A highly conserved aspartic acid residue in the signature disulfide loop of the alpha 1 subunit is a determinant of gating in the glycine receptor

Ligand-gated ion channels (LGICs) mediate rapid chemical neurotransmission. This gene superfamily includes the nicotinic acetylcholine, GABAA/C, 5-hydroxytryptamine type 3, and glycine receptors. A signature disulfide loop (Cys loop) in the extracellular domain is a structural motif common to all LGIC member subunits. Here we report that a highly conserved aspartic acid residue within the Cys loop at position 148 (Asp-148) of the glycine receptor alpha1 subunit is critical in the process of receptor activation. Mutation of this acidic residue to the basic amino acid lysine produces a large decrease in the potency of glycine, produces a decrease in the Hill slope, and converts taurine from a full agonist to a partial agonist; these data are consistent with a molecular defect in the receptor gating mechanism. Additional mutation of Asp-148 shows that alterations in the EC50 for agonists are dependent upon the charge of the side chain at this position and not molecular volume, polarity, or hydropathy. This study implicates negative charge at position Asp-148 as a critical component of the process in which agonist binding is coupled to channel gating. This finding adds to an emerging body of evidence supporting the involvement of the Cys loop in the gating mechanism of the LGICs.     

Chimeric mutations in the M2 segment of the 5-hydroxytryptamine-gated chloride channel MOD-1 define a minimal determinant of anion/cation permeability

The ionic selectivity of ligand-gated ion channels (LGICs) determines whether receptor activation produces an excitatory or inhibitory response. The determinants of anion/cation selectivity were investigated for a new member of the LGIC superfamily, MOD-1, a serotonin-gated chloride channel cloned from the nematode Caenorhabditis elegans. In common with other anionic LGICs (glycine receptors and GABA(A) receptors), the selectivity triple mutant in the pore-forming M2 segment (proline insertion, Ala --> Glu substitution at the central ring, and Thr --> Val at the hydrophobic ring) converted the selectivity of MOD-1 from anionic to cationic. Unlike other LGICs, however, this mutant in MOD-1 was highly selective for K+ over other cations. Subsets of this selectivity triple mutant were studied to define the minimal change required for conversion from anion-permeable to cation-permeable. The double mutant at the central ring of charge (deltaPro-269/A270E) produced a non-selective cation channel. Charge reversal at the central ring alone (A270E) was sufficient to convert MOD-1 to cation-permeable. These results refine the determinants of ion-charge selectivity in LGICs and demonstrate the critical role of the central ring of charge formed by the M2 segments.     

LGICdb: the ligand-gated ion channel database

Ligand-Gated Ion Channels (LGIC) are polymeric transmembrane proteins involved in the fast response to numerous neurotransmitters. All these receptors are formed by homologous subunits and the last two decades revealed an unexpected wealth of genes coding for these subunits. The Ligand-Gated Ion Channel database (LGICdb) has been developed to handle this increasing amount of data. The database aims to provide only one entry for each gene, containing annotated nucleic acid and protein sequences. The repository is carefully structured and the entries can be retrieved by various criteria. In addition to the sequences, the LGICdb provides multiple sequence alignments, phylogenetic analyses and atomic coordinates when available. The database is accessible via the World Wide Web (http://www.pasteur.fr/recherche/banques/LGIC /LGIC.html), where it is continuously updated. The version 16 (September 2000) available for download contained 333 entries covering 34 species.     

Evidence for a Diverse Cys-Loop Ligand-Gated Ion Channel Superfamily in Early Bilateria

