InteractiveFly: GeneBrief

subdued: Biological Overview | References

Transmembrane protein (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 Ca(2+)-activated Cl(-) channels (CaCCs), whereas mammalian TMEM16F, fungal afTMEM16, and nhTMEM are moonlighting (multifunctional) proteins with both Ca(2+)-activated phospholipid scramblase (CaPLSase; a protein responsible for the translocation of phospholipids between the two monolayers of a lipid bilayer of a cell membrane) and Ca(2+)-activated, nonselective ion channel (CAN) activities. To further understand the biological functions of the enigmatic TMEM proteins in different organisms, this study combined an improved annexin V-based CaPLSase-imaging assay with inside-out patch clamp technique to thoroughly characterized Subdued, a Drosophila TMEM ortholog. 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, this functional characterization and mutagenesis studies revealed that Subdued is a bona fide CaPLSase. The finding that Subdued is a moonlighting TMEM expands understanding of the molecular mechanisms of TMEM proteins and their evolution and physiology in both Drosophila and humans (Le, 2019).

The ground-breaking discoveries of TMEM16A and TMEM16B as the long-sought CaCCs advanced the understanding of a novel membrane protein superfamily that includes the TMEM family and its closely related OSCA, TMEM and TMC membrane protein families. TMEM proteins have been found in fungi, amoeboids, insects and vertebrates. The unexpected findings of mammalian TMEM16F as a moonlighting protein, a special type of proteins that can perform two or more distinct functions without gene fusions, multiple RNA splice variants or multiple proteolytic fragments, advanced understanding of the enigmatic TMEM family. Serving as a bona fide CaPLSase and a small-conductance CAN (SCAN) channel, TMEM16F has evolved the capability to passively transport phospholipids and ions, two structurally distinct classes of permeants, down their chemical gradients (Le, 2019).

Upon Ca2+ binding, TMEM16FCaPLSase mediates the rapid flip-flopping of phospholipids across cell membranes and thus dissipates the asymmetric distribution of membrane phospholipids. During platelet activation, TMEM16F-CaPLSase-induced phosphatidylserine (PS) externalization is essential for prothrombinase assembly, subsequent thrombin generation and blood coagulation. Consistent with its importance in blood coagulation, both the Scott syndrome patients who carried TMEM16F loss-of-function mutations and TMEM16F deficient mice exhibited prolonged bleeding phenotype. Despite the known physiological function of TMEM16F-CaPLSase in blood coagulation, it is unclear whether and how TMEM16F's ion channel activity can participate in this process (Le, 2019).

Recent structural and functional studies elegantly revealed that the fungal nhTMEM16, afTMEM and mammalian TMEM16E were also moonlighting proteins with CaPLSase and channel activities. Interestingly, the mammalian TMEM16A and TMEM16B CaCCs only displayed ion channel activities, while an amoebozoa TMEM homolog from Dictyostelium discoideum only showed CaPLSase activity when heterologously expressed in HEK cells. In order to understand the biological functions of TMEM moonlighting, there is an urgent need to have an in-depth understanding of TMEM evolution and function in different kingdoms ranging from Protozoa, Fungi to Animalia (Le, 2019).

TMEM moonlighting proteins have not been identified thus far in insects, despite a recent study that clearly demonstrated the physiological importance of CaPLSase in the degeneration of Drosophila sensory neurons (Sapar, 2018). However, the molecular identity of the Drosophila CaPLSase responsible for the observed scramblase activities remains elusive. Among the five Drosophila TMEM homologs, Subdued is the only protein that has been thoroughly characterized using electrophysiological tools (Wong, 2013). When heterologously expressed in HEK293T cells, whole-cell patch clamp recordings suggested that Subdued was a CaCC. Interestingly, Subdued-deficient Drosophila exhibited severe defects in host defense when challenged with the pathogenic bacterium Serratia marcescens. It remains, however, unclear how Subdued CaCC function is involved in Drosophila's immunity (Le, 2019).

