Role of Ca(2+) 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 Ca(2+)-dependent phospholipid scramblase at the plasma membrane. However, how Ca(2+) activates TMEM16F's phospholipid-scramblase activity has not been elucidated. Here we found that in the presence of Ca(2+), TMEM16K (whose function is unknown) directly binds Ca(2+) to form a stable complex that can be detected by blue-native polyacrylamide gel electrophoresis. In the absence of Ca(2+), TMEM16K and TMEM16F aggregated, suggesting that their structure is stabilized by Ca(2+). Comprehensive mutagenesis of acidic residues in TMEM16K's cytoplasmic and transmembrane regions identified five residues that are critical for binding Ca(2+). These residues were well conserved between TMEM16F and 16K, and point mutations of these residues in TMEM16F reduced its ability to support Ca(2+)-dependent phospholipid scrambling. Our results suggest that Ca(2+) binds TMEM16F directly and induces conformational changes that support its stability and function.