Explore the vast range of titles available.

Electron cryo-microscopy density maps of mouse TMEM16A reconstituted in nanodiscs or solubilized in detergent reveal two functional states of calcium-activated chloride channels. TMEM16A structure solved The diverse TMEM16 membrane protein family contains Ca( II )-activated chloride channels, lipid scramblases and cation channels. TMEM16A mediates chloride-ion permeation, which controls neuronal signalling, muscle contraction and numerous other physiological functions. In this issue of Nature , two groups have solved the structure of TMEM16A by using cryo-electron microscopy, providing insights into the function of this channel. Unlike other ligand-gated ion channels, the Ca( II ) ion interacts with the pore directly, where a glycine residue acts as a flexible hinge to adjust calcium sensitivity. Raimund Dutzler and colleagues report the structure of the protein in both Ca( II )-free and Ca( II )-bound states, which shows how calcium binding facilitates the structural rearrangements involved in channel activation. In the second Letter, Lily Jan and colleagues present two functional states of TMEM16A in the glycolipid LMNG and in nanodiscs, with one and two Ca( II ) ions bound, respectively. The closed conformation observed in nanodiscs is proposed to show channel rundown after prolonged Ca( II ) activation. Calcium-activated chloride channels (CaCCs) encoded by TMEM16A 1 , 2 , 3 control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system 4 , 5 , 6 , 7 . To understand how CaCCs mediate and control anion permeation to fulfil these physiological functions, knowledge of the mammalian TMEM16A structure and identification of its pore-lining residues are essential. TMEM16A forms a dimer with two pores 8 , 9 . Previous CaCC structural analyses have relied on homology modelling of a homologue (nhTMEM16) from the fungus Nectria haematococca that functions primarily as a lipid scramblase 10 , 11 , 12 , as well as subnanometre-resolution electron cryo-microscopy 12 . Here we present de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol as determined by single-particle electron cryo-microscopy. These structures reveal the ion permeation pore and represent different functional states. The structure in lauryl maltose neopentyl glycol has one Ca 2+ ion resolved within each monomer with a constricted pore; this is likely to correspond to a closed state, because a CaCC with a single Ca 2+ occupancy requires membrane depolarization in order to open (C.J.P. et al ., manuscript submitted). The structure in nanodiscs has two Ca 2+ ions per monomer and its pore is in a closed conformation; this probably reflects channel rundown, which is the gradual loss of channel activity that follows prolonged CaCC activation in 1 mM Ca 2+ . Our mutagenesis and electrophysiological studies, prompted by analyses of the structures, identified ten residues distributed along the pore that interact with permeant anions and affect anion selectivity, as well as seven pore-lining residues that cluster near pore constrictions and regulate channel gating. Together, these results clarify the basis of CaCC anion conduction.

The binding of cytoplasmic Ca 2+ to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck of an hourglass-shaped pore. By combining mutagenesis, electrophysiology, and cryo-electron microscopy, we identified three hydrophobic residues at the intracellular entrance of the neck as constituents of this gate. Mutation of each of these residues increases the potency of Ca 2+ and results in pronounced basal activity. The structure of an activating mutant shows a conformational change of an α-helix that contributes to Ca 2+ binding as a likely cause for the basal activity. Although not in physical contact, the three residues are functionally coupled to collectively contribute to the stabilization of the gate in the closed conformation of the pore, thus explaining the low open probability of the channel in the absence of Ca 2+ . The binding of cytoplasmic Ca 2+ to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck. Here authors use cryo-EM and electrophysiology to identify three hydrophobic residues at the intracellular entrance of the neck as constituents of this gate.

The anion channel TMEM16A is activated by intracellular Ca 2+ in a highly cooperative process. By combining electrophysiology and autocorrelation analysis, we investigated the mechanism of channel activation and the concurrent rearrangement of the gate in the narrow part of the pore. Features in the fluctuation characteristics of steady-state current indicate the sampling of intermediate conformations that are successively occupied during gating. The initial step is related to conformational changes induced by Ca 2+ binding, which is ensued by rearrangements that open the pore. Mutations in the gate shift the equilibrium of transitions in a manner consistent with a progressive destabilization of this region during pore opening. We come up with a mechanism of channel activation where the binding of Ca 2+ induces conformational changes in the protein that, in a sequential manner, propagate from the binding site and couple to the gate in the narrow pore to allow ion permeation. The anion channel TMEM16A is activated by intracellular Ca 2+ in a highly cooperative process. Here authors combine electrophysiology and autocorrelation analysis to observe the sampling of intermediate conformations during gating.

