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• W2961986906 abstract "•Ca2+ binding enhances lipid association with TMEM16F calcium-activated scramblase•TMEM16F in nanodiscs supplemented with PIP2 is stably bound to a PS-like lipid•PIP2 supplement leads to membrane distortion that varies with TMEM16F conformation•Lipid binding residues outside of the protein enclosed pore affect lipid scrambling As a Ca2+-activated lipid scramblase and ion channel that mediates Ca2+ influx, TMEM16F relies on both functions to facilitate extracellular vesicle generation, blood coagulation, and bone formation. How a bona fide ion channel scrambles lipids remains elusive. Our structural analyses revealed the coexistence of an intact channel pore and PIP2-dependent protein conformation changes leading to membrane distortion. Correlated to the extent of membrane distortion, many tightly bound lipids are slanted. Structure-based mutagenesis studies further reveal that neutralization of some lipid-binding residues or those near membrane distortion specifically alters the onset of lipid scrambling, but not Ca2+ influx, thus identifying features outside of channel pore that are important for lipid scrambling. Together, our studies demonstrate that membrane distortion does not require open hydrophilic grooves facing the membrane interior and provide further evidence to suggest separate pathways for lipid scrambling and ion permeation. As a Ca2+-activated lipid scramblase and ion channel that mediates Ca2+ influx, TMEM16F relies on both functions to facilitate extracellular vesicle generation, blood coagulation, and bone formation. How a bona fide ion channel scrambles lipids remains elusive. Our structural analyses revealed the coexistence of an intact channel pore and PIP2-dependent protein conformation changes leading to membrane distortion. Correlated to the extent of membrane distortion, many tightly bound lipids are slanted. Structure-based mutagenesis studies further reveal that neutralization of some lipid-binding residues or those near membrane distortion specifically alters the onset of lipid scrambling, but not Ca2+ influx, thus identifying features outside of channel pore that are important for lipid scrambling. Together, our studies demonstrate that membrane distortion does not require open hydrophilic grooves facing the membrane interior and provide further evidence to suggest separate pathways for lipid scrambling and ion permeation. Linked to the human bleeding disorder Scott syndrome (Boisseau et al., 2018Boisseau P. Bene M.C. Besnard T. Pachchek S. Giraud M. Talarmain P. Robillard N. Gourlaouen M.A. Bezieau S. Fouassier M. A new mutation of ANO6 in two familial cases of Scott syndrome.Br. J. Haematol. 2018; 180: 750-752Crossref PubMed Scopus (12) Google Scholar, Suzuki et al., 2010Suzuki J. Umeda M. Sims P.J. Nagata S. Calcium-dependent phospholipid scrambling by TMEM16F.Nature. 2010; 468: 834-838Crossref PubMed Scopus (640) Google Scholar), TMEM16F is a Ca2+-activated scramblase as well as ion channel that mediates Ca2+ influx (Alvadia et al., 2019Alvadia C. Lim N.K. Clerico Mosina V. Oostergetel G.T. Dutzler R. Paulino C. Cryo-EM structures and functional characterization of the murine lipid scramblase TMEM16F.eLife. 