PNU-120596, a positive allosteric modulator of mammalian α7 nicotinic acetylcholine receptor, is a negative modulator of ligand-gated chloride-selective channels of the gastropod Lymnaea stagnalis.

Excitatory α7 neuronal nicotinic receptors (nAChR) are widely expressed in the central and peripheral nervous and immune systems and are important for learning, memory, and immune response regulation. Specific α7 nAChR ligands, including positive allosteric modulators are promising to treat cognitive disorders, inflammatory processes, and pain. One of them, PNU-120596, highly increased the neuron response to α7 agonists and retarded desensitization, showing selectivity for α7 as compared to heteromeric nAChRs, but was not examined at the inhibitory ligand-gated channels. We studied PNU-120596 action on anion-conducting channels using voltage-clamp techniques: it slightly potentiated the response of human glycine receptors expressed in PC12 cells, of rat GABAA receptors in cerebellar Purkinje cells and mouse GABAA Rs heterologously expressed in Xenopus oocytes. On the contrary, PNU-120596 exerted an inhibitory effect on the receptors mediating anion currents in Lymnaea stagnalis neurons: two nAChR subtypes, GABA and glutamate receptors. Acceleration of the current decay, contrary to slowing down desensitization in mammalian α7 nAChR, was observed in L. stagnalis neurons predominantly expressing one of the two nAChR subtypes. Thus, PNU-120596 effect on these anion-selective nAChRs was just opposite to the action on the mammalian cation-selective α7 nAChRs. A comparison of PNU-120596 molecule docked to the models of transmembrane domains of the human α7 AChR and two subunits of L. stagnalis nAChR demonstrated some differences in contacts with the amino acid residues important for PNU-120596 action on the α7 nAChR. Thus, our results show that PNU-120596 action depends on a particular subtype of these Cys-loop receptors.

Crystal structures of a pentameric ion channel gated by alkaline pH show a widely open pore and identify a cavity for modulation.

Pentameric ligand-gated ion channels (pLGICs) constitute a widespread class of ion channels, present in archaea, bacteria, and eukaryotes. Upon binding of their agonists in the extracellular domain, the transmembrane pore opens, allowing ions to go through, via a gating mechanism that can be modulated by a number of drugs. Even though high-resolution structural information on pLGICs has increased in a spectacular way in recent years, both in bacterial and in eukaryotic systems, the structure of the open channel conformation of some intensively studied receptors whose structures are known in a nonactive (closed) form, such as Erwinia chrysanthemi pLGIC (ELIC), is still lacking. Here we describe a gammaproteobacterial pLGIC from an endo-symbiont of Tevnia jerichonana (sTeLIC), whose sequence is closely related to the pLGIC from ELIC with 28% identity. We provide an X-ray crystallographic structure at 2.3 Å in an active conformation, where the pore is found to be more open than any current conformation found for pLGICs. In addition, two charged restriction rings are present in the vestibule. Functional characterization shows sTeLIC to be a cationic channel activated at alkaline pH. It is inhibited by divalent cations, but not by quaternary ammonium ions, such as tetramethylammonium. Additionally, we found that sTeLIC is allosterically potentiated by aromatic amino acids Phe and Trp, as well as their derivatives, such as 4-bromo-cinnamate, whose cocrystal structure reveals a vestibular binding site equivalent to, but more deeply buried than, the one already described for benzodiazepines in ELIC.

Allosteric modulation as a unifying mechanism for receptor function and regulation.

Four major receptor families enable cells to respond to chemical and physical signals from their proximal environment. The ligand- and voltage-gated ion channels, G-protein-coupled receptors, nuclear hormone receptors and receptor tyrosine kinases are all allosteric proteins that carry multiple, spatially distinct, yet conformationally linked ligand-binding sites. Recent studies point to common mechanisms governing the allosteric transitions of these receptors, including the impact of oligomerization, pre-existing and functionally distinct conformational ensembles, intrinsically disordered regions, and the occurrence of allosteric modulatory sites. Importantly, synthetic allosteric modulators are being discovered for these receptors, providing an enriched, yet challenging, landscape for novel therapeutics.

Ketamine Inhibition of the Pentameric Ligand-Gated Ion Channel GLIC.