The genome sequences of Caenorhabditis elegans and Drosophila melanogaster reveal a diversity of cysteine-loop ligand-gated ion channels (Cys-loop LGICs) not found in vertebrates. To better understand the evolution of this gene superfamily, I compared all Cys-loop LGICs from rat, the primitive chordate Ciona intestinalis, Drosophila, and C. elegans. There are two clades of GABA receptor subunits that include both verterbate and invertebrate orthologues. In addition, I identified nine clades of anion channel subunits found only in invertebrates, including three that are specific to C. elegans and two found only in Drosophila. One well-defined clade of vertebrate cation channel subunits, the α7 nicotinic acetylcholine receptor subunits (nAChR), includes invertebrate orthologues. There are two clades of invertebrate nAChRs, one of α-type subunits and one of non-α subunits, that are most similar to the two clades of vertebrate neuronal and muscle α and non-α subunits. There is a large group of divergent C. elegans nAChR-like subunits partially resolved into clades but no orthologues of 5HT3-type serotonin receptors in the invertebrates. The topology of the trees suggests that most of the invertebrate-specific Cys-loop LGIC clades were present in the common ancestor of chordates and ecdysozoa. Many of these disappeared from the chordates. Subsequently, selected subunit genes expanded to form large subfamilies. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Rafael Zardoya]     

Predicting the transmembrane secondary structure of ligand-gated ion channels

Recent mutational analyses of ligand-gated ion channels (LGICs) have demonstrated a plausible site of anesthetic action within their transmembrane domains. Although there is a consensus that the transmembrane domain is formed from four membrane-spanning segments, the secondary structure of these segments is not known. We utilized 10 state-of-the-art bioinformatics techniques to predict the transmembrane topology of the tetrameric regions within six members of the LGIC family that are relevant to anesthetic action. They are the human forms of the GABA alpha 1 receptor, the glycine alpha 1 receptor, the 5HT3 serotonin receptor, the nicotinic AChR alpha 4 and alpha 7 receptors and the Torpedo nAChR alpha 1 receptor. The algorithms utilized were HMMTOP, TMHMM, TMPred, PHDhtm, DAS, TMFinder, SOSUI, TMAP, MEMSAT and TOPPred2. The resulting predictions were superimposed on to a multiple sequence alignment of the six amino acid sequences created using the CLUSTAL W algorithm. There was a clear statistical consensus for the presence of four alpha helices in those regions experimentally thought to span the membrane. The consensus of 10 topology prediction techniques supports the hypothesis that the transmembrane subunits of the LGICs are tetrameric bundles of alpha helices.     

LiGIoNs: A computational method for the detection and classification of ligand-gated ion channels

Ligand-Gated Ion Channels (LGICs) is one of the largest groups of transmembrane proteins. Due to their major role in synaptic transmission, both in the nervous system and the somatic neuromuscular junction, LGICs present attractive therapeutic targets. During the last few years, several computational methods for the detection of LGICs have been developed. These methods are based on machine learning approaches utilizing features extracted solely from the amino acid composition. Here we report the development of LiGIoNs, a profile Hidden Markov Model (pHMM) method for the prediction and ligand-based classification of LGICs. The method consists of a library of 10 pHMMs, one per LGIC subfamily, built from the alignment of representative LGIC sequences. In addition, 14 Pfam pHMMs are used to further annotate and classify unknown protein sequences into one of the 10 LGIC subfamilies. Evaluation of the method showed that it outperforms existing methods in the detection of LGICs. On top of that, LiGIoNs is the only currently available method that classifies LGICs into subfamilies.The method is available online at http://bioinformatics.biol.uoa.gr/ligions/.     

The Ligand Gated Ion Channel database: an example of a sequence database in neuroscience.

Multiple comparisons of receptor sequences, or receptor subunit sequences, has proved to be an invaluable tool in modern pharmacological investigations. Although of outstanding importance, general sequence databases suffer from several imperfections due to their size and their non-specificity. Room therefore exists for expert-maintained databases of restricted focus, where knowledge of the research field helps to filter the huge amount of data generated. Accordingly, neuroscientists have designed databases covering several types of proteins, in particular receptors for neurotransmitters. Ligand-gated ion channels are oligomeric transmembrane proteins involved in the fast response to neurotransmitters. All these receptors are formed by the assembly of homologous subunits, and an unexpected wealth of genes coding for these subunits has been revealed during the last two decades. The Ligand Gated Ion Channel database (LGICdb) has been developed to handle this growing body of information. The database aims to provide only one entry for each gene, containing annotated nucleic acid and protein sequences.     