2019; 8: e44365Crossref PubMed Scopus (77) Google Scholar, Han et al., 2019Han T.W. Ye W. Bethel N.P. Zubia M. Kim A. Li K.H. Burlingame A.L. Grabe M. Jan Y.N. Jan L.Y. Chemically induced vesiculation as a platform for studying TMEM16F activity.Proc. Natl. Acad. Sci. USA. 2019; 116: 1309-1318Crossref PubMed Scopus (16) Google Scholar, Watanabe et al., 2018Watanabe R. Sakuragi T. Noji H. Nagata S. Single-molecule analysis of phospholipid scrambling by TMEM16F.Proc. Natl. Acad. Sci. USA. 2018; 115: 3066-3071Crossref PubMed Scopus (46) Google Scholar, Yang et al., 2012Yang H. Kim A. David T. Palmer D. Jin T. Tien J. Huang F. Cheng T. Coughlin S.R. Jan Y.N. Jan L.Y. TMEM16F forms a Ca2+-activated cation channel required for lipid scrambling in platelets during blood coagulation.Cell. 2012; 151: 111-122Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). The dual function of this protein is critical for exposing phosphatidylserine (PS) (Zwaal et al., 2004Zwaal R.F. Comfurius P. Bevers E.M. Scott syndrome, a bleeding disorder caused by defective scrambling of membrane phospholipids.Biochim. Biophys. Acta. 2004; 1636: 119-128Crossref PubMed Scopus (191) Google Scholar) and generating and releasing of extracellular vesicles (EVs) by the platelets (György et al., 2011György B. Szabó T.G. Pásztói M. Pál Z. Misják P. Aradi B. László V. Pállinger E. Pap E. Kittel A. et al.Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles.Cell. Mol. Life Sci. 2011; 68: 2667-2688Crossref PubMed Scopus (1472) Google Scholar, Raposo and Stoorvogel, 2013Raposo G. Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends.J. Cell Biol. 2013; 200: 373-383Crossref PubMed Scopus (5145) Google Scholar, Sims et al., 1989Sims P.J. Wiedmer T. Esmon C.T. Weiss H.J. Shattil S.J. Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. Studies in Scott syndrome: an isolated defect in platelet procoagulant activity.J. Biol. Chem. 1989; 264: 17049-17057Abstract Full Text PDF PubMed Google Scholar, Whitlock and Hartzell, 2017Whitlock J.M. Hartzell H.C. Anoctamins/TMEM16 proteins: chloride channels flirting with lipids and extracellular vesicles.Annu. Rev. Physiol. 2017; 79: 119-143Crossref PubMed Scopus (92) Google Scholar). These processes play important roles in bone formation (Ehlen et al., 2013Ehlen H.W. Chinenkova M. Moser M. Munter H.M. Krause Y. Gross S. Brachvogel B. Wuelling M. Kornak U. Vortkamp A. Inactivation of anoctamin-6/Tmem16f, a regulator of phosphatidylserine scrambling in osteoblasts, leads to decreased mineral deposition in skeletal tissues.J. Bone Miner. Res. 2013; 28: 246-259Crossref PubMed Scopus (91) Google Scholar, Ousingsawat et al., 2015bOusingsawat J. Wanitchakool P. Schreiber R. Wuelling M. Vortkamp A. Kunzelmann K. Anoctamin-6 controls bone mineralization by activating the calcium transporter NCX1.J. Biol. Chem. 2015; 290: 6270-6280Crossref PubMed Scopus (28) Google Scholar) and anti-inflammatory response of neutrophils (Headland et al., 2015Headland S.E. Jones H.R. Norling L.V. Kim A. Souza P.R. Corsiero E. Gil C.D. Nerviani A. Dell’Accio F. Pitzalis C. et al.Neutrophil-derived microvesicles enter cartilage and protect the joint in inflammatory arthritis.Sci. Transl. Med. 2015; 7: 315ra190Crossref PubMed Scopus (196) Google Scholar) as well as blood coagulation (Fujii et al., 2015Fujii T. Sakata A. Nishimura S. Eto K. Nagata S. TMEM16F is required for phosphatidylserine exposure and microparticle release in activated mouse platelets.Proc. Natl. Acad. Sci. USA. 2015; 112: 12800-12805Crossref PubMed Scopus (142) Google Scholar, Wolf, 1967Wolf P. The nature and significance of platelet products in human plasma.Br. J. Haematol. 