Ketamine inhibits pentameric ligand-gated ion channels (pLGICs), including the bacterial pLGIC from Gloeobacter violaceus (GLIC). The crystal structure of GLIC shows R-ketamine bound to an extracellular intersubunit cavity. Here, we performed molecular dynamics simulations of GLIC in the absence and presence of R- or S-ketamine. No stable binding of S-ketamine in the original cavity was observed in the simulations, largely due to its unfavorable access to residue D154, which provides important electrostatic interactions to stabilize R-ketamine binding. Contrary to the symmetric binding shown in the crystal structure, R-ketamine moved away from some of the binding sites and was bound to GLIC asymmetrically at the end of simulations. The asymmetric binding is consistent with the experimentally measured negative cooperativity of ketamine binding to GLIC. In the presence of R-ketamine, all subunits showed changes in structure and dynamics, irrespective of binding stability; the extracellular intersubunit cavity expanded and intersubunit electrostatic interactions involved in channel activation were altered. R-ketamine binding promoted a conformational shift toward closed GLIC. Conformational changes near the ketamine-binding site were propagated to the interface between the extracellular and transmembrane domains, and further to the pore-lining TM2 through two pathways: pre-TM1 and the ß1-ß2 loop. Both signaling pathways have been predicted previously using the perturbation-based Markovian transmission model. The study provides a structural and dynamics basis for the inhibitory modulation of ketamine on pLGICs.

Structural Basis of Alcohol Inhibition of the Pentameric Ligand-Gated Ion Channel ELIC.

The structural basis for alcohol modulation of neuronal pentameric ligand-gated ion channels (pLGICs) remains elusive. We determined an inhibitory mechanism of alcohol on the pLGIC Erwinia chrysanthemi (ELIC) through direct binding to the pore. X-ray structures of ELIC co-crystallized with 2-bromoethanol, in both the absence and presence of agonist, reveal 2-bromoethanol binding in the pore near T237(6') and the extracellular domain (ECD) of each subunit at three different locations. Binding to the ECD does not appear to contribute to the inhibitory action of 2-bromoethanol and ethanol as indicated by the same functional responses of wild-type ELIC and mutants. In contrast, the ELIC-α1ß3GABAAR chimera, replacing the ELIC transmembrane domain (TMD) with the TMD of α1ß3GABAAR, is potentiated by 2-bromoethanol and ethanol. The results suggest a dominant role of the TMD in modulating alcohol effects. The X-ray structures and functional measurements support a pore-blocking mechanism for inhibitory action of short-chain alcohols.

Common Anesthetic-binding Site for Inhibition of Pentameric Ligand-gated Ion Channels.

BACKGROUND: Identifying functionally relevant anesthetic-binding sites in pentameric ligand-gated ion channels (pLGICs) is an important step toward understanding the molecular mechanisms underlying anesthetic action. The anesthetic propofol is known to inhibit cation-conducting pLGICs, including a prokaryotic pLGIC from Erwinia chrysanthemi (ELIC), but the sites responsible for functional inhibition remain undetermined. METHODS: We photolabeled ELIC with a light-activated derivative of propofol (AziPm) and performed fluorine-19 nuclear magnetic resonance experiments to support propofol binding to a transmembrane domain (TMD) intrasubunit pocket. To differentiate sites responsible for propofol inhibition from those that are functionally irrelevant, we made an ELIC-γ-aminobutyric acid receptor (GABAAR) chimera that replaced the ELIC-TMD with the α1ß3GABAAR-TMD and compared functional responses of ELIC-GABAAR and ELIC with propofol modulations. RESULTS: Photolabeling showed multiple AziPm-binding sites in the extracellular domain (ECD) but only one site in the TMD with labeled residues M265 and F308 in the resting state of ELIC. Notably, this TMD site is an intrasubunit pocket that overlaps with binding sites for anesthetics, including propofol, found previously in other pLGICs. Fluorine-19 nuclear magnetic resonance experiments supported propofol binding to this TMD intrasubunit pocket only in the absence of agonist. Functional measurements of ELIC-GABAAR showed propofol potentiation of the agonist-elicited current instead of inhibition observed on ELIC. CONCLUSIONS: The distinctly different responses of ELIC and ELIC-GABAAR to propofol support the functional relevance of propofol binding to the TMD. Combining the newly identified TMD intrasubunit pocket in ELIC with equivalent TMD anesthetic sites found previously in other cationic pLGICs, we propose this TMD pocket as a common site for anesthetic inhibition of pLGICs.

Direct Pore Binding as a Mechanism for Isoflurane Inhibition of the Pentameric Ligand-gated Ion Channel ELIC.

