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

Abstract

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...

Structural basis of Ca2+-dependent activation and lipid transport by a TMEM16 scramblase

“…The bound Ca 2+ ions are coordinated by five conserved acidic residues (E445 on TM6, D511 and E514 on TM7, and E534 and D547 on TM8), three polar residues (Q438, Q518, N539), and the unpaired main chain carbonyl of G441 (Figure 4A–C). This coordination is similar to that seen in the nhTMEM16 and hTMEM16K scramblases as well as in the TMEM16A channel, consistent with the evolutionary conservation of the Ca 2+ -binding residues (Figure 4C) (Yu et al, 2012; Malvezzi et al, 2013; Terashima et al, 2013; Brunner et al, 2014; Tien et al, 2014; Lim et al, 2016; Dang et al, 2017; Paulino et al, 2017a; Bushell et al, 2018). In the absence of Ca 2+ , the binding site is disrupted by the movement of TM6 which displaces the three residues participating in the site (Q438, G441 and E445) (Figure 4D–F, Video 3).…”

“…We used single-particle cryo-EM to determine the structures of nanodisc-incorporated afTMEM16 in the presence and absence of Ca 2+ to resolutions of 4.0 and 3.9 Å, respectively (Figure 1). In both conditions, afTMEM16 adopts the TMEM16 fold (Figure 1; Figure 1—figure supplement 1–6), where each monomer in the dimeric protein comprises a cytosolic domain organized into a 6 α-helix (α1-α6)/3 β-strand (β1-β3) ferredoxin fold and a transmembrane region encompassing 10 α-helices (TM1-TM10) (Figure 1—figure supplement 5) (Brunner et al, 2014; Dang et al, 2017; Paulino et al, 2017a; Bushell et al, 2018). The two monomers are related by a twofold axis of symmetry at the dimer interface, formed by TM10 and the cytosolic domain (Figure 1E).…”

“…In the presence of the functionally saturating concentration of 0.5 mM Ca 2+ (Malvezzi et al, 2013), afTMEM16 adopts a conformation similar to that of Ca 2+ -bound nhTMEM16 and hTMEM16K in detergent (Brunner et al, 2014; Bushell et al, 2018) (Figure 1—figure supplement 5). The TM3-6 from each monomer form a peripheral hydrophilic cavity that opens to the membrane with a minimum diameter of ~5 Å.…”

Predominant localization of phosphatidylserine at the cytoplasmic leaflet of the ER, and its TMEM16K-dependent redistribution

“…Among them, the tissue-specifically expressed TMEM16E and ubiquitously expressed TMEM16K have been suggested to have a Ca 2+ -dependent scrambling activity, based on analyses of their putative scramblase domain (9, 10) and on molecular dynamic simulation experiments (11). More recently, the purified TMEM16K was shown to have scramblase activity in a reconstituted liposome system (12). Nevertheless, whether these molecules actually function as a scramblase inside cells has not been addressed, because the phospholipids in intracellular membranes are not directly accessible and thus are more difficult to study than those in the plasma membrane.…”

Whole-exome sequencing reveals ANO8 as a genetic risk factor for intrahepatic cholestasis of pregnancy

“…Therefore, it is of great importance to identify a large number of ICP susceptibility genes that remain undiscovered. The anoctamin family contains 10 members (ANO1-10) with two major functions: Ca 2+ -dependent ion channels (ANO1 and ANO2) and/or Ca 2+ -activated lipid scramblases with nonselective ion channel activity (ANO3-4, ANO6-8) [16][17][18]. The ANO protein family is widely expressed in eukaryotes, exhibits diverse functions in cells throughout the body and is associated with several human diseases [19].…”