1967; 13: 269-288Crossref PubMed Scopus (1106) Google Scholar, Yang et al., 2012Yang H. Kim A. David T. Palmer D. Jin T. Tien J. Huang F. Cheng T. Coughlin S.R. Jan Y.N. Jan L.Y. TMEM16F forms a Ca2+-activated cation channel required for lipid scrambling in platelets during blood coagulation.Cell. 2012; 151: 111-122Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, Zwaal et al., 2004Zwaal R.F. Comfurius P. Bevers E.M. Scott syndrome, a bleeding disorder caused by defective scrambling of membrane phospholipids.Biochim. Biophys. Acta. 2004; 1636: 119-128Crossref PubMed Scopus (191) Google Scholar). For a channel that is activated by Ca2+ and mediates Ca2+ influx—a positive feedback that needs to be held in check to prevent excessive increase of intracellular Ca2+ level, TMEM16F has a small single-channel conductance (Yang et al., 2012Yang H. Kim A. David T. Palmer D. Jin T. Tien J. Huang F. Cheng T. Coughlin S.R. Jan Y.N. Jan L.Y. TMEM16F forms a Ca2+-activated cation channel required for lipid scrambling in platelets during blood coagulation.Cell. 2012; 151: 111-122Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). Moreover, TMEM16F channel activity is reduced by Ca2+-induced degradation of phosphatidylinositol-(4, 5)-bisphosphate (PIP2) (Ye et al., 2018Ye W. Han T.W. Nassar L.M. Zubia M. Jan Y.N. Jan L.Y. Phosphatidylinositol-(4, 5)-bisphosphate regulates calcium gating of small-conductance cation channel TMEM16F.Proc. Natl. Acad. Sci. USA. 2018; 115: E1667-E1674Crossref PubMed Scopus (47) Google Scholar), and its ion selectivity shifts dynamically with rising Ca2+ levels (Ye et al., 2019Ye W. Han T.W. He M. Jan Y.N. Jan L.Y. Dynamic change of electrostatic field in TMEM16F permeatioin pathway shifts its ion selectivity.bioRxiv. 2019; https://doi.org/10.1101/515569Crossref Google Scholar); this dynamic shift may contribute to the time-dependent change and variations of ion selectivity revealed in whole-cell recordings (Bricogne et al., 2019Bricogne C. Fine M. Pereira P.M. Sung J. Tijani M. Wang Y. Henriques R. Collins M.K. Hilgemann D.W. TMEM16F activation by Ca2+ triggers plasma membrane expansion and directs PD-1 trafficking.Sci. Rep. 2019; 9: 619Crossref PubMed Scopus (23) Google Scholar, Grubb et al., 2013Grubb S. Poulsen K.A. Juul C.A. Kyed T. Klausen T.K. Larsen E.H. Hoffmann E.K. TMEM16F (Anoctamin 6), an anion channel of delayed Ca(2+) activation.J. Gen. Physiol. 2013; 141: 585-600Crossref PubMed Scopus (92) Google Scholar, Nguyen et al., 2019Nguyen D.M. Chen L.S. Yu W.P. Chen T.Y. Comparison of ion transport determinants between a TMEM16 chloride channel and phospholipid scramblase.J. Gen. Physiol. 2019; 151: 518-531Crossref PubMed Scopus (12) Google Scholar, Ousingsawat et al., 2015aOusingsawat J. Wanitchakool P. Kmit A. Romao A.M. Jantarajit W. Schreiber R. Kunzelmann K. Anoctamin 6 mediates effects essential for innate immunity downstream of P2X7 receptors in macrophages.Nat. Commun. 2015; 6: 6245Crossref PubMed Scopus (100) Google Scholar, Ousingsawat et al., 2015bOusingsawat J. Wanitchakool P. Schreiber R. Wuelling M. Vortkamp A. Kunzelmann K. Anoctamin-6 controls bone mineralization by activating the calcium transporter NCX1.J. Biol. Chem. 2015; 290: 6270-6280Crossref PubMed Scopus (28) Google Scholar, Shimizu et al., 2013Shimizu T. Iehara T. Sato K. Fujii T. Sakai H. Okada Y. TMEM16F is a component of a Ca2+-activated Cl- channel but not a volume-sensitive outwardly rectifying Cl- channel.Am. J. Physiol. Cell Physiol. 2013; 304: C748-C759Crossref PubMed Scopus (101) Google Scholar, Yu et al., 2015Yu K. Whitlock J.M. Lee K. Ortlund E.A. Cui Y.Y. Hartzell H.C. Identification of a lipid scrambling domain in ANO6/TMEM16F.eLife. 2015; 4: e06901Crossref PubMed Scopus (122) Google Scholar). The structural basis of TMEM16F modulation by Ca2+ and PIP2 remains an open and interesting question. TMEM16F is functionally distinct from its closely related family member TMEM16A (Dang et al., 2017Dang S. Feng S. Tien J. Peters C.J. Bulkley D. Lolicato M. Zhao J. Zuberbühler K. Ye W. Qi L. et al.Cryo-EM structures of the TMEM16A calcium-activated chloride channel.Nature. 