title: Role of Ca(2+) in the Stability and Function of TMEM16F and 16K.
abstract: 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.
authors.affiliation: Biochemistry & Immunology, Immunology Frontier Research Center, Osaka University , 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Ishihara
authors.affiliation: Biochemistry & Immunology, Immunology Frontier Research Center, Osaka University , 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Suzuki
authors.affiliation: Biochemistry & Immunology, Immunology Frontier Research Center, Osaka University , 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
authors.forename: Shigekazu
authors.initials: S
authors.lastname: Nagata
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000075368:Anoctamins; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D018345:Mice, Knockout; D049294:Phospholipid Transfer Proteins / Q000737:chemistry* / Q000502:physiology*; D017354:Point Mutation; D055550:Protein Stability; D017386:Sequence Homology, Amino Acid
chemical_list: C576687:ANO6 protein, mouse; D000075368:Anoctamins; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; D002118:Calcium