gpDB: a database of GPCRs, G-proteins, effectors and their interactions

gpDB is a publicly accessible, relational database, containing information about G-proteins, G-protein coupled receptors (GPCRs) and effectors, as well as information concerning known interactions between these molecules. The sequences are classified according to a hierarchy of different classes, families and subfamilies based on literature search. The main innovation besides the classification of G-proteins, GPCRs and effectors is the relational model of the database, describing the known coupling specificity of GPCRs to their respective alpha subunits of G-proteins, and also the specific interaction between G-proteins and their effectors, a unique feature not available in any other database. AVAILABILITY: http://bioinformatics.biol.uoa.gr/gpDB CONTACT: shamodr@biol.uoa.gr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Molecular Cloning and Characterization of Novel Glutamate-Gated Chloride Channel Subunits from Schistosoma mansoni

Cys-loop ligand-gated ion channels (LGICs) mediate fast ionotropic neurotransmission. They are proven drug targets in nematodes and arthropods, but are poorly characterized in flatworms. In this study, we characterized the anion-selective, non-acetylcholine-gated Cys-loop LGICs from Schistosoma mansoni. Full-length cDNAs were obtained for SmGluCl-1 (Smp_096480), SmGluCl-2 (Smp_015630) and SmGluCl-3 (Smp_104890). A partial cDNA was retrieved for SmGluCl-4 (Smp_099500/Smp_176730). Phylogenetic analyses suggest that SmGluCl-1, SmGluCl-2, SmGluCl-3 and SmGluCl-4 belong to a novel clade of flatworm glutamate-gated chloride channels (GluCl) that includes putative genes from trematodes and cestodes. The flatworm GluCl clade was distinct from the nematode-arthropod and mollusc GluCl clades, and from all GABA receptors. We found no evidence of GABA receptors in S. mansoni. SmGluCl-1, SmGluCl-2 and SmGluCl-3 subunits were characterized by two-electrode voltage clamp (TEVC) in Xenopus oocytes, and shown to encode Cl⁻-permeable channels gated by glutamate. SmGluCl-2 and SmGluCl-3 produced functional homomers, while SmGluCl-1 formed heteromers with SmGluCl-2. Concentration-response relationships revealed that the sensitivity of SmGluCl receptors to L-glutamate is among the highest reported for GluCl receptors, with EC₅₀ values of 7–26 µM. Chloride selectivity was confirmed by current-voltage (I/V) relationships. SmGluCl receptors are insensitive to 1 µM ivermectin (IVM), indicating that they do not belong to the highly IVM-sensitive GluClα subtype group. SmGluCl receptors are also insensitive to 10 µM meclonazepam, a schistosomicidal benzodiazepine. These results provide the first molecular evidence showing the contribution of GluCl receptors to L-glutamate signaling in S. mansoni, an unprecedented finding in parasitic flatworms. Further work is needed to elucidate the roles of GluCl receptors in schistosomes and to explore their potential as drug targets.     

A novel site on gamma 3 subunits important for assembly of GABA(A) receptors

Gamma-aminobutyric acid, type A (GABA(A)) receptors are ligand-gated chloride channels and are the major inhibitory transmitter receptors in the central nervous system. The majority of these receptors is composed of two alpha, two beta, and one gamma subunits. To identify sequences important for subunit assembly, we generated C-terminally truncated and chimeric gamma(3) constructs. From their ability to associate with full-length alpha(1) and beta(3) subunits, we concluded that amino acid sequence gamma(3)(70-84) either directly interacts with alpha(1) or beta(3) subunits or stabilizes a contact site elsewhere in the protein. The observation that this sequence contains amino acid residues homologous to gamma(2) residues contributing to the benzodiazepine-binding site at the alpha(1)/gamma(2) interface suggested that in alpha(1)beta(3)gamma(3) receptors the sequence gamma(3)(70-84) is located at the alpha(1)/gamma(3) interface. In the absence of alpha(1) subunits this sequence might allow assembly of beta(3) with gamma(3) subunits. Other experiments indicated that sequences gamma(3)(86-95) and gamma(3)(94-107), which are homologous to previously identified sequences important for assembly of gamma(2) subunits, are also important for assembly of gamma(3) subunits. This indicates that during assembly of the GABA(A) receptor, more than one N-terminal sequence is important for binding to the same neighboring subunit. Whether the three sequences investigated are involved in direct interaction or stabilize other regions involved in intersubunit contacts has to be further studied.     