2017; 552: 426-429Crossref PubMed Scopus (186) Google Scholar, Paulino et al., 2017Paulino C. Kalienkova V. Lam A.K.M. Neldner Y. Dutzler R. Activation mechanism of the calcium-activated chloride channel TMEM16A revealed by cryo-EM.Nature. 2017; 552: 421-425Crossref PubMed Scopus (157) Google Scholar), which is a Ca2+-activated Cl− channel with no scramblase activity (Yu et al., 2015Yu K. Whitlock J.M. Lee K. Ortlund E.A. Cui Y.Y. Hartzell H.C. Identification of a lipid scrambling domain in ANO6/TMEM16F.eLife. 2015; 4: e06901Crossref PubMed Scopus (122) Google Scholar) but somewhat similar to the fungal scramblases nhTMEM16 and afTMEM16 (Brunner et al., 2014Brunner J.D. Lim N.K. Schenck S. Duerst A. Dutzler R. X-ray structure of a calcium-activated TMEM16 lipid scramblase.Nature. 2014; 516: 207-212Crossref PubMed Scopus (303) Google Scholar, Falzone et al., 2019Falzone M.E. Rheinberger J. Lee B.C. Peyear T. Sasset L. Raczkowski A.M. Eng E.T. Di Lorenzo A. Andersen O.S. Nimigean C.M. Accardi A. Structural basis of Ca2+-dependent activation and lipid transport by a TMEM16 scramblase.eLife. 2019; 8: e43229Crossref PubMed Scopus (43) Google Scholar, Kalienkova et al., 2019Kalienkova V. Clerico Mosina V. Bryner L. Oostergetel G.T. Dutzler R. Paulino C. Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM.eLife. 2019; 8: e44364Crossref PubMed Scopus (58) Google Scholar) that may permeate both cations and anions (Falzone et al., 2018Falzone M.E. Malvezzi M. Lee B.C. Accardi A. Known structures and unknown mechanisms of TMEM16 scramblases and channels.J. Gen. Physiol. 2018; 150: 933-947Crossref PubMed Scopus (70) Google Scholar). Structures of these fungal scramblases have a half-open subunit cavity with a hydrophilic groove open to the interior of the lipid bilayer. Such configuration is compatible with the lipid-scrambling model, in which the charged headgroup of lipid goes through this open groove while being scrambled, the so-called “credit card” model (Pomorski and Menon, 2006Pomorski T. Menon A.K. Lipid flippases and their biological functions.Cell. Mol. Life Sci. 2006; 63: 2908-2921Crossref PubMed Scopus (217) Google Scholar) for lipid scrambling (Bethel and Grabe, 2016Bethel N.P. Grabe M. Atomistic insight into lipid translocation by a TMEM16 scramblase.Proc. Natl. Acad. Sci. USA. 2016; 113: 14049-14054Crossref PubMed Scopus (72) Google Scholar, Jiang et al., 2017Jiang T. Yu K. Hartzell H.C. Tajkhorshid E. Lipids and ions traverse the membrane by the same physical pathway in the nhTMEM16 scramblase.eLife. 2017; 6: e28671Crossref PubMed Scopus (64) Google Scholar, Lee et al., 2018Lee B.C. Khelashvili G. Falzone M. Menon A.K. Weinstein H. Accardi A. Gating mechanism of the extracellular entry to the lipid pathway in a TMEM16 scramblase.Nat. Commun. 2018; 9: 3251Crossref PubMed Scopus (51) Google Scholar, Whitlock and Hartzell, 2016Whitlock J.M. Hartzell H.C. A Pore Idea: the ion conduction pathway of TMEM16/ANO proteins is composed partly of lipid.Pflugers Arch. 2016; 468: 455-473Crossref PubMed Scopus (51) Google Scholar). Furthermore, membrane distortion was observed at the site of this open groove (Falzone et al., 2019Falzone M.E. Rheinberger J. Lee B.C. Peyear T. Sasset L. Raczkowski A.M. Eng E.T. Di Lorenzo A. Andersen O.S. Nimigean C.M. Accardi A. Structural basis of Ca2+-dependent activation and lipid transport by a TMEM16 scramblase.eLife. 2019; 8: e43229Crossref PubMed Scopus (43) Google Scholar, Kalienkova et al., 2019Kalienkova V. Clerico Mosina V. Bryner L. Oostergetel G.T. Dutzler R. Paulino C. Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM.eLife. 