Dependent Phospholipid Scramblase Research Articles

Dysregulation of ADAM10 shedding activity in naked mole-rat fibroblasts is due to deficient phosphatidylserine externalization.

The naked mole-rat (NMR, Heterocephalus glaber) is of significant interest to biogerontological research, rarely developing age-associated diseases, such as cancer. The transmembrane glycoprotein CD44 is upregulated in certain cancers and CD44 cleavage by a disintegrin and metalloproteinase 10 (ADAM10) regulates cellular migration. Here we provide evidence that mature ADAM10 is expressed in NMR primary skin fibroblasts (NPSF), and that ionomycin increases cell surface ADAM10 localization. However, we observed an absence of ADAM10 mediated CD44 cleavage, as well as shedding of exogenous and overexpressed betacellulin in NPSF, whereas in mouse primary skin fibroblastsionomycin induced ADAM10-dependent cleavage of both CD44 and betacellulin. Overexpressing a hyperactive form of the Ca2+ -dependent phospholipid scramblase ANO6 in NPSF increased phosphatidylserine (PS) externalization, which rescued the ADAM10 sheddase activity and promoted cell migration in NPSF in an ADAM10-dependent manner. These findings suggest that dysregulation of ADAM10 shedding activity is due to a deficient PS externalization in NMR.

Abstract C047: Phospholipid scramblase TMEM16F in cancer associated fibroblasts regulates trogocytosis to sustain critical dependency of pancreatic cancer cells on exogenous lipids

Abstract Pancreatic ductal adenocarcinoma (PDAC) cells derive their resistance to therapy and aggressive clinical course from the symbiotic signaling and metabolic interactions with cancer-associated fibroblasts (CAFs). Trogocytosis is a process of “nibbling” of plasma membranes between two living cells. Here, we demonstrate CAFs are the primary recipients of exogenous lipids which they transfer to metabolically “parasitic” PDAC cells via a contact-mediated trogocytosis. Whereas trogocytosis has been described in immune system and in normal development, the biochemical and signaling regulators of trogocytosis between CAFs and PDAC cells have not been defined. Results: We determine that trogocytosis occurs through heterotypic CAF-PDAC cell contacts: membrane blebbing (readily observed in activated CAFs and exaggerated by release of Ca2+ from the ER stores) results in the blebs uptake by the PDAC cells. The CAF blebs express externalized phosphatidylserine (PtdSer), and blockade of PtdSer in vitro transiently deters trogocytic uptake of CAF membranes. Probing a short list of candidate targets involved in regulation of cholesterol trafficking, membrane fusion and membrane protrusions using CRISPRi, has shown that CAFs deficient in CD81, ARF6 or TMEM16F exhibit markedly reduced ability to support the viability of cholesterol-auxotrophic PDAC cells in lipid-poor media. As a promising therapy target, TMEM16F is a Ca2+-regulated scramblase increasing PtdSer on the outer leaflet of the plasma membrane. TMEM16F protein is highly expressed in human PDAC CAFs compared to fibroblasts isolated from the adjacent non-malignant pancreatic tissues. The TMEM16F-null CAFs are unable to sustain of cholesterol-auxotrophic PDAC cells in lipid-poor co-cultures, and do not support the growth of PDAC cells in orthotopic co-implantation model. Furthermore, several clinically available antibiotics used to treat tapeworms are selective TMEM16F inhibitors. One of the widely available TMEM16F inhibitors, niclozamide, is effective in blocking cholesterol transfer from CAFs to PDAC cells in vivo. Conclusion: Our overall model is that activated CAFs initiate trogocytosis by expressing PtdSer as “eat me” signals on the surfaces of their membrane blebs. This process is regulated by Ca2+-dependent phospholipid scramblase TMEM16F which is an attractive drug-amenable target to dismantle the critical metabolic dependency in PDAC on the exogenous lipids. Re-purposing of clinically available TMEM16 inhibitors will make a tangible impact on treatment of PDAC patients in the near term. Citation Format: Charline Ogier, Alena Klochkova, Linara Gabitova-Cornell, Battuya Bayarmagnai, Diana Restifo, Aizhan Surumbayeva, Debora Barbosa Vendramini-Costa, Ralph Francescone, Janusz Franco-Barraza, Jaye Gardiner, Emmanuelle Nicolas, Andrei Efimov, Elizabeth A. Handorf, Kathy Q. Cai, Bojana Gligorijevic, Edna Cukierman, Igor Astsaturov. Phospholipid scramblase TMEM16F in cancer associated fibroblasts regulates trogocytosis to sustain critical dependency of pancreatic cancer cells on exogenous lipids [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C047.

