Abstract
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.
Highlights
TMEM16A and -16F function as a Ca2ϩ-dependent ClϪ channel and phospholipid scramblase, respectively
We assayed the ClϪ channel activity by patch clamp analysis with human 293T cells that had been transfected with a TMEM16 expression vector [10]
293T cells expressing TMEM16A but not -16F showed ClϪ channel activity, and the A23187-induced PS-exposing activity was observed in TMEM16F-expressing but not 16A-expressing TMEM16FϪ/Ϫ immortalized fetal thymocytes (IFETs) transformants. These observations confirmed that TMEM16A has Ca2ϩ-dependent ClϪ channel activity, whereas 16F functions as a Ca2ϩ-dependent phospholipid scramblase
Summary
TMEM16A and -16F function as a Ca2ϩ-dependent ClϪ channel and phospholipid scramblase, respectively. 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 epigallocatechin3-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. We found that some chemicals preferentially inhibit the ClϪ channel activity of TMEM16A, whereas others preferentially inhibit the scramblase activity of TMEM16F (16F) These results indicate that TMEM16A and -16F use a similar mechanism to stabilize and localize them at plasma membranes, but their functional domains are significantly different
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