Pentameric ligand-gated ion channels (pLGICs) are targets of general anesthetics, but molecular mechanisms underlying anesthetic action remain debatable. We found that ELIC, a pLGIC from Erwinia chrysanthemi, can be functionally inhibited by isoflurane and other anesthetics. Structures of ELIC co-crystallized with isoflurane in the absence or presence of an agonist revealed double isoflurane occupancies inside the pore near T237(6') and A244(13'). A pore-radius contraction near the extracellular entrance was observed upon isoflurane binding. Electrophysiology measurements with a single-point mutation at position 6' or 13' support the notion that binding at these sites renders isoflurane inhibition. Molecular dynamics simulations suggested that isoflurane binding was more stable in the resting than in a desensitized pore conformation. This study presents compelling evidence for a direct pore-binding mechanism of isoflurane inhibition, which has a general implication for inhibitory action of general anesthetics on pLGICs.

Seeking structural specificity: direct modulation of pentameric ligand-gated ion channels by alcohols and general anesthetics.

Alcohols and other anesthetic agents dramatically alter neurologic function in a wide range of organisms, yet their molecular sites of action remain poorly characterized. Pentameric ligand-gated ion channels, long implicated in important direct effects of alcohol and anesthetic binding, have recently been illuminated in renewed detail thanks to the determination of atomic-resolution structures of several family members from lower organisms. These structures provide valuable models for understanding and developing anesthetic agents and for allosteric modulation in general. This review surveys progress in this field from function to structure and back again, outlining early evidence for relevant modulation of pentameric ligand-gated ion channels and the development of early structural models for ion channel function and modulation. We highlight insights and challenges provided by recent crystal structures and resulting simulations, as well as opportunities for translation of these newly detailed models back to behavior and therapy.

Inhibition versus potentiation of ligand-gated ion channels can be altered by a single mutation that moves ligands between intra- and intersubunit sites.

Pentameric ligand-gated ion channels (pLGICs) are similar in structure but either inhibited or potentiated by alcohols and anesthetics. This dual modulation has previously not been understood, but the determination of X-ray structures of prokaryotic GLIC provides an ideal model system. Here, we show that a single-site mutation at the F14' site in the GLIC transmembrane domain turns desflurane and chloroform from inhibitors to potentiators, and that this is explained by competing allosteric sites. The F14'A mutation opens an intersubunit site lined by N239 (15'), I240 (16'), and Y263. Free energy calculations confirm this site is the preferred binding location for desflurane and chloroform in GLIC F14'A. In contrast, both anesthetics prefer an intrasubunit site in wild-type GLIC. Modulation is therefore the net effect of competitive binding between the intersubunit potentiating site and an intrasubunit inhibitory site. This provides direct evidence for a dual-site model of allosteric regulation of pLGICs.

Novel approaches to drug design for the treatment of schizophrenia.

INTRODUCTION: Schizophrenia is an important health issue affecting almost 1% of the population with significant unmet medical needs. The classical drug targets for the treatment of schizophrenia are dopamine D2 receptors. Second-generation ('atypical') drugs block more receptors of the G-protein-coupled receptor (GPCR) class 1 (e.g., clozapine is a D(2)-5HT(2) antagonist). AREAS COVERED: In this article, the author presents the new targets for GPCR as well as ligand-gated ion. Furthermore, the author reviews the opportunities for drug design offered by the structures solved recently. EXPERT OPINION: For drug design, the availability of these protein structures, or the possibility to build high quality models, allows to shift the paradigm from ligand-based to target-based drug design. The analysis of the drugs, both on the market and in development, shows that numerous targets are being considered which may reveal an ambiguity on the ideal drug target. This situation may be simplified, in the future, owing to recent integrative projects: the 'Human Brain Project' and the 'Brain Activity Map' that aim at modeling the brain as well as the Allen Atlas.

Identification of cinnamic acid derivatives as novel antagonists of the prokaryotic proton-gated ion channel GLIC.

Pentameric ligand gated ion channels (pLGICs) mediate signal transduction. The binding of an extracellular ligand is coupled to the transmembrane channel opening. So far, all known agonists bind at the interface between subunits in a topologically conserved "orthosteric site" whose amino acid composition defines the pharmacological specificity of pLGIC subtypes. A striking exception is the bacterial proton-activated GLIC protein, exhibiting an uncommon orthosteric binding site in terms of sequence and local architecture. Among a library of Gloeobacter violaceus metabolites, we identified a series of cinnamic acid derivatives, which antagonize the GLIC proton-elicited response. Structure-activity analysis shows a key contribution of the carboxylate moiety to GLIC inhibition. Molecular docking coupled to site-directed mutagenesis support that the binding pocket is located below the classical orthosteric site. These antagonists provide new tools to modulate conformation of GLIC, currently used as a prototypic pLGIC, and opens new avenues to study the signal transduction mechanism.