2019; 8: e44364Crossref PubMed Scopus (58) Google Scholar). Such distortion is likely caused by the open groove and could reduce the energy barrier of scrambling a lipid from one leaflet to the other, further strengthening the credit card model. However, recent findings that the fungal afTMEM16 scramblase can flip lipids with headgroups larger than the width of the open groove raises the possibility of “out-of-the-groove” lipid scrambling (Malvezzi et al., 2018Malvezzi M. Andra K.K. Pandey K. Lee B.C. Falzone M.E. Brown A. Iqbal R. Menon A.K. Accardi A. Out-of-the-groove transport of lipids by TMEM16 and GPCR scramblases.Proc. Natl. Acad. Sci. USA. 2018; 115: E7033-E7042Crossref PubMed Scopus (33) Google Scholar), which is likely also facilitated by the membrane distortion (Falzone et al., 2019Falzone M.E. Rheinberger J. Lee B.C. Peyear T. Sasset L. Raczkowski A.M. Eng E.T. Di Lorenzo A. Andersen O.S. Nimigean C.M. Accardi A. Structural basis of Ca2+-dependent activation and lipid transport by a TMEM16 scramblase.eLife. 2019; 8: e43229Crossref PubMed Scopus (43) Google Scholar). The dual functionality of channel and scramblase of TMEM16F and its fungal homologs (Falzone et al., 2018Falzone M.E. Malvezzi M. Lee B.C. Accardi A. Known structures and unknown mechanisms of TMEM16 scramblases and channels.J. Gen. Physiol. 2018; 150: 933-947Crossref PubMed Scopus (70) Google Scholar) raises intriguing questions: how might an open groove permeate ions? If TMEM16F has a channel pore similar to the enclosed pore of TMEM16A, how might the membrane be distorted to facilitate lipid scrambling without an open hydrophilic groove facing the membrane interior? We combined structural and mutagenesis studies to explore the possibility that lipid scrambling by TMEM16F does not require an open hydrophilic groove. Using single-particle electron cryo-microscopy (cryo-EM), we determined four structures of TMEM16F under different physiological conditions. Our structural analyses reveal an enclosed channel pore in all these structures for different functional states and PIP2-dependent membrane distortion and thinning. Such membrane distortions vary with different protein conformations, and they are accompanied with tightly bound lipids that are in slanted orientations. Mutations of some residues that either bind lipids or are associated with membrane distortion impact the onset of lipid scrambling, but not Ca2+ influx, thus establishing a connection between membrane distortion and lipid scrambling. The specific effects of these mutations further suggest that lipid scrambling and ion permeation are not always correlated. Together, our studies reveal that membrane distortion and lipid scrambling may take place without an open groove and further suggest that lipid scrambling and ion permeation do not share the same pathway. Recombinant mouse TMEM16F was expressed in HEK293 cells and purified in digitonin with added lipids. We determined two cryo-EM structures of TMEM16F in digitonin, without and with Ca2+ (Figures 1 and S1; Table S1). The overall resolutions of both structures are better than 4 Å. The dimeric architecture of TMEM16F resembles that of TMEM16A with ten transmembrane segments (TM1–10). Unexpectedly, a single Ca2+ ion is resolved in the density map of Ca2+-bound TMEM16F, which is coordinated by N621 (and likely E624 as well) on TM6, E670 on TM7, and D703 on TM8 (Figure 1C). Comparison of these structures revealed that Ca2+ binding induced a conformational change in the TM6, from a helix that is slightly bent at G615 to a straight helix (Figure 1G). Another noticeable change involves the extracellular loops (Figure 1H); Ca2+ binding facilitates the stable binding of lipids to clusters of basic residues on the TM9-TM10 loop (Figures 1I and 1J). When the Ca2+-free and Ca2+-bound TMEM16F maps are compared at the same resolution, it appears that the changes of TMEM16F conformation induced by Ca2+ binding facilitate the