The plasma membrane inner leaflet PI(4,5)P2 is essential for the activation of proton-activated chloride channels.

Proton-activated chloride (PAC) channels, ubiquitously expressed in tissues, regulate intracellular Cl- levels and cell death following acidosis. However, molecular mechanisms and signaling pathways involved in PAC channel modulation are largely unknown. Herein, we determine that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] of the plasma membrane inner leaflet is essential for the proton activation of PAC channels. PI(4,5)P2 depletion by activating phosphatidylinositol 5-phosphatases or Gq protein-coupled muscarinic receptors substantially inhibits human PAC currents. In excised inside-out patches, PI(4,5)P2 application to the cytoplasmic side increases the currents. Structural simulation reveals that the putative PI(4,5)P2-binding site is localized within the cytosol in resting state but shifts to the cell membrane's inner surface in an activated state and interacts with inner leaflet PI(4,5)P2. Alanine neutralization of basic residues near the membrane-cytosol interface of the transmembrane helice 2 significantly attenuates PAC currents. Overall, our study uncovers a modulatory mechanism of PAC channel through inner membrane PI(4,5)P2.

Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells.

The Ca2+-activated Cl- channel regulator CLCA1 potentiates the activity of the Ca2+-activated Cl- channel (CaCC) TMEM16A by directly engaging the channel at the cell surface, inhibiting its reinternalization and increasing Ca2+-dependent Cl- current (ICaCC) density. We now present evidence of functional pairing between two other CLCA and TMEM16 protein family members, namely CLCA4 and the CaCC TMEM16B. Similar to CLCA1, (i) CLCA4 is a self-cleaving metalloprotease, and the N-terminal portion (N-CLCA4) is secreted; (ii) the von Willebrand factor type A (VWA) domain in N-CLCA4 is sufficient to potentiate ICaCC in HEK293T cells; and (iii) this is mediated by the metal ion-dependent adhesion site motif within VWA. The results indicate that, despite the conserved regulatory mechanism and homology between CLCA1 and CLCA4, CLCA4-dependent ICaCC are carried by TMEM16B, rather than TMEM16A. Our findings show specificity in CLCA/TMEM16 interactions and suggest broad physiological and pathophysiological links between these two protein families.

In or out of the groove? Mechanisms of lipid scrambling by TMEM16 proteins.

Phospholipid scramblases mediate the rapid movement of lipids between membrane leaflets, a key step in establishing and maintaining membrane homeostasis of the membranes of all eukaryotic cells and their organelles. Thus, impairment of lipid scrambling can lead to a variety of pathologies. How scramblases catalyzed the transbilayer movement of lipids remains poorly understood. Despite the availability of direct structural information on three unrelated families of scramblases, the TMEM16s, the Xkrs, and ATG-9, a unifying mechanism has failed to emerge thus far. Among these, the most extensively studied and best understood are the Ca2+ activated TMEM16s, which comprise ion channels and/or scramblases. Early work supported the view that these proteins provided a hydrophilic, membrane-exposed groove through which the lipid headgroups could permeate. However, structural, and functional experiments have since challenged this mechanism, leading to the proposal that the TMEM16s distort and thin the membrane near the groove to facilitate lipid scrambling. Here, we review our understanding of the structural and mechanistic underpinnings of lipid scrambling by the TMEM16s and discuss how the different proposals account for the various experimental observations.

VSI: The anoctamins: Structure and function: "Intracellular" anoctamins.

Plasma membrane localized anoctamin 1, 2 and 6 (TMEM16A, B, F) have been examined in great detail with respect to structure and function, but much less is known about the other seven intracellular members of this exciting family of proteins. This is probably due to their limited accessibility in intracellular membranous compartments, such as the endoplasmic reticulum (ER) or endosomes. However, these so-called intracellular anoctamins are also found in the plasma membrane (PM) which adds to the confusion regarding their cellular role. Probably all intracellular anoctamins except of ANO8 operate as intracellular phospholipid (PL) scramblases, allowing for Ca2+-activated, passive transport of phospholipids like phosphatidylserine between both membrane leaflets. Probably all of them also conduct ions, which is probably part of their physiological function. In this brief overview, we summarize key findings on the biological functions of ANO3, 4, 5, 7, 8, 9 and 10 (TMEM16C, D, E, G, H, J, K) that are gradually coming to light. Compartmentalized regulation of intracellular Ca2+ signals, tethering of the ER to specific PM contact sites, and control of intracellular vesicular trafficking appear to be some of the functions of intracellular anoctamins, while loss of function and abnormal expression are the cause for various diseases.