Ion channels as drug targets in central nervous system disorders.

Ion channel targeted drugs have always been related with either the central nervous system (CNS), the peripheral nervous system, or the cardiovascular system. Within the CNS, basic indications of drugs are: sleep disorders, anxiety, epilepsy, pain, etc. However, traditional channel blockers have multiple adverse events, mainly due to low specificity of mechanism of action. Lately, novel ion channel subtypes have been discovered, which gives premises to drug discovery process led towards specific channel subtypes. An example is Na(+) channels, whose subtypes 1.3 and 1.7-1.9 are responsible for pain, and 1.1 and 1.2 - for epilepsy. Moreover, new drug candidates have been recognized. This review is focusing on ion channels subtypes, which play a significant role in current drug discovery and development process. The knowledge on channel subtypes has developed rapidly, giving new nomenclatures of ion channels. For example, Ca(2+)s channels are not any more divided to T, L, N, P/Q, and R, but they are described as Ca(v)1.1-Ca(v)3.3, with even newer nomenclature α1A-α1I and α1S. Moreover, new channels such as P2X1-P2X7, as well as TRPA1-TRPV1 have been discovered, giving premises for new types of analgesic drugs.

Inhibition of the prokaryotic pentameric ligand-gated ion channel ELIC by divalent cations.

The modulation of pentameric ligand-gated ion channels (pLGICs) by divalent cations is believed to play an important role in their regulation in a physiological context. Ions such as calcium or zinc influence the activity of pLGIC neurotransmitter receptors by binding to their extracellular domain and either potentiate or inhibit channel activation. Here we have investigated by electrophysiology and X-ray crystallography the effect of divalent ions on ELIC, a close prokaryotic pLGIC homologue of known structure. We found that divalent cations inhibit the activation of ELIC by the agonist cysteamine, reducing both its potency and, at higher concentrations, its maximum response. Crystal structures of the channel in complex with barium reveal the presence of several distinct binding sites. By mutagenesis we confirmed that the site responsible for divalent inhibition is located at the outer rim of the extracellular domain, at the interface between adjacent subunits but at some distance from the agonist binding region. Here, divalent cations interact with the protein via carboxylate side-chains, and the site is similar in structure to calcium binding sites described in other proteins. There is evidence that other pLGICs may be regulated by divalent ions binding to a similar region, even though the interacting residues are not conserved within the family. Our study provides structural and functional insight into the allosteric regulation of ELIC and is of potential relevance for the entire family.

Structure of the pentameric ligand-gated ion channel GLIC bound with anesthetic ketamine.

Pentameric ligand-gated ion channels (pLGICs) are targets of general anesthetics, but a structural understanding of anesthetic action on pLGICs remains elusive. GLIC, a prokaryotic pLGIC, can be inhibited by anesthetics, including ketamine. The ketamine concentration leading to half-maximal inhibition of GLIC (58 µM) is comparable to that on neuronal nicotinic acetylcholine receptors. A 2.99 Å resolution X-ray structure of GLIC bound with ketamine revealed ketamine binding to an intersubunit cavity that partially overlaps with the homologous antagonist-binding site in pLGICs. The functional relevance of the identified ketamine site was highlighted by profound changes in GLIC activation upon cysteine substitution of the cavity-lining residue N152. The relevance is also evidenced by changes in ketamine inhibition upon the subsequent chemical labeling of N152C. The results provide structural insight into the molecular recognition of ketamine and are valuable for understanding the actions of anesthetics and other allosteric modulators on pLGICs.

The pharmacological profile of ELIC, a prokaryotic GABA-gated receptor.

The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of vertebrate Cys-loop ligand-gated ion channels. It is activated by GABA, and this property, combined with its structural similarity to GABA(A) and other Cys-loop receptors, makes it potentially an excellent model to probe their structure and function. Here we characterise the pharmacological profile of ELIC, examining the effects of compounds that could activate or inhibit the receptor. We confirm that a range of amino acids and classic GABA(A) receptor agonists do not elicit responses in ELIC, and we show the receptor can be at least partially activated by 5-aminovaleric acid and γ-hydroxybutyric acid, which are weak agonists. A range of GABA(A) receptor non-competitive antagonists inhibit GABA-elicited ELIC responses including α-endosulfan (IC50 = 17 µM), dieldrin (IC50 = 66 µM), and picrotoxinin (IC50 = 96 µM) which were the most potent. Docking suggested possible interactions at the 2' and 6' pore-lining residues, and mutagenesis of these residues supports this hypothesis for α-endosulfan. A selection of compounds that act at Cys-loop and other receptors also showed some efficacy at blocking ELIC responses, but most were of low potency (IC50 > 100 µM). Overall our data show that a number of compounds can inhibit ELIC, but it has limited pharmacological similarity to GLIC and to Cys-loop receptors.