binding of a greater number of lipids (Figures 1A and 1D). Considering that the channel and lipid scrambling activities of TMEM16F are Ca2+ dependent, having more stably associated lipids in Ca2+-bound condition may be related to these activities. First, we test the functional roles of residues involved in Ca2+-induced conformational changes, such as N621 on the TM6 involved in Ca2+ binding (Figures 1C and S2A), R542 on the TM4-TM5 loop that contacts the TM2-TM3 loop only in Ca2+-free TMEM16F (Figures S2C and S2D), and K706 on TM8 that is in the proximity of E624 solely in the Ca2+-bound TMEM16F (Figures S2A and S2B). We used Ca2+ imaging to assay TMEM16F-dependent Ca2+ influx and live imaging of PS exposure to assay the lipid scrambling activity. We also examined giant plasma membrane vesicle (GPMV) generation, which requires both TMEM16F-dependent Ca2+ influx and lipid scrambling (Han et al., 2019Han T.W. Ye W. Bethel N.P. Zubia M. Kim A. Li K.H. Burlingame A.L. Grabe M. Jan Y.N. Jan L.Y. Chemically induced vesiculation as a platform for studying TMEM16F activity.Proc. Natl. Acad. Sci. USA. 2019; 116: 1309-1318Crossref PubMed Scopus (16) Google Scholar). Time-lapse imaging of 500–1,000 cells with 10× magnification was performed to concurrently monitor GPMV formation and Ca2+ influx, and PS exposure was monitored in separate experiments involving time-lapse imaging of individual cells viewed with 60× magnification (Figures 2 and S3). All measurements were normalized by the expression levels (Figures S3G–S3I; Table S2). Control experiments revealed that, whereas TMEM16F is not required for the initial chemically induced Ca2+ release from internal stores, TMEM16F is essential for the subsequent Ca2+ influx (Figures S3A–S3F); HEK293 cells without heterologous expression of TMEM16F do not exhibit this Ca2+ influx, PS exposure, or GPMV formation (Han et al., 2019Han T.W. Ye W. Bethel N.P. Zubia M. Kim A. Li K.H. Burlingame A.L. Grabe M. Jan Y.N. Jan L.Y. Chemically induced vesiculation as a platform for studying TMEM16F activity.Proc. Natl. Acad. Sci. USA. 2019; 116: 1309-1318Crossref PubMed Scopus (16) Google Scholar). Because the initiation of Ca2+ influx and PS exposure depends primarily on TMEM16F functions, we focused on the time of onset, which is the maximum of the second derivative of the curve (maximal acceleration; see STAR Methods). N621A and K706A mutations significantly reduced Ca2+ influx (Figure 2A) and GPMV formation (Figure 2C). N621A also suppressed PS exposure (Figure 2B). In contrast, R542A accelerated the onset of Ca2+ influx, PS exposure, and GPMV generation (Figures 2G–2I). These results support the notion that Ca2+ binding is important for both lipid scrambling and channel activities. Next, we tested the functional roles of two clusters of basic residues on the TM9-TM10 loop that bind lipids in Ca2+-bound TMEM16F (Figures 1I and 1J). Remarkably, the R753A/H768A double mutation specifically delayed the onset of PS exposure (Figures 2E and 2H) without affecting the onset of Ca2+ influx (Figures 2D and 2G), and the R813A/K823A/H824A triple mutation slowed Ca2+ influx, PS exposure, as well as GPMV generation (Figures 2D–2I). The finding that the lipid binding residues R753 and H768 specifically affect the onset of PS exposure, but not Ca2+ influx, indicates that they are functionally important for lipid scrambling rather than channel function. We further reconstituted digitonin-solubilized TMEM16F into lipid nanodiscs for structure determinations (Figure S4; Table S1). We determined structures of Ca2+-bound TMEM16F in lipid nanodiscs without PIP2 to ∼7 Å and with supplement of PIP2. From the latter, we classified particles into two classes with distinct