Mechanical activation opens a lipid-lined pore in OSCA ion channels.

OSCA/TMEM63 channels are the largest known family of mechanosensitive channels1-3, playing critical roles in plant4-7 and mammalian8,9 mechanotransduction. Here we determined 44 cryogenic electron microscopy structures of OSCA/TMEM63 channels in different environments to investigate the molecular basis of OSCA/TMEM63 channel mechanosensitivity. In nanodiscs, we mimicked increased membrane tension and observed a dilated pore with membrane access in one of the OSCA1.2 subunits. In liposomes, we captured the fully open structure of OSCA1.2 in the inside-in orientation, in which the pore shows a large lateral opening to the membrane. Unusually for ion channels, structural, functional and computational evidence supports the existence of a 'proteo-lipidic pore' in which lipids act as a wall of the ion permeation pathway. In the less tension-sensitive homologue OSCA3.1, we identified an 'interlocking' lipid tightly bound in the central cleft, keeping the channel closed. Mutation of the lipid-coordinating residues induced OSCA3.1 activation, revealing a conserved open conformation of OSCA channels. Our structures provide a global picture of the OSCA channel gating cycle, uncover the importance of bound lipids and show that each subunit can open independently. This expands both our understanding of channel-mediated mechanotransduction and channel pore formation, with important mechanistic implications for the TMEM16 and TMC protein families.

The allosteric mechanism leading to an open-groove lipid conductive state of the TMEM16F scramblase.

TMEM16F is a Ca2+-activated phospholipid scramblase in the TMEM16 family of membrane proteins. Unlike other TMEM16s exhibiting a membrane-exposed hydrophilic groove that serves as a translocation pathway for lipids, the experimentally determined structures of TMEM16F shows the groove in a closed conformation even under conditions of maximal scramblase activity. It is currently unknown if/how TMEM16F groove can open for lipid scrambling. Here we describe the analysis of ~400 µs all-atom molecular dynamics (MD) simulations of the TMEM16F revealing an allosteric mechanism leading to an open-groove, lipid scrambling competent state of the protein. The groove opens into a continuous hydrophilic conduit that is highly similar in structure to that seen in other activated scramblases. The allosteric pathway connects this opening to an observed destabilization of the Ca2+ ion bound at the distal site near the dimer interface, to the dynamics of specific protein regions that produces the open-groove state to scramble phospholipids.

Activation of TMEM16F by inner gate charged mutations and possible lipid/ion permeation mechanisms.

Transmembrane protein 16F (TMEM16F) is a ubiquitously expressed Ca2+-activated phospholipid scramblase that also functions as a largely non-selective ion channel. Though recent structural studies have revealed the closed and intermediate conformations of mammalian TMEM16F (mTMEM16F), the open and conductive state remains elusive. Instead, it has been proposed that an open hydrophilic pathway may not be required for lipid scrambling. We previously identified an inner activation gate, consisting of F518, Y563, and I612, and showed that charged mutations of the inner gate residues led to constitutively active mTMEM16F scrambling. Herein, atomistic simulations show that lysine substitution of F518 and Y563 can indeed lead to spontaneous opening of the permeation pore in the Ca2+-bound state of mTMEM16F. Dilation of the pore exposes hydrophilic patches in the upper pore region, greatly increases the pore hydration level, and enables lipid scrambling. The putative open state of mTMEM16F resembles the active state of fungal scramblases and is a meta-stable state for the wild-type protein in the Ca2+-bound state. Therefore, mTMEM16F may be capable of supporting the canonical in-groove scrambling mechanism in addition to the out-of-groove one. Further analysis reveals that the in-groove phospholipid and ion transduction pathways of mTMEM16F overlap from the intracellular side up to the inner gate but diverge from each other with different exits to the extracellular side of membrane.

Structures of tweety homolog proteins TTYH2 and TTYH3 reveal a Ca2+-dependent switch from intra- to intermembrane dimerization.