Abstract 1571: Cancer associated fibroblasts sustain critical dependency of pancreatic cancer cells on exogenous lipids

Abstract Pancreatic ductal adenocarcinoma (PDAC) cells derive their resistance to therapy and aggressive clinical course from the symbiotic signaling and metabolic interactions with cancer-associated fibroblasts (CAFs). CAFs have been shown to provide nutrients to "parasitic" PDAC cells including water soluble glucose and amino acids. In hypoxic tumor microenvironments, aggressive PDAC are functional auxotroph for lipids, and scavenge exogenous lipids to build cellular membranes. However, the mechanism by which PDAC cells obtain these water-insoluble essential membrane building materials remains poorly understood. To gain insights into the mechanism of lipid scavenging, we generated cholesterol-auxotrophic human and mouse PDAC PDAC cells since cholesterol is the major constituent of lipid cellular membranes. Here, we discovered that PDAC cells utilize CAFs as a main source of lipids in vivo through direct heterotypic cellular contacts. In this process, PDAC cells directly acquire the CAF plasma membrane (PM) via trogocytosis. In response to yet unidentified paracrine “feed me” signals activating CAF-dependent trogocytosis, CAFs upregulate phosphatidylserine (PtdSer) on the outer leaflet of the PM. Consequently, blockade of PtdSer on CAFs partially deterred trogocytic transfer of CAF membranes to PDAC cells in vitro. Furthermore, Ca2+-dependent phospholipid scramblase TMEM16F is a critical regulator of PtdSer externalization. TMEM16F is highly expressed in human PDAC CAFs compared to fibroblasts isolated from the matching adjacent non-malignant pancreatic tissues CAFs deficient in TMEM16F scramblase exhibited markedly reduced ability to support the growth of cholesterol-auxotrophic PDAC cells cultured in lipid depleted media. We propose trogocytosis as a new mode of lipid scavenging by PDAC cells from CAFs involving activation of Ca2+-dependent phospholipid scramblase TMEM16F in CAFs and increased expression of PtdSer as “eat me” signals on CAF PM. To inactivate trogocytosis, we nominate TMEM16F as a plausible PDAC therapy target using clinically available TMEM16 inhibitors with a potential for impact on treatment of PDAC patients in the near term. Citation Format: Charline Ogier, Alena Klochkova, Battuya Bayarmagnai, Linara Gabitova, Diana Restifo, Aizhan Surumbayeva, Janusz Franco-Barraza, Debora Vendramini Costa, Ralph Francescone, Jaye Gardiner, Emmanuelle Nicolas, Elizabeth A. Handorf, Kathy Q. Cai, Edna Cukierman, Igor Astsaturov. Cancer associated fibroblasts sustain critical dependency of pancreatic cancer cells on exogenous lipids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1571.

Abstract PR-015: Cancer-associated fibroblasts sustain critical dependency of pancreatic cancer cells on exogenous lipids