The calcium-activated chloride channel TMEM16A is a member of a conserved protein family that comprises ion channels and lipid scramblases. Although the structure of the scramblase nhTMEM16 has defined the architecture of the family, it was unknown how a channel has adapted to cope with its distinct functional properties. Here we have addressed this question by the structure determination of mouse TMEM16A by cryo-electron microscopy and a complementary functional characterization. The protein shows a similar organization to nhTMEM16, except for changes at the site of catalysis. There, the conformation of transmembrane helices constituting a membrane-spanning furrow that provides a path for lipids in scramblases has changed to form an enclosed aqueous pore that is largely shielded from the membrane. Our study thus reveals the structural basis of anion conduction in a TMEM16 channel and it defines the foundation for the diverse functional behavior in the TMEM16 family. Cell membranes are made up of two layers of oily molecules, called lipids, embedded with a variety of proteins. Each type of membrane protein carries out a particular activity for the cell, and many are involved in transporting other molecules from one side of the membrane to the other. The TMEM16 proteins are a large family of membrane proteins. Most are known as lipid scramblases and move lipids between the two layers of the membrane. However, some TMEM16 proteins transport ions in or out of the cell, and are instead called ion channels. TMEM16 proteins are found in animals, plants and fungi but not bacteria, and play key roles in many biological activities that keep these organisms alive. For example, in humans, ion channels belonging to the TMEM16 family help keep the lining of the lung moist, and allow muscles in the gut to contract. The structure of a scramblase shows that two protein units interact, with each unit containing a furrow that spans the membrane, through which lipids can move from one layer to the other. However, to date, the shape of a TMEM16 ion channel has not been determined. It was therefore not clear how a protein with features that let it transport large, oily molecules like lipids had evolved to transport small, charged particles instead. TMEM16A is a member of the TMEM16 family that transports negatively charged chloride ions. Using a technique called cryo-electron microscopy, Paulino et al. have determined the three-dimensional shape of the version of TMEM16A from a mouse. Overall, TMEM16A is organized similarly to the lipid scramblase. However, some parts of the TMEM16A protein have undergone rearrangements such that the membrane-exposed furrow that provides a path for lipids in scramblases is now partially sealed in TMEM16A. This results in an enclosed pore that is largely shielded from the oily membrane and through which ions can pass. Additionally, biochemical analysis suggests that TMEM16A forms a narrow pore that may widen towards the side facing the inside of the cell, though further work is needed to understand if this is relevant to the protein’s activity. The three-dimensional structure of TMEM16A reveals how the protein’s architecture differs from other family members working as lipid scramblases. It also gives insight into how TMEM16 proteins might work as ion channels. These findings can now form a strong basis for future studies into the activity of TMEM16 proteins.

Bestrophins form Ca 2+ -activated Cl − channels and regulate intracellular Ca 2+ signaling. We demonstrate that bestrophin 1 is localized in the endoplasmic reticulum (ER), where it interacts with stromal interacting molecule 1, the ER-Ca 2+ sensor. Intracellular Ca 2+ transients elicited by stimulation of purinergic P2Y 2 receptors in HEK293 cells were augmented by hBest1. The p21-activated protein kinase Pak2 was found to phosphorylate hBest1, thereby enhancing Ca 2+ signaling and activation of Ca 2+ -dependent Cl − (TMEM16A) and K + (SK4) channels. Lack of bestrophin 1 expression in respiratory epithelial cells of mBest1 knockout mice caused expansion of ER cisterns and induced Ca 2+ deposits. hBest1 is, therefore, important for Ca 2+ handling of the ER store and may resemble the long-suspected counterion channel to balance transient membrane potentials occurring through inositol triphosphate (IP 3 )-induced Ca 2+ release and store refill. Thus, bestrophin 1 regulates compartmentalized Ca 2+ signaling that plays an essential role in Best macular dystrophy, inflammatory diseases such as cystic fibrosis, as well as proliferation.

Caveolin (Cav)‐1 is an integral membrane protein of caveolae playing a crucial role in various signal transduction pathways. Caveolae represent the sites for calcium entry and storage especially in smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). Cav‐1−/− mice lack caveolae and show abnormalities in pacing and contractile activity of the small intestine. Presently, we investigated, by transmission electron microscopy (TEM) and immunohistochemistry, whether the absence of Cav‐1 in Cav‐1−/− mouse small intestine affects ICC, SMC and neuronal morphology, the expression of NK1 and NK2 receptors, and of Ano1 (also called Dog1 or TMEM16A), an essential molecule for slow wave activity in gastrointestinal muscles. ICC were also labelled with c‐Kit and tachykinergic neurons with Substance P (SP). In Cav‐1−/− mice: (i) ICC were Ano1‐negative but maintained c‐Kit expression, (ii) NK1 and NK2 receptor immunoreactivity was more intense and, in the SMC, mainly intracytoplasmatic, (iii) SP‐immunoreactivity was significantly reduced. Under TEM: (i) ICC, SMC and telocytes lacked typical caveolae but had few and large flask‐shaped vesicles we called large‐sized caveolae; (ii) SMC and ICC contained an extraordinary high number of mitochondria, (iii) neurons were unchanged. To maintain intestinal motility, loss of caveolae and reduced calcium availability in Cav‐1–knockout mice seem to be balanced by a highly increased number of mitochondria in ICC and SMC. Loss of Ano‐1 expression, decrease of SP content and consequently overexpression of NK receptors suggest that all these molecules are Cav‐1–associated proteins.