Gating and Regulatory Mechanisms of TMEM16 Ion Channels and Scramblases.
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SUBMITTER: Le SC
PROVIDER: S-EPMC8640346 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The TMEM16 family of membrane proteins is composed of both Ca2+-gated Cl- channels and Ca2+-dependent phospholipid scramblases. The functional diversity of TMEM16s underlies their involvement in numerous signal transduction pathways that connect changes in cytosolic Ca2+ levels to cellular signaling networks. Indeed, defects in the function of several TMEM16s cause a variety of genetic disorders, highlighting their fundamental pathophysiological importance. Here, we review how our mechanistic understanding of TMEM16 function has been shaped by recent functional and structural work. Remarkably, the recent determination of near-atomic-resolution structures of TMEM16 proteins of both functional persuasions has revealed how relatively minimal rearrangements in the substrate translocation pathway are sufficient to precipitate the dramatic functional differences that characterize the family. These structures, when interpreted in the light of extensive functional analysis, point to an unusual mechanism for Ca2+-dependent activation of TMEM16 proteins in which substrate permeation is regulated by a combination of conformational rearrangements and electrostatics. These breakthroughs pave the way to elucidate the mechanistic bases of ion and lipid transport by the TMEM16 proteins and unravel the molecular links between these transport activities and their function in human pathophysiology.
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Project description: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.
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Project description:Acid-sensing ion channels (ASICs) are trimeric, proton-gated and sodium-selective members of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily of ion channels and are expressed throughout vertebrate central and peripheral nervous systems. Gating of ASICs occurs on a millisecond time scale and the mechanism involves three conformational states: high pH resting, low pH open and low pH desensitized. Existing X-ray structures of ASIC1a describe the conformations of the open and desensitized states, but the structure of the high pH resting state and detailed mechanisms of the activation and desensitization of the channel have remained elusive. Here we present structures of the high pH resting state of homotrimeric chicken (Gallus gallus) ASIC1a, determined by X-ray crystallography and single particle cryo-electron microscopy, and present a comprehensive molecular mechanism for proton-dependent gating in ASICs. In the resting state, the position of the thumb domain is further from the three-fold molecular axis, thereby expanding the 'acidic pocket' in comparison to the open and desensitized states. Activation therefore involves 'closure' of the thumb into the acidic pocket, expansion of the lower palm domain and an iris-like opening of the channel gate. Furthermore, we demonstrate how the β11-β12 linkers that demarcate the upper and lower palm domains serve as a molecular 'clutch', and undergo a simple rearrangement to permit rapid desensitization.
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Project description:The phospholipid bilayer has evolved to be a protective and selective barrier by which the cell maintains high concentrations of life sustaining organic and inorganic material. As gatekeepers responsible for an immense amount of bidirectional chemical traffic between the cytoplasm and extracellular milieu, ion channels have been studied in detail since their postulated existence nearly three-quarters of a century ago. Over the past fifteen years, we have begun to understand how selective permeability can be achieved for both cationic and anionic ions. Our mechanistic knowledge has expanded recently with studies of a large family of anion channels, the Formate Nitrite Transport (FNT) family. This family has proven amenable to structural studies at a resolution high enough to reveal intimate details of ion selectivity and gating. With five representative members having yielded a total of 15 crystal structures, this family represents one of the richest sources of structural information for anion channels.
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Project description:Ion channels open and close in response to diverse stimuli, and the molecular events underlying these processes are extensively modulated by ligands of both endogenous and exogenous origin. In the past decade, high-resolution structures of several channel types have been solved, providing unprecedented details of the molecular architecture of these membrane proteins. Intrinsic conformational flexibility of ion channels critically governs their functions. However, the dynamics underlying gating mechanisms and modulations are obscured in the information from crystal structures. While nuclear magnetic resonance spectroscopic methods allow direct measurements of protein dynamics, they are limited by the large size of these membrane protein assemblies in detergent micelles or lipid membranes. Electron paramagnetic resonance (EPR) spectroscopy has emerged as a key biophysical tool to characterize structural dynamics of ion channels and to determine stimulus-driven conformational transition between functional states in a physiological environment. This review will provide an overview of the recent advances in the field of voltage- and ligand-gated channels and highlight some of the challenges and controversies surrounding the structural information available. It will discuss general methods used in site-directed spin labeling and EPR spectroscopy and illustrate how findings from these studies have narrowed the gap between high-resolution structures and gating mechanisms in membranes, and have thereby helped reconcile seemingly disparate models of ion channel function.
