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Na + /Cl – -coupled biogenic amine transporters are the primary targets of therapeutic and abused drugs, ranging from antidepressants to the psychostimulants cocaine and amphetamines, and to their cognate substrates. Here we determine X-ray crystal structures of the Drosophila melanogaster dopamine transporter (dDAT) bound to its substrate dopamine, a substrate analogue 3,4-dichlorophenethylamine, the psychostimulants d -amphetamine and methamphetamine, or to cocaine and cocaine analogues. All ligands bind to the central binding site, located approximately halfway across the membrane bilayer, in close proximity to bound sodium and chloride ions. The central binding site recognizes three chemically distinct classes of ligands via conformational changes that accommodate varying sizes and shapes, thus illustrating molecular principles that distinguish substrates from inhibitors in biogenic amine transporters. Here the X-ray crystal structures of the Drosophila dopamine transporter bound to dopamine, D -amphetamine, methamphetamine and cocaine are solved; these structures show how a neurotransmitter, small molecule stimulants and cocaine bind to a biogenic amine transporter, and are examples of how the ligand binding site of a neurotransmitter transporter can remodel itself to accommodate structurally unrelated small molecules that are different in shape, size and polarity or charge. Dopamine transporter structures The dopamine transporter is a membrane protein that removes the neurotransmitter dopamine from the synaptic cleft and imports it into the cytosol of surrounding cells, thereby terminating the neurotransmitter signal. Eric Gouaux and colleagues have solved X-ray crystal structures of the Drosophila dopamine transporter bound to various small molecules, including cocaine, D -amphetamine, methamphetamine, dopamine and two antidepressants. As well as providing the first glimpses of how a neurotransmitter binds to a biogenic amine transporter and how cocaine binds to a biogenic amine transporter, this structure is a good example of how the ligand-binding site of a protein can remodel itself to bind to accommodate structurally unrelated small molecules that are different shapes and sizes.

Antidepressants targeting Na + /Cl − -coupled neurotransmitter uptake define a key therapeutic strategy to treat clinical depression and neuropathic pain. However, identifying the molecular interactions that underlie the pharmacological activity of these transport inhibitors, and thus the mechanism by which the inhibitors lead to increased synaptic neurotransmitter levels, has proven elusive. Here we present the crystal structure of the Drosophila melanogaster dopamine transporter at 3.0 Å resolution bound to the tricyclic antidepressant nortriptyline. The transporter is locked in an outward-open conformation with nortriptyline wedged between transmembrane helices 1, 3, 6 and 8, blocking the transporter from binding substrate and from isomerizing to an inward-facing conformation. Although the overall structure of the dopamine transporter is similar to that of its prokaryotic relative LeuT, there are multiple distinctions, including a kink in transmembrane helix 12 halfway across the membrane bilayer, a latch-like carboxy-terminal helix that caps the cytoplasmic gate, and a cholesterol molecule wedged within a groove formed by transmembrane helices 1a, 5 and 7. Taken together, the dopamine transporter structure reveals the molecular basis for antidepressant action on sodium-coupled neurotransmitter symporters and elucidates critical elements of eukaryotic transporter structure and modulation by lipids, thus expanding our understanding of the mechanism and regulation of neurotransmitter uptake at chemical synapses. The X-ray crystal structure of the Drosophila dopamine transporter bound to the antidepressant drug nortriptyline is presented, providing the first crystal structure of a eukaryotic neurotransmitter sodium symporter. Dopamine transport protein structure The dopamine transporter (DAT) is a membrane protein that removes the neurotransmitter dopamine from the synaptic cleft and imports it into the cytosol of surrounding cells, thereby terminating the signal of the neurotransmitter. Eric Gouaux and colleagues report the X-ray structure of the Drosophila DAT bound to the tricyclic antidepressant nortriptyline. This is the first crystal structure of a eukaryotic neurotransmitter sodium symporter to have been determined. The overall structure of Drosophila DAT is similar to that of LeuT, but the authors identify several differences that may have important roles in the transport mechanism of the eukaryotic protein and its regulation by phosphorylation.