Structure of the pentameric ligand-gated ion channel ELIC cocrystallized with its competitive antagonist acetylcholine.

ELIC, the pentameric ligand-gated ion channel from Erwinia chrysanthemi, is a prototype for Cys-loop receptors. Here we show that acetylcholine is a competitive antagonist for ELIC. We determine the acetylcholine-ELIC cocrystal structure to a 2.9-Å resolution and find that acetylcholine binding to an aromatic cage at the subunit interface induces a significant contraction of loop C and other structural rearrangements in the extracellular domain. The side chain of the pore-lining residue F247 reorients and the pore size consequently enlarges, but the channel remains closed. We attribute the inability of acetylcholine to activate ELIC primarily to weak cation-π and electrostatic interactions in the pocket, because an acetylcholine derivative with a simple quaternary-to-tertiary ammonium substitution activates the channel. This study presents a compelling case for understanding the structural underpinning of the functional relationship between agonism and competitive antagonism in the Cys-loop receptors, providing a new framework for developing novel therapeutic drugs.

From toxins targeting ligand gated ion channels to therapeutic molecules.

Ligand-gated ion channels (LGIC) play a central role in inter-cellular communication. This key function has two consequences: (i) these receptor channels are major targets for drug discovery because of their potential involvement in numerous human brain diseases; (ii) they are often found to be the target of plant and animal toxins. Together this makes toxin/receptor interactions important to drug discovery projects. Therefore, toxins acting on LGIC are presented and their current/potential therapeutic uses highlighted.

Intraretinal calcium channels and retinal morbidity in experimental retinopathy of prematurity.

PURPOSE: To test the hypothesis that intraretinal calcium channels participate in retinal morbidity in a variable oxygen (VO) model of retinopathy of prematurity. METHODS: In control and VO Long Evans (LE) rats, either untreated or treated with voltage- or ligand-gated calcium channel antagonists, we measured retinal neovascular (NV) incidence and severity (adenosine diphosphatase staining), and retinal thickness and intraretinal ion channel activity (manganese-enhanced magnetic resonance imaging). Comparisons with the commonly studied Sprague Dawley rats were performed. Visual performance (optokinetic tracking) in untreated VO LE rats was also evaluated. RESULTS: In control LE rats, specific L-type voltage calcium channel antagonism, but not ligand-gated channel blockers, suppressed retinal manganese accumulation, while the inhibition of L-type channels normalized intraretinal uptake in VO LE rats. VO LE rats developed more severe NV than VO Sprague Dawley rats. Following VO, both strains demonstrated significant and similar degrees of retinal thinning and supernormal intraretinal manganese uptake. However, over time, intraretinal uptake remained elevated only in VO LE rats. Visual performance was subnormal in VO LE rats. L-type voltage-gated calcium channel antagonism reduced NV severity by 28% (p<0.05) in experimental LE rats compared to that in the control group. CONCLUSIONS: Abnormal intraretinal calcium channel activity is linked with retinal morbidity in experimental retinopathy of prematurity.

Modulation of ligand-gated ion channels as a novel pharmacological principle.

The present study investigated the functional antagonism of different antidepressants on 5-HT (3) receptor function and the role of lipid rafts for these modulatory effects. Electrophysiological recordings of 5-HT evoked cation currents were recorded with N1E-115 and HEK-5-HT (3A) cells and hippocampal neurons. The characterization of the antagonism of antidepressants was made by the displacement of [ (3)H]GR65630 binding. For membrane fractionation, sucrose density gradient centrifugation was used. Gradient fractions were assayed for antidepressant concentrations by HPLC; 5-HT (3) receptor membrane distribution was determined by Western blot. Colocalization experiments were performed by means of immunocytochemistry. Most antidepressants acted as non-competitive antagonists at the 5-HT (3) receptor. Moreover, some of these compounds were enriched within lipid rafts. Cholesterol depletion impaired lipid raft integrity thereby affecting 5-HT (3) receptor function, whereas the antagonistic effects of antidepressants were not altered.In conclusion, most antidepressants directly antagonize 5-HT (3) receptor activity. 5-HT (3) receptor function PER SE appears to depend on lipid raft integrity, which is, however, not a prerequisite for the modulatory potency of antidepressants at this receptor.