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Project description:TMEM16 scramblases dissipate the plasma membrane lipid asymmetry to activate multiple eukaryotic cellular pathways. Scrambling was proposed to occur with lipid headgroups moving between leaflets through a membrane-spanning hydrophilic groove. Direct information on lipid-groove interactions is lacking. We report the 2.3 Å resolution cryogenic electron microscopy structure of the nanodisc-reconstituted Ca2+-bound afTMEM16 scramblase showing how rearrangement of individual lipids at the open pathway results in pronounced membrane thinning. Only the groove's intracellular vestibule contacts lipids, and mutagenesis suggests scrambling does not require specific protein-lipid interactions with the extracellular vestibule. We find scrambling can occur outside a closed groove in thinner membranes and is inhibited in thicker membranes, despite an open pathway. Our results show afTMEM16 thins the membrane to enable scrambling and that an open hydrophilic pathway is not a structural requirement to allow rapid transbilayer movement of lipids. This mechanism could be extended to other scramblases lacking a hydrophilic groove.
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Project description:Ion channels play a central role in the regulation of nearly every cellular process. Dating back to the classic 1952 Hodgkin-Huxley model of the generation of the action potential, ion channels have always been thought of as independent agents. A myriad of recent experimental findings exploiting advances in electrophysiology, structural biology, and imaging techniques, however, have posed a serious challenge to this long-held axiom, as several classes of ion channels appear to open and close in a coordinated, cooperative manner. Ion channel cooperativity ranges from variable-sized oligomeric cooperative gating in voltage-gated, dihydropyridine-sensitive CaV1.2 and CaV1.3 channels to obligatory dimeric assembly and gating of voltage-gated NaV1.5 channels. Potassium channels, transient receptor potential channels, hyperpolarization cyclic nucleotide-activated channels, ryanodine receptors (RyRs), and inositol trisphosphate receptors (IP3Rs) have also been shown to gate cooperatively. The implications of cooperative gating of these ion channels range from fine-tuning excitation-contraction coupling in muscle cells to regulating cardiac function and vascular tone, to modulation of action potential and conduction velocity in neurons and cardiac cells, and to control of pacemaking activity in the heart. In this review, we discuss the mechanisms leading to cooperative gating of ion channels, their physiological consequences, and how alterations in cooperative gating of ion channels may induce a range of clinically significant pathologies.
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Project description:Biological ion channels are nanoscale transmembrane pores. When water and ions are enclosed within the narrow confines of a sub-nanometer hydrophobic pore, they exhibit behavior not evident from macroscopic descriptions. At this nanoscopic level, the unfavorable interaction between the lining of a hydrophobic pore and water may lead to stochastic liquid-vapor transitions. These transient vapor states are "dewetted", i.e. effectively devoid of water molecules within all or part of the pore, thus leading to an energetic barrier to ion conduction. This process, termed "hydrophobic gating", was first observed in molecular dynamics simulations of model nanopores, where the principles underlying hydrophobic gating (i.e., changes in diameter, polarity, or transmembrane voltage) have now been extensively validated. Computational, structural, and functional studies now indicate that biological ion channels may also exploit hydrophobic gating to regulate ion flow within their pores. Here we review the evidence for this process and propose that this unusual behavior of water represents an increasingly important element in understanding the relationship between ion channel structure and function.
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Project description:Phospholipid scramblases externalize phosphatidylserine to facilitate numerous physiological processes. Several members of the structurally unrelated TMEM16 and G protein-coupled receptor (GPCR) protein families mediate phospholipid scrambling. The structure of a TMEM16 scramblase shows a membrane-exposed hydrophilic cavity, suggesting that scrambling occurs via the ‟credit-card" mechanism where lipid headgroups permeate through the cavity while their tails remain associated with the membrane core. Here we show that afTMEM16 and opsin, representatives of the TMEM16 and GCPR scramblase families, transport phospholipids with polyethylene glycol headgroups whose globular dimensions are much larger than the width of the cavity. This suggests that transport of these large headgroups occurs outside rather than within the cavity. These large lipids are scrambled at rates comparable to those of normal phospholipids and their presence in the reconstituted vesicles promotes scrambling of normal phospholipids. This suggests that both large and small phospholipids can move outside the cavity. We propose that the conformational rearrangements underlying TMEM16- and GPCR-mediated credit-card scrambling locally deform the membrane to allow transbilayer lipid translocation outside the cavity and that both mechanisms underlie transport of normal phospholipids.
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Project description:Our laboratory has developed bioinformatic strategies for identifying distant phylogenetic relationships and characterizing families and superfamilies of transport proteins. Results using these tools suggest that the Anoctamin Superfamily of cation and anion channels, as well as lipid scramblases, includes three functionally characterized families: the Anoctamin (ANO), Transmembrane Channel (TMC) and Ca2+-permeable Stress-gated Cation Channel (CSC) families; as well as four families of functionally uncharacterized proteins, which we refer to as the Anoctamin-like (ANO-L), Transmembrane Channel-like (TMC-L), and CSC-like (CSC-L1 and CSC-L2) families. We have constructed protein clusters and trees showing the relative relationships among the seven families. Topological analyses suggest that the members of these families have essentially the same topologies. Comparative examination of these homologous families provides insight into possible mechanisms of action, indicates the currently recognized organismal distributions of these proteins, and suggests drug design potential for the disease-related channel proteins.
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