Characterization of the scrambling domain of the TMEM16 family

The TMEM16 protein family has 10 members, each of which carries 10 transmembrane segments. TMEM16A and 16B are Ca²⁺-activated Cl^- channels. Several other members, including TMEM16F, promote phospholipid scrambling between the inner and outer leaflets of a cell membrane in response to intracellular Ca²⁺ However, the mechanism by which TMEM16 proteins translocate phospholipids in plasma membranes remains elusive. Here we show that Ca²⁺-activated, TMEM16F-supported phospholipid scrambling proceeds at 4 °C. Similar to TMEM16F and 16E, seven TMEM16 family members were found to carry a domain (SCRD; scrambling domain) spanning the fourth and fifth transmembrane segments that conferred scrambling ability to TMEM16A. By introducing point mutations into TMEM16F, we found that a lysine in the fourth transmembrane segment of the SCRD as well as an arginine in the third and a glutamic acid in the sixth transmembrane segment were important for exposing phosphatidylserine from the inner to the outer leaflet. However, their role in internalizing phospholipids was limited. Our results suggest that TMEM16 provides a cleft containing hydrophilic "stepping stones" for the outward translocation of phospholipids.