Glycine receptors (GlyRs) are pentameric, ‘Cys-loop’ receptors that form chloride-permeable channels and mediate fast inhibitory signalling throughout the central nervous system 1 , 2 . In the spinal cord and brainstem, GlyRs regulate locomotion and cause movement disorders when mutated 2 , 3 . However, the stoichiometry of native GlyRs and the mechanism by which they are assembled remain unclear, despite extensive investigation 4 – 8 . Here we report cryo-electron microscopy structures of native GlyRs from pig spinal cord and brainstem, revealing structural insights into heteromeric receptors and their predominant subunit stoichiometry of 4α:1β. Within the heteromeric pentamer, the β(+)–α(−) interface adopts a structure that is distinct from the α(+)–α(−) and α(+)–β(−) interfaces. Furthermore, the β-subunit contains a unique phenylalanine residue that resides within the pore and disrupts the canonical picrotoxin site. These results explain why inclusion of the β-subunit breaks receptor symmetry and alters ion channel pharmacology. We also find incomplete receptor complexes and, by elucidating their structures, reveal the architectures of partially assembled α-trimers and α-tetramers. Cryo-electron microscopy structures of pig glycine receptors indicate that they are predominantly assembled with 4α:1β stoichiometry via α-homotrimer and homotetramer intermediates.

P2X receptors are trimeric ATP-activated ion channels permeable to Na + , K + and Ca 2+ . The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X 4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body β-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents. The X-ray crystal structure of the zebrafish P2X 4 receptor in the presence and absence of ATP is determined, revealing an ATP-binding site and an open ion channel pore. ATP-gated P2X receptor structures P2X receptors are ATP-activated ion channels that are permeable to Na + , K + and Ca 2+ ions. These proteins are involved in a broad range of physiological processes, including the modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. In this study, the authors report X-ray crystal structures of the zebrafish P2X 4 receptor in the presence and absence of ATP. The ATP-bound structure reveals a previously unseen ATP-binding motif, and comparison of the two structures indicates that ATP binding leads to a radial expansion of the extracellular vestibule, which causes an iris-like expansion of the transmembrane helices, opening the ion channel.

Neurotransmitter sodium symporters are integral membrane proteins that remove chemical transmitters from the synapse and terminate neurotransmission mediated by serotonin, dopamine, noradrenaline, glycine and GABA (γ-aminobutyric acid). Crystal structures of the bacterial homologue, LeuT, in substrate-bound outward-occluded and competitive inhibitor-bound outward-facing states have advanced our mechanistic understanding of neurotransmitter sodium symporters but have left fundamental questions unanswered. Here we report crystal structures of LeuT mutants in complexes with conformation-specific antibody fragments in the outward-open and inward-open states. In the absence of substrate but in the presence of sodium the transporter is outward-open, illustrating how the binding of substrate closes the extracellular gate through local conformational changes: hinge-bending movements of the extracellular halves of transmembrane domains 1, 2 and 6, together with translation of extracellular loop 4. The inward-open conformation, by contrast, involves large-scale conformational changes, including a reorientation of transmembrane domains 1, 2, 5, 6 and 7, a marked hinge bending of transmembrane domain 1a and occlusion of the extracellular vestibule by extracellular loop 4. These changes close the extracellular gate, open an intracellular vestibule, and largely disrupt the two sodium sites, thus providing a mechanism by which ions and substrate are released to the cytoplasm. The new structures establish a structural framework for the mechanism of neurotransmitter sodium symporters and their modulation by therapeutic and illicit substances. The X-ray crystal structure of LeuT, the bacterial homologue of the neurotransmitter sodium symporter family, is reported in the outward-open and inward-open states. Ins and outs of neurotransmitter channels Chemical neurotransmission in the central nervous system is terminated through re-uptake of neurotransmitters from the synapse into surrounding neuronal and glial cells. Chemical transmitters are removed from the synaptic cleft by neurotransmitter sodium symporters (NSSs). The X-ray crystal structures of LeuT, a bacterial homologue of the NSS family, have now been determined in the outward-open and inward-open states. The structure of the outward-open state illustrates how substrate binding leads to the closure of the extracellular gate. The switch to the inward-open form requires large-scale conformational changes in the membrane protein. These structures establish a framework for the mechanism of neurotransmitter sodium symporters and their modulation by therapeutic and illicit drugs.