Combining an improved Annexin V-based CaPLSase imaging assay with inside-out patch clamping technique, this study has discovered that Subdued is also a moonlighting TMEM16 protein in Drosophila. Notably, it was also found that Subdued harbors biophysical features that strikingly resembled those of the mammalian TMEM16F, which has been unambiguously shown to function as a CaPLSase and a CAN channel. These results thus support the notion that TMEM moonlighting could be an ancient feature of TMEM family, which is conserved in fungi, insects and vertebrates. These study provided new insights into understanding the evolution of TMEM family, the molecular mechanisms of their ion and phospholipid permeation, as well as TMEM physiological functions in Drosophila (Le, 2019).

Protein moonlighting as both ion channels and phospholipid scramblases has been observed in mammal and fungal TMEM proteins. By using patch clamp electrophysiology and an improved phospholipid scrambling assay, these studies reveal that Drosophila Subdued, an insect TMEM16, is also a moonlighting protein that can serve as both a CAN channel and a CaPLSase (Le, 2019).

In this study, it was also shown that the widely used HEK293T cell line had endogenous TMEM16F expression and strong CaPLSase activity. The endogenous CaPLSase activity can interfere with characterization of exogenous TMEM CaPLSases and complicate subsequent interpretation. To circumvent this complication, CRISPR-Cas method was applied to generate a TMEM16F KO HEK293T cell line, which lacks endogenous CaPLSase activity and thereby can serve as an ideal heterologous expression system to characterize CaPLSase activities. When Subdued was heterologously expressed in this KO cell line, robust Subdued-mediated CaPLSase activity was observed. Disrupting a key conserved residue at the extracellular entrance of Subdued abolished its CaPLSase activity. In addition, when one of the conserved Ca2+-binding residues was replaced with a positively charged Arg residue, the mutant Subdued failed to scramble phospholipids\. Collectively, these data show that Subdued is a bona fide CaPLSase (Le, 2019).

Inside-out excised patch clamp recordings demonstrated that Subdued is a CAN channel with higher cation permeability than chloride (PNa/PCl = 5.83) in μM intracellular Ca2+. This conclusion stands in stark contrast to a previous study, which reported that Subdued functioned as a CaCC (PNa/PCl = 0.16) based on whole-cell patch recordings (Wong, 2013). It is postulated that this discrepancy might be derived from the inherent differences between the two patch clamp configurations. First, infusion of pipette solution with high micromolar Ca2+ into cytosol could disrupt intracellular environment, which might subsequently alter channel activity. In the case of Subdued, channel current run-up was observed in whole-cell recording (Wong, 2013). When whole-cell recording was used to measure TMEM16F current, a to 15-minute delay of channel activation has been frequently observed after membrane break-in. Under inside-out configuration, both Subdued and TMEM16F current can be immediately recorded after membrane excision. Without the long delay to obtain stable current, the reversal potential measured using inside-out configuration may reflect the intrinsic channel selectivity. Second, whole-cell patch clamp may suffer from larger leak current during recording, especially when infusing with high micromolar Ca2+ into the cytosol. The potential leak current could confound the reversible potential measurement. Third, measuring the reversal potential requires exchanging solutions with drastically different ionic concentrations. Whole-cell recording usually requires whole-chamber solution exchange, which can induce large liquid junction potential to complicate reversal potential measurement. In the current inside-out patch clamp experiments, a pressurized focal perfusion system was used to achieve rapid solution exchange directly to the excised patch membrane. As this process is fast and only requires a small volume of solution, the impact of liquid junction potential is negligible (Le, 2019).