Tweety homologs (TTYHs) comprise a conserved family of transmembrane proteins found in eukaryotes with three members (TTYH1-3) in vertebrates. They are widely expressed in mammals including at high levels in the nervous system and have been implicated in cancers and other diseases including epilepsy, chronic pain, and viral infections. TTYHs have been reported to form Ca2+- and cell volume-regulated anion channels structurally distinct from any characterized protein family with potential roles in cell adhesion, migration, and developmental signaling. To provide insight into TTYH family structure and function, we determined cryo-EM structures of Mus musculus TTYH2 and TTYH3 in lipid nanodiscs. TTYH2 and TTYH3 adopt a previously unobserved fold which includes an extended extracellular domain with a partially solvent exposed pocket that may be an interaction site for hydrophobic molecules. In the presence of Ca2+, TTYH2 and TTYH3 form homomeric cis-dimers bridged by extracellularly coordinated Ca2+. Strikingly, in the absence of Ca2+, TTYH2 forms trans-dimers that span opposing membranes across a ~130 Å intermembrane space as well as a monomeric state. All TTYH structures lack ion conducting pathways and we do not observe TTYH2-dependent channel activity in cells. We conclude TTYHs are not pore forming subunits of anion channels and their function may involve Ca2+-dependent changes in quaternary structure, interactions with hydrophobic molecules near the extracellular membrane surface, and/or association with additional protein partners.

P2X7 Receptors and TMEM16 Channels Are Functionally Coupled with Implications for Macropore Formation and Current Facilitation.

P2X7 receptors (P2X7) are cationic channels involved in many diseases. Following their activation by extracellular ATP, distinct signaling pathways are triggered, which lead to various physiological responses such as the secretion of pro-inflammatory cytokines or the modulation of cell death. P2X7 also exhibit unique behaviors, such as "macropore" formation, which corresponds to enhanced large molecule cell membrane permeability and current facilitation, which is caused by prolonged activation. These two phenomena have often been confounded but, thus far, no clear mechanisms have been resolved. Here, by combining different approaches including whole-cell and single-channel recordings, pharmacological and biochemical assays, CRISPR/Cas9 technology and cell imaging, we provide evidence that current facilitation and macropore formation involve functional complexes comprised of P2X7 and TMEM16, a family of Ca2+-activated ion channel/scramblases. We found that current facilitation results in an increase of functional complex-embedded P2X7 open probability, a result that is recapitulated by plasma membrane cholesterol depletion. We further show that macropore formation entails two distinct large molecule permeation components, one of which requires functional complexes featuring TMEM16F subtype, the other likely being direct permeation through the P2X7 pore itself. Such functional complexes can be considered to represent a regulatory hub that may orchestrate distinct P2X7 functionalities.

ANO7: Insights into topology, function, and potential applications as a biomarker and immunotherapy target.

Anoctamin 7 (ANO7) is a member of the transmembrane protein TMEM16 family. It has a conservative topology similar to other members in this family, such as the typical eight-transmembrane domain, but it also has unique features. Although the ion channel role of ANO7 has been well accepted, evolutionary analyses and relevant studies suggest that ANO7 may be a multi-facet protein in function. Studies have shown that ANO7 may also function as a scramblase. ANO7 is highly expressed in prostate cancer as well as normal prostate tissues. A considerable amount of evidence has confirmed that ANO7 is associated with human physiology and pathology, particularly with the development of prostate cancer, which makes ANO7 a good candidate as a diagnostic and prognostic biomarker. In addition, ANO7 may be a potential target for prostate cancer immunotherapy. Antibody-based or T cell-mediated immunotherapies against prostate cancer by targeting ANO7 have been highly anticipated. ANO7 may also correlate with several other types of cancers or diseases, where further studies are warranted.

Molecular mechanisms of ion conduction and ion selectivity in TMEM16 lipid scramblases.

TMEM16 lipid scramblases transport lipids and also operate as ion channels with highly variable ion selectivities and various physiological functions. However, their molecular mechanisms of ion conduction and selectivity remain largely unknown. Using computational electrophysiology simulations at atomistic resolution, we identified the main ion-conductive state of TMEM16 lipid scramblases, in which an ion permeation pathway is lined by lipid headgroups that directly interact with permeating ions in a voltage polarity-dependent manner. We found that lipid headgroups modulate the ion-permeability state and regulate ion selectivity to varying degrees in different scramblase isoforms, depending on the amino-acid composition of the pores. Our work has defined the structural basis of ion conduction and selectivity in TMEM16 lipid scramblases and uncovered the mechanisms responsible for the direct effects of membrane lipids on the conduction properties of ion channels.

Prediction of Functional Consequences of Missense Mutations in ANO4 Gene.