Abstract Pancreatic ductal adenocarcinoma (PDAC) cells derive their resistance to therapy and aggressive clinical course from the symbiotic signaling and metabolic interactions with cancer-associated fibroblasts (CAFs). CAFs have been shown to provide to metabolically “parasitic” PDAC cells with water-soluble metabolites such as glucose and amino acids, or bulk protein via micropinocytosis. To meet the elevated demands for cellular membrane lipids, cancer cells rely on uptake of the exogenous lipids. However, the mechanism by which PDAC cells obtain water-insoluble essential lipids, such as cholesterol, remains poorly understood. Here, we define CAFs as a main source of lipids for PDAC cells by direct “feeding” of the CAF plasma membrane (PM) to cancer cells via heterotypic cellular contacts, a phenomenon known as trogocytosis. To gain insights into the mechanisms of regulation of CAF trogocytosis, we engineered cholesterol-auxotrophic human and mouse PDAC cells. In the absence of exogenously provided cholesterol, these cancer cells undergo apoptosis, which is completely rescued in CAF co-cultures. Using CRISPRi-mediated depletion in CAFs of select genes involved in cholesterol trafficking, membrane fusion and membrane protrusions, we found that CAFs deficient in CD81, TMEM16F, or ARF6 exhibited markedly reduced ability to support the viability of cholesterol-auxotrophic PDAC cells in lipid-poor media. As a promising therapy target, TMEM16F is a Ca2+-regulated scramblase increasing phosphatidylserine (PtdSer) on the outer leaflet of the PM. TMEM16F protein is highly expressed in human PDAC CAFs compared to fibroblasts isolated from the adjacent non-malignant pancreatic tissues. The TMEM16F-null CAFs are unable to sustain of cholesterol-auxotrophic PDAC cells in lipid-poor co-cultures. Our overall model is that, to initiate trogocytosis, PDAC cells release paracrine “feed me” signals activating Ca2+-dependent phospholipid scramblase TMEM16F. As the result, increased outward expression of PtdSer on CAF PM is a hallmark “eat me” signal that is recognized by the trogocytic PDAC cells. We conclude that trogocytosis is the critical metabolic dependency in PDAC, and nominated TMEM16F as a plausible PDAC therapy target. Re-purposing of clinically available TMEM16 inhibitors will make a tangible impact on treatment of PDAC patients in the near term. Citation Format: Charline Ogier, Alena Klochkova, Linara Gabitova, Battuya Bayarmagnai, Diana Restifo, Aizhan Surumbayeva, Janusz Franco-Barraza, Ralph Francescone, Debora B. Barbosa Vendramini-Costa, Jaye Gardiner, Emmanuelle Nicolas, Elizabeth A. Handorf, Kathy Q. Cai, Edna Cukierman, Igor Astsaturov. Cancer-associated fibroblasts sustain critical dependency of pancreatic cancer cells on exogenous lipids [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-015.

Anion and Cation Permeability of the Mouse TMEM16F Calcium-Activated Channel.

TMEM16F is involved in several physiological processes, such as blood coagulation, bone development and virus infections. This protein acts both as a Ca2+-dependent phospholipid scramblase and a Ca2+-activated ion channel but several studies have reported conflicting results about the ion selectivity of the TMEM16F-mediated current. Here, we have performed a detailed side-by-side comparison of the ion selectivity of TMEM16F using the whole-cell and inside-out excised patch configurations to directly compare the results. In inside-out configuration, Ca2+-dependent activation was fast and the TMEM16F-mediated current was activated in a few milliseconds, while in whole-cell recordings full activation required several minutes. We determined the relative permeability between Na+ and Cl¯ (PNa/PCl) using the dilution method in both configurations. The TMEM16F-mediated current was highly nonselective, but there were differences depending on the configuration of the recordings. In whole-cell recordings, PNa/PCl was approximately 0.5, indicating a slight preference for Cl¯ permeation. In contrast, in inside-out experiments the TMEM16F channel showed a higher permeability for Na+ with PNa/PCl reaching 3.7. Our results demonstrate that the time dependence of Ca2+ activation and the ion selectivity of TMEM16F depend on the recording configuration.

Temperature-dependent increase in the calcium sensitivity and acceleration of activation of ANO6 chloride channel variants

Anoctamin-6 (ANO6) belongs to a family of calcium (Ca2+)-activated chloride channels (CaCCs), with three splicing variants (V1, V2, and V5) showing plasma membrane expression. Unlike other CaCCs, ANO6 requires a non-physiological intracellular free calcium concentration ([Ca2+]i > 1 μM) and several minutes for full activation under a whole-cell patch clamp. Therefore, its physiological role as an ion channel is uncertain and it is more commonly considered a Ca2+-dependent phospholipid scramblase. Here, we demonstrate that physiological temperature (37 °C) increases ANO6 Ca2+ sensitivity under a whole-cell patch clamp; V1 was activated by 1 μM [Ca2+]i, whereas V2 and V5 were activated by 300 nM [Ca2+]i. Increasing the temperature to 42 °C led to activation of all ANO6 variants by 100 nM [Ca2+]i. The delay time for activation of the three variants was significantly shortened at 37 °C. Notably, the temperature-dependent Ca2+-sensitisation of ANO6 became insignificant under inside-out patch clamp, suggesting critical roles of unknown cytosolic factors. Unlike channel activity, 27 °C but not 37 °C (physiological temperature) induced the scramblase activity of ANO6 at submicromolar [Ca2+]i (300 nM), irrespective of variant type. Our results reveal a physiological ion conducting property of ANO6 at 37 °C and suggest that ANO6 channel function acts separately from its scramblase activity.