This study also found that Subdued ion permeability resembles that of the mammalian TMEM16F-SCAN. Similar to TMEM16F-SCAN (Yang, 2012), common CaCC blockers such as niflumic acid (NFA), flufenamic acid (FFA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) cannot block Subdued current (Wong, 2013), further supporting that Subdued channel is different from TMEM16A-CaCC. Interestingly, the fungal afTMEM and nhTMEM channels also exhibit substantial cation permeability. The non-selective nature of the moonlighting TMEM proteins towards phospholipids and ions suggests that their ancestors may have experienced low selective pressure during evolution, so that they could mediate simultaneous permeation of different ions and phospholipids without expanding the genome size. Interestingly, TMEM16A and TMEM16B are more selective to anions and lack CaPLSase function. It is hoped that the current findings can shine new lights on understanding TMEM evolution and molecular mechanisms for substrate selectivity. This study also provides new insights into understanding the physiological functions of TMEM moonlighting proteins. Previous studies have shown that Subdued knocked-out or knocked-down Drosophila strains harbored defects in their host defense and exhibited lethality upon ingestion of the pathogenic bacteria S. marcescens (Wong, 2013; Cronin, 2009). It is thus far not clear whether Subdued's CaPLSase function and/or ion channel function play a major role in participating host defense. Interestingly, the moonlighting protein TMEM16F has also been reported to express in immune cells and play an important role in immune responses. Considering the fact that channel activation of both Subdued and TMEM CAN current requires both high Ca2+ and membrane depolarization, both of which conditions are unlikely to be achieved under physiological conditions, it is likely that their CaPLSase functions might play the major role in immunity. A recent study suggested that PS externalization induced by overexpressing mammalian TMEM16F- CaPLSase played an important role in controlling neurite degeneration in Drosophila sensory neurons (Sapar, 2018). The current finding that Subdued as a bona fide CaPLSase might help identify the CaPLSase that is responsible for Drosophila neuronal degeneration (Le, 2019).

The Anoctamin family channel Subdued mediates thermal nociception in Drosophila

Calcium-permeable and thermo-sensitive transient receptor potential (TRP) channels mediate the nociceptive transduction of noxious temperature in Drosophila nociceptors; yet the underlying molecular mechanisms are not completely understood. This study found that Subdued, a calcium-activated chloride channel of the Drosophila anoctamin family, functions in conjunction with the thermo-TRPs in thermal nociception. Genetic analysis with deletion and the RNAi-mediated reduction of subdued show that subdued is required for thermal nociception in nociceptors. Further genetic analysis of subdued mutant and thermo-TRP mutants show that they interact functionally in thermal nociception. Subdued expressed in heterologous cells mediates a strong chloride conductance in the presence of both heat and calcium ions. Thus this analysis suggests that Subdued channels may amplify the nociceptive neuronal firing that is initiated by thermo-TRP channels in response to thermal stimuli (Jang, 2014).

Drosophila larvae sense noxious temperatures and show nocifensive behaviors when touched with a heated substance. These behaviors are mediated by class IV md neurons, which fire at temperatures above 40 °C. This study showed that Subdued channels opened at temperatures above 40°C, matching the threshold temperature of nociceptive neuronal activation. Subdued RNAi reduction in class IV md neurons was found to impair the larval nociceptive behavior in response to a heated probe. The same was true for subdued-null mutants. This finding suggests that chloride conduits through the Subdued channel are excitatory to sensory nociceptors, similar to mammalian dorsal root ganglion neurons. The expression of Subdued in sensory nociceptive neurons rescues the subdued mutant phenotype. Consistently, the subdued enhancer-trap (c240-Gal4) was expressed in class IV md neurons. Taken together, these data suggest that Subdued functions in sensory nociceptive neurons to transduce noxious thermal stimuli (Jang, 2014).

