Functional swapping between transmembrane proteins TMEM16A and TMEM16F.
The transmembrane proteins TMEM16A and -16F each carry eight transmembrane regions with cytoplasmic N and C termini. TMEM16A carries out Ca(2+)-dependent Cl(-) ion transport, and TMEM16F is responsible for Ca(2+)-dependent phospholipid scrambling. Here we established assay systems for the Ca(2+)-dependent Cl(-) channel activity using 293T cells and for the phospholipid scramblase activity using TMEM16F(-/-) immortalized fetal thymocytes. Chemical cross-linking analysis showed that TMEM16A and -16F form homodimers in both 293T cells and immortalized fetal thymocytes. Successive deletion from the N or C terminus of both proteins and the swapping of regions between TMEM16A and -16F indicated that their cytoplasmic N-terminal (147 amino acids for TMEM16A and 95 for 16F) and C-terminal (88 amino acids for TMEM16A and 68 for 16F) regions were essential for their localization at plasma membranes and protein stability, respectively, and could be exchanged. Analyses of TMEM16A and -16F mutants with point mutations in the pore region (located between the fifth and sixth transmembrane regions) indicated that the pore region is essential for both the Cl(-) channel activity of TMEM16A and the phospholipid scramblase activity of TMEM16F. Some chemicals such as epigallocatechin-3-gallate and digallic acid inhibited the Cl(-) channel activity of TMEM16A and the scramblase activity of TMEM16F with an opposite preference. These results indicate that TMEM16A and -16F use a similar mechanism for sorting to plasma membrane and protein stabilization, but their functional domains significantly differ.
From the Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan and.
Takayuki
T
Suzuki
Jun
J
Suzuki
Shigekazu
S
Nagata
D000595:Amino Acid Sequence; D000818:Animals; D000075369:Anoctamin-1; D000075368:Anoctamins; D002118:Calcium / Q000737:chemistry*; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics; D002712:Chlorides / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D004594:Electrophysiology; D017353:Gene Deletion; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics; D049294:Phospholipid Transfer Proteins / Q000737:chemistry* / Q000235:genetics; D010743:Phospholipids / Q000737:chemistry*; D017354:Point Mutation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D060168:Thymocytes / Q000378:metabolism
C476213:ANO1 protein, human; C531181:ANO1 protein, mouse; C576687:ANO6 protein, mouse; C555078:ANO6 protein, human; D000075369:Anoctamin-1; D000075368:Anoctamins; D018118:Chloride Channels; D002712:Chlorides; D003432:Cross-Linking Reagents; D009363:Neoplasm Proteins; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D002118:Calcium
Chloride Channels; Enzyme Mutation; Phosphatidylserine; Plasma Membrane; Protein Domains