Abstract
Transmembrane protein 16 (TMEM16) family members play numerous important physiological roles, ranging from controlling membrane excitability and secretion to mediating blood coagulation and viral infection. These diverse functions are largely due to their distinct biophysical properties. Mammalian TMEM16A and TMEM16B are Ca2+-activated Cl- channels (CaCCs), whereas mammalian TMEM16F, fungal afTMEM16, and nhTMEM16 are moonlighting (multifunctional) proteins with both Ca2+-activated phospholipid scramblase (CaPLSase) and Ca2+-activated, nonselective ion channel (CAN) activities. To further understand the biological functions of the enigmatic TMEM16 proteins in different organisms, here, by combining an improved annexin V-based CaPLSase-imaging assay with inside-out patch clamp technique, we thoroughly characterized Subdued, a Drosophila TMEM16 ortholog. We show that Subdued is also a moonlighting transport protein with both CAN and CaPLSase activities. Using a TMEM16F-deficient HEK293T cell line to avoid strong interference from endogenous CaPLSases, our functional characterization and mutagenesis studies revealed that Subdued is a bona fide CaPLSase. Our finding that Subdued is a moonlighting TMEM16 expands our understanding of the molecular mechanisms of TMEM16 proteins and their evolution and physiology in both Drosophila and humans.
Highlights
Transmembrane protein 16 (TMEM16) family members play numerous important physiological roles, ranging from controlling membrane excitability and secretion to mediating blood coagulation and viral infection
Combining an improved annexin V-based CaPLSase imaging assay with inside-out patch clamping technique, we discovered that Subdued is a moonlighting TMEM16 protein in Drosophila
Without membrane depolarization, Subdued and TMEM16F channels could barely open even in the presence of 100 M Ca2ϩ (Fig. 1, B and C, and Fig. S1, C and D). Both Subdued and TMEM16F channels are less sensitive to Ca2ϩ than TMEM16A–Ca2؉-activated Cl؊ channels (CaCCs), as evidenced by the rightward shift of their Ca2ϩ dose-response curves compared with TMEM16A– CaCC (Fig. 1, D and E), in addition to minimal activation under 0.39 M Ca2ϩ (Fig. 1, B and C, and Fig. S1, C and D)
Summary
Transmembrane protein 16 (TMEM16) family members play numerous important physiological roles, ranging from controlling membrane excitability and secretion to mediating blood coagulation and viral infection. These diverse functions are largely due to their distinct biophysical properties. To further understand the biological functions of the enigmatic TMEM16 proteins in different organisms, here, by combining an improved annexin V-based CaPLSase-imaging assay with inside-out patch clamp technique, we thoroughly characterized Subdued, a Drosophila TMEM16 ortholog. Combining an improved annexin V-based CaPLSase imaging assay with inside-out patch clamping technique, we discovered that Subdued is a moonlighting TMEM16 protein in Drosophila. We hope our findings can provide new insights into understanding the evolution of TMEM16 family, the molecular mechanisms of their ion and phospholipid permeation, and TMEM16 physiological functions in Drosophila
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