X-ray and cryo-electron microscopy structures of the acid-sensing ion channel ASIC1a reveal the molecular mechanisms of channel gating and desensitization. Acid sensors at rest Acid-sensing ion channels (ASICs) are proton-gated channels that respond to extracellular acidification from inflammation or ischemic injury. Although several structural studies have elucidated certain details about ASICs, the physiologically relevant resting state has remained elusive. Here, Eric Gouaux and colleagues report both crystallographic and cryo-electron microscopy structures of chicken ASIC1a at high pH. These data, along with biochemical studies, provide insights into the molecular-level mechanism of gating and modulation in ASICs and the epithelial sodium channel/degenerin superfamily. The structures contain an expanded acidic pocket, which collapses on exposure to protons through a linker in the palm domain. The linker acts as a clutch, disengaging the acidic pocket from the lower part of the channel. Acid-sensing ion channels (ASICs) are trimeric 1 , proton-gated 2 , 3 and sodium-selective 4 , 5 members of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily of ion channels 6 , 7 and are expressed throughout vertebrate central and peripheral nervous systems. Gating of ASICs occurs on a millisecond time scale 8 and the mechanism involves three conformational states: high pH resting, low pH open and low pH desensitized 9 . Existing X-ray structures of ASIC1a describe the conformations of the open 10 and desensitized 1 , 11 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.

Type A γ-aminobutyric acid receptors (GABA A Rs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including anaesthetics, sedatives, hypnotics and antidepressants 1 – 3 . However, our understanding of GABA A R pharmacology has been hindered by the vast number of pentameric assemblies that can be derived from 19 different subunits 4 and the lack of structural knowledge of clinically relevant receptors. Here, we isolate native murine GABA A R assemblies containing the widely expressed α1 subunit and elucidate their structures in complex with drugs used to treat insomnia (zolpidem (ZOL) and flurazepam) and postpartum depression (the neurosteroid allopregnanolone (APG)). Using cryo-electron microscopy (cryo-EM) analysis and single-molecule photobleaching experiments, we uncover three major structural populations in the brain: the canonical α1β2γ2 receptor containing two α1 subunits, and two assemblies containing one α1 and either an α2 or α3 subunit, in which the single α1-containing receptors feature a more compact arrangement between the transmembrane and extracellular domains. Interestingly, APG is bound at the transmembrane α/β subunit interface, even when not added to the sample, revealing an important role for endogenous neurosteroids in modulating native GABA A Rs. Together with structurally engaged lipids, neurosteroids produce global conformational changes throughout the receptor that modify the ion channel pore and the binding sites for GABA and insomnia medications. Our data reveal the major α1-containing GABA A R assemblies, bound with endogenous neurosteroid, thus defining a structural landscape from which subtype-specific drugs can be developed. Using cryo-EM, structures of three major assemblies of type A GABA receptors, which regulate brain excitability, are revealed in the mouse brain and provide a basis for the development of subtype-specific drugs.

Acid-sensing ion channels (ASICs) are voltage-independent, amiloride-sensitive channels involved in diverse physiological processes ranging from nociception to taste. Despite the importance of ASICs in physiology, we know little about the mechanism of channel activation. Here we show that psalmotoxin activates non-selective and Na + -selective currents in chicken ASIC1a at pH 7.25 and 5.5, respectively. Crystal structures of ASIC1a–psalmotoxin complexes map the toxin binding site to the extracellular domain and show how toxin binding triggers an expansion of the extracellular vestibule and stabilization of the open channel pore. At pH 7.25 the pore is approximately 10 Å in diameter, whereas at pH 5.5 the pore is largely hydrophobic and elliptical in cross-section with dimensions of approximately 5 by 7 Å, consistent with a barrier mechanism for ion selectivity. These studies define mechanisms for activation of ASICs, illuminate the basis for dynamic ion selectivity and provide the blueprints for new therapeutic agents. Acid-sensing ion channels (ASICs) are voltage-independent ion channels that participate in a broad range of biological processes, including nociception and mechanosensation; here X-ray crystal structures of the complexes of chicken ASIC1a with psalmotoxin, a peptide toxin from tarantula, indicate that toxin binding triggers an expansion of the extracellular vestibule and stabilization of the open channel pore. Open ASIC1a channel structure Acid-sensing ion channels (ASICs) are members of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily of voltage-independent ion channels. ENaCs, including ASICs, participate in a broad range of biological processes, such as nociception, mechanosensation and regulation of sodium-ion homeostasis. Here, Isabelle Baconguis and Eric Gouaux show that psalmotoxin, a peptide toxin from the tarantula, activates nonselective and sodium-selective currents in chicken ASIC1a. X-ray crystal structures of the chicken ASIC1a–psalmotoxin complexes indicate that toxin binding triggers an expansion of the extracellular vestibule and stabilization of the open channel pore. This view of an important type of ion channel in an open conformation is of relevance to the design of open-channel blockers that might have therapeutic promise for the treatment of pain.