The anoctamin (TMEM16) family of transmembrane protein consists of ten members in vertebrates, which act as Ca2+-dependent ion channels and/or Ca2+-dependent scramblases. ANO4 which is primarily expressed in the CNS and certain endocrine glands, has been associated with various neuronal disorders. Therefore, we focused our study on prioritizing missense mutations that are assumed to alter the structure and stability of ANO4 protein. We employed a wide array of evolution and structure based in silico prediction methods to identify potentially deleterious missense mutations in the ANO4 gene. Identified pathogenic mutations were then mapped to the modeled human ANO4 structure and the effects of missense mutations were studied on the atomic level using molecular dynamics simulations. Our data show that the G80A and A500T mutations significantly alter the stability of the mutant proteins, thus providing new perspective on the role of missense mutations in ANO4 gene. Results obtained in this study may help to identify disease associated mutations which affect ANO4 protein structure and function and might facilitate future functional characterization of ANO4.

The Groovy TMEM16 Family: Molecular Mechanisms of Lipid Scrambling and Ion Conduction.

The TMEM16 family of membrane proteins displays a remarkable functional dichotomy - while some family members function as Ca2+-activated anion channels, the majority of characterized TMEM16 homologs are Ca2+-activated lipid scramblases, which catalyze the exchange of phospholipids between the two membrane leaflets. Furthermore, some TMEM16 scramblases can also function as channels. Due to their involvement in important physiological processes, the family has been actively studied ever since their molecular identity was unraveled. In this review, we will summarize the recent advances in the field and how they influenced our view of TMEM16 family function and evolution. Structural, functional and computational studies reveal how relatively small rearrangements in the permeation pathway are responsible for the observed functional duality: while TMEM16 scramblases can adopt both ion- and lipid conductive conformations, TMEM16 channels can only populate the former. Recent data further provides the molecular details of a stepwise activation mechanism, which is initiated by Ca2+ binding and modulated by various cellular factors, including lipids. TMEM16 function and the surrounding membrane properties are inextricably intertwined, with the protein inducing bilayer deformations associated with scrambling, while the surrounding lipids modulate TMEM16 conformation and activity.

Divalent Cation Modulation of Ion Permeation in TMEM16 Proteins.

Intracellular divalent cations control the molecular function of transmembrane protein 16 (TMEM16) family members. Both anion channels (such as TMEM16A) and phospholipid scramblases (such as TMEM16F) in this family are activated by intracellular Ca2+ in the low µM range. In addition, intracellular Ca2+ or Co2+ at mM concentrations have been shown to further potentiate the saturated Ca2+-activated current of TMEM16A. In this study, we found that all alkaline earth divalent cations in mM concentrations can generate similar potentiation effects in TMEM16A when applied intracellularly, and that manipulations thought to deplete membrane phospholipids weaken the effect. In comparison, mM concentrations of divalent cations minimally potentiate the current of TMEM16F but significantly change its cation/anion selectivity. We suggest that divalent cations may increase local concentrations of permeant ions via a change in pore electrostatic potential, possibly acting through phospholipid head groups in or near the pore. Monovalent cations appear to exert a similar effect, although with a much lower affinity. Our findings resolve controversies regarding the ion selectivity of TMEM16 proteins. The physiological role of this mechanism, however, remains elusive because of the nearly constant high cation concentrations in cytosols.

Ca2+ Sensitivity of Anoctamin 6/TMEM16F Is Regulated by the Putative Ca2+-Binding Reservoir at the N-Terminal Domain.

Anoctamin 6/TMEM16F (ANO6) is a dual-function protein with Ca2+-activated ion channel and Ca2+-activated phospholipid scramblase activities, requiring a high intracellular Ca2+ concentration (e.g., half-maximal effective Ca2+ concentration [EC50] of [Ca2+]i > 10 µM), and strong and sustained depolarization above 0 mV. Structural comparison with Anoctamin 1/TMEM16A (ANO1), a canonical Ca2+- activated chloride channel exhibiting higher Ca2+ sensitivity (EC50 of 1 µM) than ANO6, suggested that a homologous Ca2+-transferring site in the N-terminal domain (Nt) might be responsible for the differential Ca2+ sensitivity and kinetics of activation between ANO6 and ANO1. To elucidate the role of the putative Ca2+-transferring reservoir in the Nt (Nt-CaRes), we constructed an ANO6-1-6 chimera in which Nt-CaRes was replaced with the corresponding domain of ANO1. ANO6- 1-6 showed higher sensitivity to Ca2+ than ANO6. However, neither the speed of activation nor the voltage-dependence differed between ANO6 and ANO6-1-6. Molecular dynamics simulation revealed a reduced Ca2+ interaction with Nt- CaRes in ANO6 than ANO6-1-6. Moreover, mutations on potentially Ca2+-interacting acidic amino acids in ANO6 Nt- CaRes resulted in reduced Ca2+ sensitivity, implying direct interactions of Ca2+ with these residues. Based on these results, we cautiously suggest that the net charge of Nt- CaRes is responsible for the difference in Ca2+ sensitivity between ANO1 and ANO6.