The thermo-TRP ion channels Painless and dTRPA1 are required for thermal nociception by class IV md neurons. Similarly, this study shows that the Subdued channel is activated by noxious temperatures and is required in sensory nociceptors for heat-induced nociceptive behaviors. This evidence suggests that nociceptors utilize both the thermo-TRPs and the anoctamin ion channel. Because the Painless and dTRPA1 ion channels are highly calcium-permeable upon activation, and because robust Subdued activation requires both calcium ions and heat, it is possible that activation of the Painless and dTRPA1 channels strengthens the subsequent activation of Subdued channels. Transheterozygotes of subdued with either painless or dTrpA1 exhibited defects in thermal nociceptive response, supporting the possibility that Subdued collaborates with thermo-TRPs in thermal transduction. Subdued participation in nociceptor depolarization, along with the thermo-TRP channels, may therefore be necessary for the efficient activation of high-threshold nociceptors in response to noxious temperatures (Jang, 2014).

The subdued mutants have defects in their immunity to ingested virulent bacteria and consume less food (Wong, 2013). Expression of Subdued, as monitored by an expression of c240-Gal4, is not visible in the gut but is visible in the proventriculus and renal tubule. The proventriculus is a valve that regulates food transport to the midgut, so Subdued might play a role in food transit through the proventriculus, which might then account for the subdued knockout phenotype of consuming less food. The renal tubule plays a role in the immune response, so the subdued defect in the immunity to virulent bacteria may be associated with Subdued expression in the renal tubule. Subdued is expressed in the epithelial cells of the oviduct that regulates egg release from the ovary to the uterus and also in the spermathecal secretory cells that play a role in sperm storage and its release into the uterus. The female sterility observed in subdued mutant flies might be due to a failure to release either egg or sperm from the ovary or spermatica, respectively, and that possibility needs to be explored further (Jang, 2014).

Subdued, a TMEM16 family Ca(2)(+)-activated Cl(-)channel in Drosophila melanogaster with an unexpected role in host defense

TMEM16A and TMEM16B are calcium-activated chloride channels (CaCCs) with important functions in mammalian physiology. Whether distant relatives of the vertebrate TMEM16 families also form CaCCs is an intriguing open question. This study reports that a TMEM16 family member from Drosophila melanogaster, Subdued (CG16718), is a CaCC. Amino acid substitutions of Subdued alter the ion selectivity and kinetic properties of the CaCC channels heterologously expressed in HEK 293T cells. This Drosophila channel displays characteristics of classic CaCCs, thereby providing evidence for evolutionarily conserved biophysical properties in the TMEM16 family. Additionally, this study shows that knockout flies lacking subdued gene activity more readily succumb to death caused by ingesting the pathogenic bacteria Serratia marcescens, suggesting that subdued has novel functions in Drosophila host defense (Wong, 2013).

Subdued is activated by internal calcium, with a lower bound of [Ca2+]in = 20 μM in whole cell patch clamp experiments in which current was observed. No significant currents were observed when an EGTA-buffered zero calcium solution was used as the internal solution. Relative to mammalian TMEM16A (Yang, 2008), TMEM16B (Pifferi, 2009) or Xenopus TMEM16A, Subdued is one to two orders of magnitude less calcium sensitive in whole cell patch clamp experiments. This could either reflect a true biophysical property of the channel or could be an indication that the non-native HEK 293T expression system used in these experiments lacks auxiliary subunits required for higher calcium sensitivity. It would be interesting to test if directed mutagenesis of Subdued can tune its calcium sensitivity within the realm of its mammalian and Xenopus counterparts (Wong, 2013).

Subdued rectifies outwardly, passing larger currents at more positive voltages. The channel permeates mainly chloride with a PNa/PCl of 0.16, and preferentially permeates larger anions relative to smaller ones, giving the selectivity series SCN- > I- > Br- > Cl-. A Y489H mutation affected the ionic selectivity of the channel, making it more permeable to Na+. This suggests that perhaps the Y489 residue is pore lining, or has an allosteric effect on the structure of the pore. The Y489H mutant also gave rise to smaller currents compared to the wild-type channel, a reflection of either decreased unitary channel conductance or decreased membrane expression. Additionally, a Q672K mutation produced a dramatic slowing of activation kinetics, a phenomenon also observed when mutating the corresponding residue in mammalian TMEM16F (Yang, 2012). The observation that mutations to Subdued alter the properties of the currents strongly points to this protein as a pore-forming subunit of the recorded CaCCs (Wong, 2013).