conformations (Figures 3, 4, and 5; Videos S1 and S2); whereas class 2 is slightly influenced by preferred orientation, both have better than 4-Å resolution (Figure S4). Major structural features, including TM helices and membrane surrounding the protein, are well resolved in all structures (Figures S4 and S5). Similar to what we observed in the digitonin structures, we found a single Ca2+ ion with similar arrangements of Ca2+-coordinating residues in the PIP2 supplemented nanodiscs (Figures S6A–S6C), at a location comparable to one of the densities for Ca2+ ions in TMEM16F in nanodiscs without PIP2 supplement (Figures S2E–S2J; Alvadia et al., 2019Alvadia C. Lim N.K. Clerico Mosina V. Oostergetel G.T. Dutzler R. Paulino C. Cryo-EM structures and functional characterization of the murine lipid scramblase TMEM16F.eLife. 2019; 8: e44365Crossref PubMed Scopus (77) Google Scholar). Interestingly, comparison of structures after filtering all density maps to the same resolution of ∼7 Å revealed that, in both classes of TMEM16F structures in nanodiscs with PIP2 supplementation, the membrane is distorted and thinned by ∼30% for class 1 and ∼40% for class 2, respectively (Figures 3A, 3B, 4A, S6F, and S6G) near the site where TM6 is kinked at P628 with its lower part unwound and veering away (Figures 3D–3I). Many lipids found stably bound to the protein near the membrane distortion site are in slanted orientations; the slant angles vary with the protein conformation of these two classes of TMEM16F, however, we cannot be sure about the correspondence of the bound lipids in the two classes (Figure 4B).Figure 4TMEM16F-Conformation-Dependent Orientation of Bound Lipids and Functional Tests of Basic Residues Associated with Membrane DistortionShow full caption(A) Transmembrane helices and loops in ribbon diagram for class 2 TMEM16F (blue) and bound lipids (magenta) shown with distorted membrane (gray). Membrane distortion is near TM3 and TM4 (red) and TM6 and pre-TM1 elbow (orange) with clusters of basic residues (encircled in dashed line) that are tested with mutagenesis.(B) Superimposition of lipids bound to class 1 (gold) and class 2 (blue) TMEM16F in PIP2-supplemented nanodiscs. Dotted circles mark the lipid headgroups that interact with extracellular loops.(C–E) Live imaging of TMEM16F-dependent Ca2+ influx (C), PS exposure (D), and GPMV generation (E). Time-lapse imaging of 500–1,000 cells with 10× magnification was performed to concurrently monitor GPMV formation and Ca2+ influx via Fluo-8 fluorescence. Time-lapse imaging of individual cells viewed with 60× magnification was performed to monitor PS exposure via pSIVA fluorescence. Data are represented as mean ± SEM.(F–H) Scattered dot plots of time of onset of TMEM16F-dependent Ca2+ influx (F), PS exposure (G), and GPMV generation (H). Time of onset could not be determined for those time courses with a linear rather than sigmoidal rise. Data are represented as mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Statistical significance of all mutants as compared to TMEM16F WT is determined by one-way ANOVA followed by Holm- Šídák multiple comparisons test.See also Figure S3, Table S2, and Videos S1 and S2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5PIP2-Dependent Lipid Binding Near Membrane Distortion and Functional Tests of Lipid-Binding ResiduesShow full caption(A) Ca2+-bound TMEM16F (class 2) in PIP2 supplemented nanodiscs. Bound lipids are in cyan, and the one shown in (B) is in magenta.(B) A phosphatidylserine (PS) (fatty acid tails in magenta and headgroup encircled with dashed line, superimposed on the sharpened electron density map in light gray) has its polar headgroup coordinated by R478 on TM3 and K590 and R592 on the TM5-TM6 loop. TMEM16Fs from class 1 (in orange, top panel) and class 2 (in blue, bottom panel) are shown with the same protein orientation.