ADAM10 sheddase activation is controlled by cell membrane asymmetry.

Dysregulation of the disintegrin-metalloproteinase ADAM10 may contribute to the development of diseases including tumorigenesis and Alzheimer’s disease. The mechanisms underlying ADAM10 sheddase activation are incompletely understood. Here, we show that transient exposure of the negatively charged phospholipid phosphatidylserine (PS) is necessarily required. The soluble PS headgroup was found to act as competitive inhibitor of substrate cleavage. Overexpression of the Ca2+-dependent phospholipid scramblase Anoctamin-6 (ANO6) led to increased PS externalization and substrate release. Transfection with a constitutively active form of ANO6 resulted in maximum sheddase activity in the absence of any stimulus. Calcium-dependent ADAM10 activation could not be induced in lymphocytes of patients with Scott syndrome harbouring a missense mutation in ANO6. A putative PS-binding motif was identified in the conserved stalk region. Replacement of this motif resulted in strong reduction of sheddase activity. In conjunction with the recently described 3D structure of the ADAM10 extracellular domain, a model is advanced to explain how surface-exposed PS triggers ADAM10 sheddase function.

Anoctamin-6 regulates ADAM sheddase function

ADAM17, a prominent member of the “Disintegrin and Metalloproteinase” (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates including TGF-alpha, Amphiregulin (AREG) and TNF-Receptor 1 (TNFR1). We recently presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. Anoctamin-6 (ANO6) has Ca2+-dependent phospholipid scramblase activity and it followed that the functions of ANO6 and ADAM17 might be linked. We report that overexpression of ANO6 in HEK293T cells led to increased Ca2+-mediated PS-exposure that was indeed accompanied by enhanced release of AREG and TGF-alpha. The effect was not observed when cells were treated with the PKC-dependent ADAM17 activator PMA. Transformation of cells with a constitutively active ANO6 mutant led to spontaneous PS-exposure and to the release of ADAM17-substrates in the absence of any stimuli. Inhibitor experiments indicated that ANO6-mediated enhancement of substrate cleavage simultaneously broadened the spectrum of participating metalloproteinases. In complementary experiments, siRNA-mediated downregulation of ANO6 was shown to decrease ionophore-mediated release of TNFR1 in human umbilical vein endothelial cells (HUVECs). We conclude that ANO6, by virtue of its scramblase activity, may play a role as an important regulator of the ADAM-network in the plasma membrane.

Known structures and unknown mechanisms of TMEM16 scramblases and channels.

The TMEM16 family of membrane proteins is composed of both Ca2+-gated Cl- channels and Ca2+-dependent phospholipid scramblases. The functional diversity of TMEM16s underlies their involvement in numerous signal transduction pathways that connect changes in cytosolic Ca2+ levels to cellular signaling networks. Indeed, defects in the function of several TMEM16s cause a variety of genetic disorders, highlighting their fundamental pathophysiological importance. Here, we review how our mechanistic understanding of TMEM16 function has been shaped by recent functional and structural work. Remarkably, the recent determination of near-atomic-resolution structures of TMEM16 proteins of both functional persuasions has revealed how relatively minimal rearrangements in the substrate translocation pathway are sufficient to precipitate the dramatic functional differences that characterize the family. These structures, when interpreted in the light of extensive functional analysis, point to an unusual mechanism for Ca2+-dependent activation of TMEM16 proteins in which substrate permeation is regulated by a combination of conformational rearrangements and electrostatics. These breakthroughs pave the way to elucidate the mechanistic bases of ion and lipid transport by the TMEM16 proteins and unravel the molecular links between these transport activities and their function in human pathophysiology.

