Gating mechanism of the extracellular entry to the lipid pathway in a TMEM16 scramblase

Lee, Byoung-Cheol; Khelashvili, George; Falzone, Maria; Menon, Anant K.; Weinstein, Harel; Accardi, Alessio

doi:10.1038/s41467-018-05724-1

Cited by 80 publications

(204 citation statements)

References 64 publications

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“…First, the residue corresponding to Subdued Glu-716 has been proposed to serve as an extracellular entrance controlling phospholipid permeation through nhTMEM16 -CaPLSase (30). Mutating this residue in fungal nhTMEM16 -and TMEM16F-CaPLSase abolished phospholipid scrambling (24,31,32). Consistent with the importance of this residue in controlling phospholipid permeation, Subdued E716A also abrogated ionomycin-induced phospholipid scrambling and PS externalization ( Fig.…”

Section: Subdued Is a Caplsasementioning

confidence: 57%

Drosophila Subdued is a moonlighting transmembrane protein 16 (TMEM16) that transports ions and phospholipids

Yang

2019

Journal of Biological Chemistry

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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 Ca 2؉ -activated Cl ؊ channels (CaCCs), whereas mammalian TMEM16F, fungal afTMEM16, and nhTMEM16 are moonlighting (multifunctional) proteins with both Ca 2؉ -activated phospholipid scramblase (CaPLSase) and Ca 2؉ -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.

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Section: Subdued Is a Caplsasementioning

confidence: 57%

Drosophila Subdued is a moonlighting transmembrane protein 16 (TMEM16) that transports ions and phospholipids

Yang

2019

Journal of Biological Chemistry

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“…4f-i). Interestingly, the TM3-5 unit forms one side of the hydrophobic dimer cavity which could provide space to accommodate these movements, potentially even allowing greater opening of the groove, as recently proposed 38 , to accommodate PEGylated lipids 39 .…”

Section: Cryo-em Structures Of Tmem16k In Low Ca 2+ Reveal a Closed Gmentioning

confidence: 88%

The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K

Bushell¹,

Pike²,

Falzone³

et al. 2018

Preprint

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Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase and ion channel activity, or specific chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident calcium-regulated lipid scramblase. Our crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional structures solved by cryo-EM reveal extensive conformational changes extending from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity. Our results suggest mechanisms by which missense variants of TMEM16K could cause SCAR10 ataxia, providing new hypotheses to explore for therapy. 4 Cells and their organelles are enclosed by lipid bilayers and the lipid composition of either side of these membranes is controlled by active transporters (flippases and floppases) and passive scramblases, which equilibrate lipids between the membrane leaflets 1 . Many lipids are synthesized on the cytoplasmic side of the endoplasmic reticulum (ER) membrane which, unlike the plasma membrane (PM), has a symmetrical lipid distribution, suggesting a role for scramblases in the ER. To date, specific ER scramblases have not been identified and characterised. The ten members of the TMEM16 scramblase/channel family of integral membrane proteins show a surprising diversity of function, being either Ca 2+ -activated chloride channels (TMEM16A and B) 2-4 , or Ca 2+ -activated lipid scramblases with nonselective ion channel activity (TMEM16C, D, F, G and J) 5-8 . While some members of the family (A,B,F)reside in the plasma membrane, others, including TMEM16K 9 may function in intracellular membranes. TMEM16K is a widely distributed 10 , but relatively unstudied member of the TMEM16K family. Truncations and missense variants of TMEM16K (ANO10) are associated with the autosomal recessive spinocerebellar ataxia SCAR10 11,12 (as known as ARCA3 13-15 or ATX-ANO10 16 ). SCAR10 causing cerebellar ataxia, epilepsy and cognitive impairment with cerebellar atrophy noted on MRI brain and coenzyme Q10 deficiency found in muscle biopsy, fibroblasts and cerebrospinal fluid 11,12,17,18 . Some patients also have epilepsy and cognitive impairment 13,14 . Knockout studies in Drosophila 19 and mice 20 have suggested that loss of TMEM16K homologue function affects spindle formation 19 , Ca 2+ signalling 20 and apoptosis 19,20 .Structural studies have gone some way towards explaining how TMEM16 family members function as channels or lipid scrambla...

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“…The cleft is also consistent with the proposed hydrophobic gate model 7 with a central role for the conserved isoleucine of the PISL motif 14 , although the conformation of TM1-2 in E2P active (and the other E2P sub-states reported here) and earlier homology models are different. Interestingly, while the putative lipid entry pathway is reminiscent of that described for scramblases 10,34 , it does not extend to span both leaflets of the membrane, thus highlighting a fundamental requirement for an alternating access mechanism of lipid movement against its gradient.…”

Section: Introductionmentioning

confidence: 93%

Structure and autoregulation of a P4-ATPase lipid flippase

Timcenko

Lyons

Januliene

et al. 2019

Preprint

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SummaryP4-ATPases are lipid flippases that drive active transport of phospholipids from the exoplasmic or lumenal to the cytosolic leaflets of eukaryotic membranes to maintain their asymmetric lipid composition. The molecular architecture of P4-ATPases and how they work in lipid recognition and transport has remained elusive. Using cryo-electron microscopy we have determined the structures of a P4-ATPase, specifically of theSaccharomyces cerevisiaeDrs2p-Cdc50p, which is a phosphatidylserine and phosphatidylethanolamine specific lipid flippase. Drs2p-Cdc50p is autoinhibited by the Drs2p C-terminal tail and activated by phosphatidylinositol-4 phosphate (PI4P). We present three structures representing an autoinhibited, an intermediate, and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of auto-inhibition and PI4P-dependent activation. We observe the opening of a putative flippase pathway engaging conserved residues Ile508 of transmembrane segment 4 and Lys1018 and polar residues of transmembrane segment 5 in the centre of the lipid bilayer.

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Gating mechanism of the extracellular entry to the lipid pathway in a TMEM16 scramblase

Cited by 80 publications

References 64 publications

Drosophila Subdued is a moonlighting transmembrane protein 16 (TMEM16) that transports ions and phospholipids

Drosophila Subdued is a moonlighting transmembrane protein 16 (TMEM16) that transports ions and phospholipids

The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K

Structure and autoregulation of a P4-ATPase lipid flippase

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