(C–E) Live imaging of TMEM16F-dependent Ca2+ influx (C), PS exposure (D), and GPMV generation (E). Time-lapse imaging of 500–1,000 cells with 10× magnification was performed to concurrently monitor GPMV formation and Ca2+ influx via Fluo-8 fluorescence. Time-lapse imaging of individual cells viewed with 60× magnification was performed to monitor PS exposure via pSIVA fluorescence. Data are represented as mean ± SEM.(F–H) Scattered dot plots of time of onset of TMEM16F-dependent Ca2+ influx (F), PS exposure (G), and GPMV generation (H). Time of onset could not be determined for those time courses with a linear rather than sigmoidal rise. Data are represented as mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Statistical significance of all mutants as compared to TMEM16F WT is determined by one-way ANOVA followed by Holm- Šídák multiple comparisons test.See also Figure S3 and Table S2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Transmembrane helices and loops in ribbon diagram for class 2 TMEM16F (blue) and bound lipids (magenta) shown with distorted membrane (gray). Membrane distortion is near TM3 and TM4 (red) and TM6 and pre-TM1 elbow (orange) with clusters of basic residues (encircled in dashed line) that are tested with mutagenesis. (B) Superimposition of lipids bound to class 1 (gold) and class 2 (blue) TMEM16F in PIP2-supplemented nanodiscs. Dotted circles mark the lipid headgroups that interact with extracellular loops. (C–E) Live imaging of TMEM16F-dependent Ca2+ influx (C), PS exposure (D), and GPMV generation (E). Time-lapse imaging of 500–1,000 cells with 10× magnification was performed to concurrently monitor GPMV formation and Ca2+ influx via Fluo-8 fluorescence. Time-lapse imaging of individual cells viewed with 60× magnification was performed to monitor PS exposure via pSIVA fluorescence. Data are represented as mean ± SEM. (F–H) Scattered dot plots of time of onset of TMEM16F-dependent Ca2+ influx (F), PS exposure (G), and GPMV generation (H). Time of onset could not be determined for those time courses with a linear rather than sigmoidal rise. Data are represented as mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Statistical significance of all mutants as compared to TMEM16F WT is determined by one-way ANOVA followed by Holm- Šídák multiple comparisons test. See also Figure S3, Table S2, and Videos S1 and S2. (A) Ca2+-bound TMEM16F (class 2) in PIP2 supplemented nanodiscs. Bound lipids are in cyan, and the one shown in (B) is in magenta. (B) A phosphatidylserine (PS) (fatty acid tails in magenta and headgroup encircled with dashed line, superimposed on the sharpened electron density map in light gray) has its polar headgroup coordinated by R478 on TM3 and K590 and R592 on the TM5-TM6 loop. TMEM16Fs from class 1 (in orange, top panel) and class 2 (in blue, bottom panel) are shown with the same protein orientation. (C–E) Live imaging of TMEM16F-dependent Ca2+ influx (C), PS exposure (D), and GPMV generation (E). Time-lapse imaging of 500–1,000 cells with 10× magnification was performed to concurrently monitor GPMV formation and Ca2+" @default.
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• W2961986906 date "2019-07-01" @default.
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• W2961986906 title "Cryo-EM Studies of TMEM16F Calcium-Activated Ion Channel Suggest Features Important for Lipid Scrambling" @default.
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• W2961986906 doi "https://doi.org/10.1016/j.celrep.2019.06.023" @default.
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