Pharmacologically, Subdued is not blocked by the CaCC blockers NFA, FFA, NPPB or T16Ainh-A01. This might arise from structural differences in Subdued, perhaps in the pore, relative to its mammalian and Xenopus counterparts. However benzbromarone blocks Subdued significantly and could potentially be used to interrogate the location and properties of the channel pore. In conclusion, as a distantly related TMEM16 family member, Subdued will be useful as a tool in structure/function studies to parse out conserved or divergent biophysical properties such as calcium- and voltage-dependent gating, permeation and ion selectivity (Wong, 2013).

To study the function of the channel in Drosophila, subdued knockout strains were generated. Confirming results from a previous genome-wide RNAi study, the knockout was found to be more susceptible to gut infection by a strain of Serratia marcescens, Db11. An earlier study proposed that the cause of lethality is bacterial proliferation leading to invasion of the gut tissue and subsequent gut distension and escape of bacteria into the hemolymph (Wong, 2013).

In the case of the subdued knockout, susceptibility arises, at least in part, from deficient host defense, since in vivo proliferation of Db11 was higher in knockout fly guts as well as in the whole animal as compared to wild-type flies. Additionally, slightly but significantly less food dye was recovered from the guts of the knockout flies. This might arise from lower food consumption by knockout flies, but could also be an indication of increased gut tissue damage due to greater numbers of Db11 in the gut, leading to leakage and diffusion of food dye into the hemolymph. It remains to be determined if higher Db11 titers in the whole animal also result from defective immune responses within the hemolymph. Tissue-specific RNAi of subdued using gut or hemocytes drivers did not recapitulate the whole animal RNAi phenotype, suggesting that Subdued is likely to exert its protective function in a multitude of tissues (Wong, 2013).

One potential function for Subdued is in the regulation of the secretion of cationic antimicrobial peptides (AMPs), a process that occurs widely on epithelial surfaces and is known to play critical roles in host defense. This hypothesis is consistent with the abundant mRNA expression of subdued in various epithelial tissues. The susceptibility observed in the subdued knockout flies could also be a consequence of a deficiency in dual oxidase (DUOX)-mediated immunity. The DUOX system is reported to be critical in generating reactive oxygen species (ROS) in Drosophila gut epithelia. This study reported that strong antimicrobial ROS species are generated by the peroxidase homology domain (PHD) of Drosophila DUOX in a chloride-dependent manner. These ROS species are likely to be the highly reactive hypohalites OCl or OSCN, the in vivo production of which requires trans-epithelial anion transport. Additionally, the Drosophila DUOX system has also been shown to mobilize downstream of the Gαq-coupled signaling pathway, implicating other calcium-dependent responses in the Drosophila immune response. Following Db11 infection of subdued knockouts, it is possible that Gαq receptor stimulation fails to elicit sufficient amounts of halide transport onto gut epithelia due to a deficiency in CaCCs, reducing PHD-mediated generation of antimicrobial hypohalites and leading to increased bacterial proliferation and higher lethality (Wong, 2013).

There remains the possibility that subtle structural deficits also contribute to the susceptibility of subdued knockouts to Db11 infection. Developmental defects in gut epithelial integrity or the peritrophic matrix lining the gut might result in the susceptibility phenotype. The subdued knockout flies did not have significant defects in gut epithelial polarity and integrity under basal conditions as assessed by immunostaining for Armadillo and Discs Large (Dlg) to observe adherens and septate junction structure. However, Subdued might function in gut epithelial or peritrophic matrix integrity only upon Db11 challenge to the gut, a possibility that will be explored in future study (Wong, 2013).

