblase. The function of other TMEM16 members is unknown. The phospholipid distribution between the outer and inner leaflets is not disrupted; ATP-dependent translocase inactivation alone does not appear sufficient to cause the rapid PS exposure seen in apoptotic cell death and platelet activation. Confirming that TMEM16F is a Ca2ϩ-dependent phospholipid scramblase, recessive TMEM16F mutations were identified in human patients with Scott syndrome [20, 21], which is known to result from a phospholipid-scrambling defect; these patients suffer from impaired blood clotting It is not clear whether TMEM16F is involved in other processes, such as apoptotic cell death or cell fusion. Two family members, TMEM16A and 16B, but not others showed Ca2ϩ-dependent ClϪ channel activity These results, together with their tissue-specific expression, suggest that the TMEM16 family members have distinct functions in lipid scrambling and ClϪ channel functions in various biological processes
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