Single-molecule analysis of phospholipid scrambling by TMEM16F

Transmembrane protein 16F (TMEM16F) is a Ca2+-dependent phospholipid scramblase that translocates phospholipids bidirectionally between the leaflets of the plasma membrane. Phospholipid scrambling of TMEM16F causes exposure of phosphatidylserine in activated platelets to induce blood clotting and in differentiated osteoblasts to promote bone mineralization. Despite the importance of TMEM16F-mediated phospholipid scrambling in various biological reactions, the fundamental features of the scrambling reaction remain elusive due to technical difficulties in the preparation of a platform for assaying scramblase activity in vitro. Here, we established a method to express and purify mouse TMEM16F as a dimeric molecule by constructing a stable cell line and developed a microarray containing membrane bilayers with asymmetrically distributed phospholipids as a platform for single-molecule scramblase assays. The purified TMEM16F was integrated into the microarray, and monitoring of phospholipid translocation showed that a single TMEM16F molecule transported phospholipids nonspecifically between the membrane bilayers in a Ca2+-dependent manner. Thermodynamic analysis of the reaction indicated that TMEM16F transported 4.5 × 104 lipids per second at 25 °C, with an activation free energy of 47 kJ/mol. These biophysical features were similar to those observed with channels, which transport substrates by facilitating diffusion, and supported the stepping-stone model for the TMEM16F phospholipid scramblase.

Activation of the phospholipid scramblase TMEM16F by nanosecond pulsed electric fields (nsPEF) facilitates its diverse cytophysiological effects

Nanosecond pulsed electric fields (nsPEF) are emerging as a novel modality for cell stimulation and tissue ablation. However, the downstream protein effectors responsible for nsPEF bioeffects remain to be established. Here we demonstrate that nsPEF activate TMEM16F (or Anoctamin 6), a protein functioning as a Ca2+-dependent phospholipid scramblase and Ca2+-activated chloride channel. Using confocal microscopy and patch clamp recordings, we investigated the relevance of TMEM16F activation for several bioeffects triggered by nsPEF, including phosphatidylserine (PS) externalization, nanopore-conducted currents, membrane blebbing, and cell death. In HEK 293 cells treated with a single 300-ns pulse of 25.5 kV/cm, Tmem16f expression knockdown and TMEM16F-specific inhibition decreased nsPEF-induced PS exposure by 49 and 42%, respectively. Moreover, the Tmem16f silencing significantly decreased Ca2+-dependent chloride channel currents activated in response to the nanoporation. Tmem16f expression also affected nsPEF-induced cell blebbing, with only 20% of the silenced cells developing blebs compared with 53% of the control cells. This inhibition of cellular blebbing correlated with a 25% decrease in cytosolic free Ca2+ transient at 30 s after nanoporation. Finally, in TMEM16F-overexpressing cells, a train of 120 pulses (300 ns, 20 Hz, 6 kV/cm) decreased cell survival to 34% compared with 51% in control cells (*, p < 0.01). Taken together, these results indicate that TMEM16F activation by nanoporation mediates and enhances the diverse cellular effects of nsPEF.

Extracellular sphingomyelinase activity impairs TNF-α-induced endothelial cell death via ADAM17 activation and TNF receptor 1 shedding.

ADAM17, a prominent member of the “Disintegrin and Metalloproteinase” (ADAM) family, is an important regulator of endothelial cell proliferation and cell survival. The protease controls vital cellular functions through cleavage of growth factors, cytokines and their receptors including transforming growth factor-alpha (TGF-α), tumor necrosis factor-alpha (TNF-α) and TNF-α receptor 1 (TNFR1). TNF-α is the major inducer of endothelial cell death in cardiovascular diseases. The latter are also characterized by elevated plasma and tissue levels of extracellular sphingomyelinase (SMase). Whether the SMase affects ADAM activity and thus endothelial cell function has not been addressed to date. Here, we analyzed the effect of SMase on ADAM17-mediated shedding in COS7 cells and in human umbilical vein endothelial cells (HUVECs). Exposure to SMase significantly increased ADAM17-mediated release of alkaline-phosphatase (AP)-tagged TGF-α in COS7 cells and shedding of endogenously expressed TNFR1 in HUVECs. We previously presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase function. We found that SMase treatment led to PS externalization in both cell types. Transient non-apoptotic PS exposure is often mediated by Ca2+-dependent phospholipid scramblases. Accordingly, the Ca2+-chelator EGTA markedly reduced the breakdown of phospholipid asymmetry and shedding of TGF-α and TNFR1. Moreover, sheddase activity was significantly diminished in the presence of the competing PS-headgroup OPLS. SMase-stimulated TNFR1 shedding strikingly diminished TNF-α-induced signalling cascades and endothelial cell death. Taken together, our data suggest that SMase activity might act as protective factor for endothelial cells in cardiovascular diseases.