Acid-sensing ion channels (ASICs) are proton-gated members of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily of ion channels and are expressed throughout the central and peripheral nervous systems. The homotrimeric splice variant ASIC1a has been implicated in nociception, fear memory, mood disorders and ischemia. Here, we extract full-length chicken ASIC1 (cASIC1) from cell membranes using styrene maleic acid (SMA) copolymer, elucidating structures of ASIC1 channels in both high pH resting and low pH desensitized conformations by single-particle cryo-electron microscopy (cryo-EM). The structures of resting and desensitized channels reveal a reentrant loop at the amino terminus of ASIC1 that includes the highly conserved ‘His-Gly’ (HG) motif. The reentrant loop lines the lower ion permeation pathway and buttresses the ‘Gly-Ala-Ser’ (GAS) constriction, thus providing a structural explanation for the role of the His-Gly dipeptide in the structure and function of ASICs.

This study solved structures of the glutamate-gated chloride channel (GluCl), a Cys-loop receptor from C. elegans , in an apo , closed state and in a lipid-bound state — comparison of these structures with a previously published structure of GluCl in an ivermectin-bound state reveals what conformational changes probably occur as this membrane protein transitions from the closed/resting state towards an open/activated state. Resting-to-activated state of a Cys-loop receptor Depending on their ligand and ion selectivity, Cys-loop receptors are neurotransmitter-activated ion channels that mediate either excitatory or inhibitory neurotransmission. In this paper the authors solve the structures of the glutamate-gated chloride channel (GluCl), a Cys-loop receptor from Caenorhabditis elegans , in an apo or closed state and in a lipid-bound state. Comparison of these structures with the previously published structure of GluCl in an ivermectin-bound state reveals the conformational changes involved as this membrane protein transitions between the closed/resting and an open/activated state. Cys-loop receptors are neurotransmitter-gated ion channels that are essential mediators of fast chemical neurotransmission and are associated with a large number of neurological diseases and disorders, as well as parasitic infections 1 , 2 , 3 , 4 . Members of this ion channel superfamily mediate excitatory or inhibitory neurotransmission depending on their ligand and ion selectivity. Structural information for Cys-loop receptors comes from several sources including electron microscopic studies of the nicotinic acetylcholine receptor 5 , high-resolution X-ray structures of extracellular domains 6 and X-ray structures of bacterial orthologues 7 , 8 , 9 , 10 . In 2011 our group published structures of the Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in complex with the allosteric partial agonist ivermectin, which provided insights into the structure of a possibly open state of a eukaryotic Cys-loop receptor, the basis for anion selectivity and channel block, and the mechanism by which ivermectin and related molecules stabilize the open state and potentiate neurotransmitter binding 11 . However, there remain unanswered questions about the mechanism of channel opening and closing, the location and nature of the shut ion channel gate, the transitions between the closed/resting, open/activated and closed/desensitized states, and the mechanism by which conformational changes are coupled between the extracellular, orthosteric agonist binding domain and the transmembrane, ion channel domain. Here we present two conformationally distinct structures of C. elegans GluCl in the absence of ivermectin. Structural comparisons reveal a quaternary activation mechanism arising from rigid-body movements between the extracellular and transmembrane domains and a mechanism for modulation of the receptor by phospholipids.