Search PubMed for articles about Drosophila Subdued

Cronin, S. J., Nehme, N. T., Limmer, S., Liegeois, S., Pospisilik, J. A., Schramek, D., Leibbrandt, A., Simoes Rde, M., Gruber, S., Puc, U., Ebersberger, I., Zoranovic, T., Neely, G. G., von Haeseler, A., Ferrandon, D., and Penninger, J. M. (2009) Genome-wide RNAi screen identifies genes involved in intestinal pathogenic bacterial infection. Science 325: 340-343. PubMed ID: 19520911

Huang, J., Lam, H., Koziol-White, C., Limjunyawong, N., Kim, D., Kim, N., Karmacharya, N., Rajkumar, P., Firer, D., Dalesio, N. M., Jude, J., Kurten, R. C., Pluznick, J. L., Deshpande, D. A., Penn, R. B., Liggett, S. B., Panettieri, R. A., Jr., Dong, X. and An, S. S. (2020). The odorant receptor OR2W3 on airway smooth muscle evokes bronchodilation via a cooperative chemosensory tradeoff between TMEM16A and CFTR. Proc Natl Acad Sci U S A 117(45): 28485-28495. PubMed ID: 33097666

Jang, W., Kim, J. Y., Cui, S., Jo, J., Lee, B. C., Lee, Y., Kwon, K. S., Park, C. S. and Kim, C. (2014). The Anoctamin family channel Subdued mediates thermal nociception in Drosophila. J Biol Chem 290(4):2521-8. PubMed ID: 25505177

Ko, W., Jung, S. R., Kim, K. W., Yeon, J. H., Park, C. G., Nam, J. H., Hille, B. and Suh, B. C. (2020). Allosteric modulation of alternatively spliced Ca(2+)-activated Cl(-) channels TMEM16A by PI(4,5)P2 and CaMKII. Proc Natl Acad Sci U S A. PubMed ID: 33199590

Le, T., Le, S. C. and Yang, H. (2019). Drosophila Subdued is a moonlighting transmembrane protein (TMEM16) that transports ions and phospholipids. J Biol Chem 294(12): 4529-4537. PubMed ID: 30700552

Pifferi, S., Dibattista, M. and Menini, A. (2009). TMEM16B induces chloride currents activated by calcium in mammalian cells. Pflugers Arch 458(6): 1023-1038. PubMed ID: 19475416

Sapar, M. L., Ji, H., Wang, B., Poe, A. R., Dubey, K., Ren, X., Ni, J. Q., and Han, C. (2018). Phosphatidylserine externalization results from and causes neurite degeneration in Drosophila. Cell Rep 24: 2273-2286. PubMed ID: 30157423

Sui, J., Zhang, C., Fang, X., Wang, J., Li, Y., Wang, J., Wang, L., Dong, J., Zhou, Z., Li, C., Chen, J., Ma, T. and Chen, D. (2020). Dual role of Ca(2+)-activated Cl(-) channel transmembrane member 16A in lipopolysaccharide-induced intestinal epithelial barrier dysfunction in vitro. Cell Death Dis 11(5): 404. PubMed ID: 32472021

Wong, X. M., Younger, S., Peters, C. J., Jan, Y. N., and Jan, L. Y. (2013). Subdued, a TMEM16 family Ca(2)(+)-activated Cl(-)channel in Drosophila melanogaster with an unexpected role in host defense. Elife 2: e00862. PubMed ID: 24192034

Yang, Y. D., Cho, H., Koo, J. Y., Tak, M. H., Cho, Y., Shim, W. S., Park, S. P., Lee, J., Lee, B., Kim, B. M., Raouf, R., Shin, Y. K. and Oh, U. (2008). TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature 455(7217): 1210-1215. PubMed ID: 18724360

Yang, H., Kim, A., David, T., Palmer, D., Jin, T., Tien, J., Huang, F., Cheng, T., Coughlin, S. R., Jan, Y. N., and Jan, L. Y. (2012). TMEM16F forms a Ca2+-activated cation channel required for lipid scrambling in platelets during blood coagulation. Cell 151: 111-122. PubMed ID: 23021219

date revised: 20 November 2020

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.