Role of Hydrophilic Groove of the TMEM16 Protein in Lipid Scrambling

The TMEM16 proteins are Ca2+-dependent ion channels and/or phospholipid scramblases that play key roles in a variety of physiological functions. Mutations in TMEM16F cause Scott syndrome, a bleeding disorder caused by impaired Ca2+-dependent externalization of phosphatidylserine (PS) in activated platelets, suggesting that this homologue functions as a scramblase. Explicit demonstration that some TMEM16 proteins are Ca2+-dependent phospholipid scramblases comes from reconstitution and assay of purified fungal TMEM16 homologs (afTMEM16 and nhTMEM16). The recently determined crystal structure of nhTMEM16, revealed a dimeric arrangement of the protein and the presence of hydrophilic groove on the opposite sides of the dimer interface exposed to the lipid membrane. Extensive mutagenesis studies have established the importance of the groove in the lipid scrambling by showing that mutations of specific residues in the groove region to either Trp or Ala significantly reduce the kinetics of lipid translocation. We have used a combination of unbiased and enhanced sampling atomistic molecular dynamics (MD) simulations of nhTMEM16 in lipid membrane and water environments to evaluate the structural context and detailed mechanisms for the measured changes in scrambling efficiency by mutant nhTMEM16 constructs. The findings center on the formation and mechanistic role of a continuous water pathway along the membrane-facing surface of the groove in the wild type protein that enables lipid translocation. The manner in which the mutations cause a significant reduction in the level of the hydration in the groove region points to a key role of the hydrophilic groove of TMEM16 in lipid scrambling.

Anoctamins/TMEM16 Proteins: Chloride Channels Flirting with Lipids and Extracellular Vesicles.

Anoctamin (ANO)/TMEM16 proteins exhibit diverse functions in cells throughout the body and are implicated in several human diseases. Although the founding members ANO1 (TMEM16A) and ANO2 (TMEM16B) are Ca2+-activated Cl- channels, most ANO paralogs are Ca2+-dependent phospholipid scramblases that serve as channels facilitating the movement (scrambling) of phospholipids between leaflets of the membrane bilayer. Phospholipid scrambling significantly alters the physical properties of the membrane and its landscape and has vast downstream signaling consequences. In particular, phosphatidylserine exposed on the external leaflet of the plasma membrane functions as a ligand for receptors vital for cell-cell communication. A major consequence of Ca2+-dependent scrambling is the release of extracellular vesicles that function as intercellular messengers by delivering signaling proteins and noncoding RNAs to alter target cell function. We discuss the physiological implications of Ca2+-dependent phospholipid scrambling, the extracellular vesicles associated with this activity, and the roles of ANOs in these processes.

Role of Ca2+ in the Stability and Function of TMEM16F and 16K

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.

Sizing up the Lipid Pathway in a TMEM16 Phospholipid Scramblase

Phospholipid scramblases collapse the plasma membrane lipid asymmetry, externalizing phosphatidylserine to trigger blood coagulation and mark apoptotic cells. Recently, members of the TMEM16 family were shown to function as Ca2+-dependent phospholipid scramblases as well as ion channels in cells and in reconstituted systems. The recent crystal structure of nhTMEM16 identified a hydrophilic groove, ∼11 A wide, exposed to the hydrophobic core of the membrane as the putative lipid pathway. It was proposed that lipid scrambling occurs via phospholipid headgroup-groove interactions. To test this mechanism and to gain insights into the properties of the lipid pathway we investigated whether there is a headgroup size cut-off. We generated NBD-labelled phospholipids with PEG molecules of increasing size (PEG1000-5000) attached to the headgroup and monitored scrambling by afTMEM16. We found that increasing the headgroup size up to PEG2000 (diameter ∼40 A) does not alter scrambling in high Ca2+, and even lipids conjugated to PEG5000 (diameter ∼50 A) are scrambled albeit less well. This suggests that the cut off is considerably larger than the width of the cavity. In the absence of Ca2+ TMEM16 scramblases show substantial activity. We found that the selectivity of the apo state differs from that of the bound state as they have different headgroup size selectivity: lipids conjugated with PEG2000 are poorly scrambled and PEG5000 is completely excluded. This is consistent with the idea that the activity in the apo state reflects an incomplete closure of the lipid pathway, which is narrower than in the Ca2+ bound state by ∼10 A. Taken together these results suggest that interactions between the groove and the lipid headgroup do not play a key role in scrambling or, alternatively, that in a membrane environment the cavity could be substantially wider than in the structure.