ANO5 ensures trafficking of annexins in wounded myofibers.

Mutations in ANO5 (TMEM16E) cause limb-girdle muscular dystrophy R12. Defective plasma membrane repair is a likely mechanism. Using myofibers from Ano5 knockout mice, we show that trafficking of several annexin proteins, which together form a cap at the site of injury, is altered upon loss of ANO5. Annexin A2 accumulates at the wound to nearly twice the level observed in WT fibers, while annexin A6 accumulation is substantially inhibited in the absence of ANO5. Appearance of annexins A1 and A5 at the cap is likewise diminished in the Ano5 knockout. These changes are correlated with an alteration in annexin repair cap fine structure and shedding of annexin-positive vesicles. We conclude that loss of annexin coordination during repair is disrupted in Ano5 knockout mice and underlies the defective repair phenotype. Although ANO5 is a phospholipid scramblase, abnormal repair is rescued by overexpression of a scramblase-defective ANO5 mutant, suggesting a novel, scramblase-independent role of ANO5 in repair.

Evidence that polyphenols do not inhibit the phospholipid scramblase TMEM16F.

TMEM16 Ca2+-activated phospholipid scramblases (CaPLSases) mediate rapid transmembrane phospholipid flip-flop and as such play essential roles in various physiological and pathological processes such as blood coagulation, skeletal development, viral infection, cell-cell fusion, and ataxia. Pharmacological tools specifically targeting TMEM16 CaPLSases are urgently needed to understand these novel membrane transporters and their contributions to health and disease. Tannic acid (TA) and epigallocatechin gallate (EGCG) were recently reported as promising TMEM16F CaPLSase inhibitors. However, our present study shows that TA and EGCG do not inhibit the phospholipid-scrambling or ion conduction activities of the dual-functional TMEM16F. Instead, we found that TA and EGCG mainly acted as fluorescence quenchers that rapidly suppress the fluorophores conjugated to annexin V, a phosphatidylserine-binding probe commonly used to report on TMEM16 CaPLSase activity. These data demonstrate the false positive effects of TA and EGCG on inhibiting TMEM16F phospholipid scrambling and discourage the use of these polyphenols as CaPLSase inhibitors. Appropriate controls as well as a combination of both fluorescence imaging and electrophysiological validation are necessary in future endeavors to develop TMEM16 CaPLSase inhibitors.

Reconstitution of Proteoliposomes for Phospholipid Scrambling and Nonselective Channel Assays.

Phospholipid scramblases catalyze the rapid trans-bilayer movement of lipids down their concentration gradients. This process is essential for numerous cellular signaling functions including cell fusion, blood coagulation, and apoptosis. The importance of scramblases is highlighted by the number of human diseases caused by mutations in these proteins. Because of their indispensable function, it is essential to understand and characterize the molecular function of phospholipid scramblases. Powerful tools to measure lipid transport in cells are available. However, these approaches provide limited mechanistic insights into the molecular bases of scrambling. Here we describe in detail an in vitro phospholipid scramblase assay and the accompanying analysis which allows for determination of the macroscopic rate constants associated with phospholipid scrambling. Notably, members of the TMEM16 family of scramblases also function as nonselective ion channels. To better understand the physiological relevance of this channel function as well as its relationship to the scrambling activity of the TMEM16s we also describe in detail an in vitro flux assay to measure nonselective channel activity. Together, these two assays can be used to investigate the dual activities of the TMEM16 scramblases/nonselective channels.

Single-Particle Cryo-EM of Membrane Proteins in Lipid Nanodiscs.

Single-particle cryo-electron microscopy has become an indispensable technique in structural biology. In particular when studying membrane proteins, it allows the use of membrane-mimicking tools, which can be crucial for a comprehensive understanding of the structure-function relationship of the protein in its native environment. In this chapter we focus on the application of nanodiscs and use our recent studies on the TMEM16 family as an example.