The TMEM16 family of proteins, also known as anoctamins, features a remarkable functional diversity. This family contains the long sought-after Ca(2+)-activated chloride channels as well as lipid scramblases and cation channels. Here we present the crystal structure of a TMEM16 family member from the fungus Nectria haematococca that operates as a Ca(2+)-activated lipid scramblase. Each subunit of the homodimeric protein contains ten transmembrane helices and a hydrophilic membrane-traversing cavity that is exposed to the lipid bilayer as a potential site of catalysis. This cavity harbours a conserved Ca(2+)-binding site located within the hydrophobic core of the membrane. Mutations of residues involved in Ca(2+) coordination affect both lipid scrambling in N. haematococca TMEM16 and ion conduction in the Cl(-) channel TMEM16A. The structure reveals the general architecture of the family and its mode of Ca(2+) activation. It also provides insight into potential scrambling mechanisms and serves as a framework to unravel the conduction of ions in certain TMEM16 proteins.

The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium-activated channels for ions and lipids. Here, we reveal features of murine TMEM16F (mTMEM16F) that underlie its function as a lipid scramblase and an ion channel. The cryo-EM data of mTMEM16F in absence and presence of Ca2+ define the ligand-free closed conformation of the protein and the structure of a Ca2+-bound intermediate. Both conformations resemble their counterparts of the scrambling-incompetent anion channel mTMEM16A, yet with distinct differences in the region of ion and lipid permeation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, both functions appear to be mediated by alternate protein conformations that are at equilibrium in the ligand-bound state.

observation that synaptic strength correlates with dendritic spine morphology leads to the hypothesis that the mushroom-like shape of dendritic spines functions as a receptor trap. We developed a mimetic system to investigate dendritic spine morphology and its effects on receptor confinement and diffusion. Giant unilamellar vesicles (GUV's) are made from lipids using electroswelling. To mimic the mushroom-shaped morphologies of dendritic spines, a micromanipulator is used to pull membrane tubes from the GUV lipid bilayer. Trapping capabilities for different spine morphologies are assessed by tracking quantum dots attached to membrane lipids, thus mimicking receptors. Results show a strong dependence of escape times on GUV morphology, as quantified by GUV radius and tube length. Instead of a trivial quadratic dependence of escape times on GUV radius we find a powerlaw dependence with an exponent of 2.85. This confirms the idea that receptors can be trapped by the morphology of a dendritic spine. Therefore the connection strength of a mushroom-shaped dendritic spine is much more stable than the strengths of stubby shaped dendritic spines.

The TMEM16 family contains dimeric membrane proteins activated by intracellular Ca2+. Realizing that lipid scramblase family members contain two independently activated subunits, Lim et al. use concatenated TMEM16A subunits to show that ion channel members contain two independently activated pores.

Human dendritic cell‐specific intercellular adhesion molecule‐1 grabbing nonintegrin, DC‐SIGN, and the sinusoidal endothelial cell receptor DC‐SIGNR or L‐SIGN, are closely related sugar‐binding receptors. DC‐SIGN acts both as a pathogen‐binding endocytic receptor and as a cell adhesion molecule, while DC‐SIGNR has only the pathogen‐binding function. In addition to differences in the sugar‐binding properties of the carbohydrate‐recognition domains in the two receptors, there are sequence differences in the adjacent neck domains, which are coiled‐coil tetramerization domains comprised largely of 23‐amino acid repeat units. A series of model polypeptides consisting of uniform repeat units have been characterized by gel filtration, differential scanning calorimetry and circular dichroism. The results demonstrate that two features characterize repeat units which form more stable tetramers: a leucine reside in the first position of the heptad pattern of hydrophobic residues that pack on the inside of the coiled coil and an arginine residue on the surface of the coiled coil that forms a salt bridge with a glutamic acid residue in the same polypeptide chain. In DC‐SIGNR from all primates, very stable repeat units predominate, so the carbohydrate‐recognition domains must be held relatively closely together. In contrast, stable repeat units are found only near the membrane in DC‐SIGN. The presence of residues that disrupt tetramer formation in repeat units near the carbohydrate‐recognition domains of DC‐SIGN would allow these domains to splay further apart. Thus, the neck domains of DC‐SIGN and DC‐SIGNR can contribute to the different functions of these receptors by presenting the sugar‐binding sites in different contexts.

The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium-activated channels for ions and lipids. Here, we reveal features of TMEM16F that underlie its function as lipid scramblase and ion channel. The cryo-EM structures of TMEM16F in Ca 2+ -bound and Ca 2+ -free states display a striking similarity to the scramblingincompetent anion channel TMEM16A, yet with distinct differences in the catalytic site and in the conformational changes upon activation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, which likely resembles an equivalent process defined in the homologue nhTMEM16, both functions appear to be mediated by alternate protein conformations, which are at equilibrium in the ligand-bound state. Keywords:TMEM16 family, lipid scramblase, membrane protein structure, transport mechanism, cryo-EM, single particle of the family, TMEM16F appears, with respect to phylogenetic relationships, as intermediate between the two proteins. Although working as lipid scramblase (Suzuki et al., 2010; Watanabe et al., 2018), it is closer related to the ion channel TMEM16A than to nhTMEM16 ( Figure S1A).Moreover, whereas scrambling-related ion conduction was found to be a feature of several family members (Lee et al., 2016;Malvezzi et al., 2018; Whitlock and Hartzell, 2016b), TMEM16F is the only lipid scramblase for which instantaneous calcium-activated currents were recorded in excised patches (Yang et al., 2012). To better understand how the small sequence differences in TMEM16F give rise to its distinct functional properties, we determined its structure by cryo-EM in Ca 2+ -bound and Ca 2+ -free states, both in a detergent and in a lipid environment. In parallel, we characterized the lipid transport properties of TMEM16F in vitro after reconstitution of the protein into liposomes, as well as ion conduction in transfected cells by electrophysiology. Collectively, our study reveals the architecture of TMEM16F, defines conformational changes upon ligand binding and suggests potential mechanisms for ion and lipid movement. In the most plausible scenario, both transport processes, although activated by the same mechanism, are mediated by distinct protein conformations which are at equilibrium in a calcium-bound state. RESULTS Functional properties of TMEM16FIn our study, we explored the relationship of the TMEM16F structure to its diverse functional properties. For this purpose, we have expressed TMEM16F in HEK293 cells and purified it in the detergent digitonin ( Figures S1B and S1C). To confirm that the purified protein has retained its activity as a lipid scramblase, we have investigated lipid transport with proteoliposomes using an assay that was previously established for fungal TMEM16 scramblases (Malvezzi et al., 2013;Ploier and Menon, 2016). Our data reveals a Ca 2+ -induced ...

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