Necrotic Cells Actively Attract Phagocytes through the Collaborative Action of Two Distinct PS-Exposure Mechanisms.

Necrosis, a kind of cell death closely associated with pathogenesis and genetic programs, is distinct from apoptosis in both morphology and mechanism. Like apoptotic cells, necrotic cells are swiftly removed from animal bodies to prevent harmful inflammatory and autoimmune responses. In the nematode Caenorhabditis elegans, gain-of-function mutations in certain ion channel subunits result in the excitotoxic necrosis of six touch neurons and their subsequent engulfment and degradation inside engulfing cells. How necrotic cells are recognized by engulfing cells is unclear. Phosphatidylserine (PS) is an important apoptotic-cell surface signal that attracts engulfing cells. Here we observed PS exposure on the surface of necrotic touch neurons. In addition, the phagocytic receptor CED-1 clusters around necrotic cells and promotes their engulfment. The extracellular domain of CED-1 associates with PS in vitro. We further identified a necrotic cell-specific function of CED-7, a member of the ATP-binding cassette (ABC) transporter family, in promoting PS exposure. In addition to CED-7, anoctamin homolog-1 (ANOH-1), the C. elegans homolog of the mammalian Ca2+-dependent phospholipid scramblase TMEM16F, plays an independent role in promoting PS exposure on necrotic cells. The combined activities from CED-7 and ANOH-1 ensure efficient exposure of PS on necrotic cells to attract their phagocytes. In addition, CED-8, the C. elegans homolog of mammalian Xk-related protein 8 also makes a contribution to necrotic cell-removal at the first larval stage. Our work indicates that cells killed by different mechanisms (necrosis or apoptosis) expose a common “eat me” signal to attract their phagocytic receptor(s); furthermore, unlike what was previously believed, necrotic cells actively present PS on their outer surfaces through at least two distinct molecular mechanisms rather than leaking out PS passively.

Anoctamin 6 mediates effects essential for innate immunity downstream of P2X7 receptors in macrophages.

Purinergic P2X7 receptors (P2X7R) are fundamental to innate immune response. In macrophages, transient stimulation of P2X7R activates several transport mechanisms and induces the scrambling of phospholipids with subsequent membrane blebbing and apoptosis. These processes support phagocytosis and subsequent killing of phagocytosed bacteria. Here we demonstrate that the stimulation of P2X7 receptors activates anoctamin 6 (ANO6, TMEM16F), a protein that functions as Ca(2+) dependent phospholipid scramblase and Ca(2+)-activated Cl(-) channel. Inhibition or knockdown of ANO6 attenuates ATP-induced cell shrinkage, cell migration and phospholipid scrambling. In mouse macrophages, Ano6 produces large ion currents by stimulation of P2X7 receptors and contributes to ATP-induced membrane blebbing and apoptosis, which is largely reduced in macrophages from Ano6-/- mice. ANO6 supports bacterial phagocytosis and killing by mouse and human THP-1 macrophages. Our data demonstrate that anoctamin 6 is an essential component of the immune defense by macrophages.

Breaking asymmetry: phospholipid scrambling by GPCRs and TMEM16 proteins (351.2)

Polar lipids must flip rapidly, and often bidirectionally, across membranes to support cellular life. For example, phospholipids are synthesized on the cytoplasmic face of the endoplasmic reticulum (ER) and must be apportioned between the luminal and cytoplasmic leaflets for uniform expansion of the ER during cell growth. Transbilayer movement of lipids is also needed for the generation, maintenance and dissipation of transbilayer lipid asymmetry at the plasma membrane. As flipping is energetically costly, specialized transporters increase its intrinsically low rate to a physiologically relevant level. While some of these transporters couple ATP hydrolysis to lipid movement, many function without any discernible metabolic energy input. The molecular identification of these latter ‘ATP‐independent flippases’, also termed ‘scramblases’, has eluded researchers for decades. We now report the first examples of this novel class of proteins: rhodopsin, a prototypical G protein‐coupled receptor, is a constitutively active, ATP‐independent phospholipid flippase (Menon et al. (2011) Curr. Biol. 21: 149), and afTMEM16, a fungal homolog of the TMEM16 family of ion channels is a Ca2+‐dependent phospholipid scramblase (Malvezzi et al. (2013) Nature Commun. 4: 2367). Both proteins break transbilayer lipid asymmetry.

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.