Role of Ca2+ in the Stability and Function of TMEM16F and 16K

There are 10 transmembrane protein (TMEM) 16-family proteins in humans and mice. Among them, TMEM16F acts as a Ca2+-dependent phospholipid scramblase at the plasma membrane. However, how Ca2+ activates TMEM16F’s phospholipid-scramblase activity has not been elucidated. Here we found that in the presence of Ca2+, TMEM16K (whose function is unknown) directly binds Ca2+ to form a stable complex that can be detected by blue-native polyacrylamide gel electrophoresis. In the absence of Ca2+, TMEM16K and TMEM16F aggregated, suggesting that their structure is stabilized by Ca2+. Comprehensive mutagenesis of acidic residues in TMEM16K’s cytoplasmic and transmembrane regions identified five residues that are critical for binding Ca2+. These residues were well conserved between TMEM16F and 16K, and point mutations of these residues in TMEM16F reduced its ability to support Ca2+-dependent phospholipid scrambling. Our results suggest that Ca2+ binds TMEM16F directly and induces conformational changes that support its ...