Intracellular trafficking of GABA(A) receptors

Some of the mechanisms that control the intracellular trafficking of GABA(A) receptors have recently been described. Following the synthesis of alpha, beta, and gamma subunits in the endoplasmic reticulum, ternary receptor complexes assemble slowly and are inefficiently inserted into surface membranes of heterologous cells. While beta3, beta4, and gamma2S subunits appear to contain polypeptide sequences that alone are sufficient for surface targeting, these sequences are neither conserved nor essential for surface expression of heteromeric GABA(A) receptors formed from alpha1beta or alpha1betagamma subunits. At the neuronal surface, native GABA(A) receptor clustering and synaptic targeting require a gamma2 subunit and the participation of gephyrin, a clustering protein for glycine receptors. A linker protein, such as the GABA(A) receptor associated protein (GABARAP), may be necessary for the formation of GABA(A) receptor aggregates containing gephyrin. A substantial fraction of surface receptors are sequestered by endocytosis, another process which apparently requires a GABA(A) receptor gamma2 subunit. In heterologous cells, constitutive endocytosis seems to predominate while, in cortical neurons, internalization is evoked when receptors are occupied by GABA(A) agonists. After constitutive endocytosis, receptors are relatively stable and can be rapidly recycled to the cell surface, a process that may be regulated by protein kinase C. On the other hand, a portion of the intracellular GABA(A) receptors derived from ligand-dependent endocytosis is apparently degraded. The clustering of GABA(A) receptors at synapses and at coated pits are two mechanisms that may compete for a pool of diffusable receptors, providing a model for plasticity at inhibitory synapses.     

Molecular and pharmacological properties of GABA-ρ subunits from white perch retina

Five γ-aminobutyric acid (GABA)-ρ subunits were cloned from a white perch retinal cDNA library and expressed in Xenopus oocytes. The deduced amino acid sequences indicated that all are highly homologous to the GABA-ρ subunits cloned from mammalian retinas; two clones (perch-ρ1A and perch-ρ1B) were in the ρ1 family, two (perch-ρ2A and perch-ρ2B) were in the ρ2 family, and one clone has been tentatively identified as a perch-ρ3 subunit. When expressed in Xenopus oocytes, all but one of the subunits (ρ3) formed functional homooligomeric receptors. However, the receptors expressed by each of the GABA-ρ subunits displayed unique response properties that distinguished one from the other. For example, receptors formed by perch-ρ1B subunits were more sensitive to GABA than the receptors formed by other GABA-ρ subunits, the dose–response curves for the various receptors revealed different Hill coefficients, and there were differences in the kinetics of the GABA-induced currents. In addition, the GABA-mediated current–voltage curve for ρ2 receptors was approximately linear, whereas the responses from ρ1 receptors showed outward rectification. A further division in the properties of the GABA-ρ subunits was revealed in their responses to imidazole-4-acetic acid (I4AA); the drug behaved as an antagonist on A-type ρ receptors and a partial agonist on the B-type ρ receptors. These results suggest that there is a large diversity of GABA_c receptors in the vertebrate retina, probably formed by homooligomeric and heterooligomeric combinations of GABA ρ subunits, that exhibit different functional properties. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 305–320, 1998     

Design and validation of a homogeneous time-resolved fluorescence cell-based assay targeting the ligand-gated ion channel 5-HT3A

Ligand-gated ion channels (LGICs) are considered as attractive protein targets in the search for new therapeutic agents. Nowadays, this strategy involves the capability to screen large chemical libraries. We present a new Tag-lite ligand binding assay targeting LGICs on living cells. This technology combines the use of suicide enzyme tags fused to channels of interest with homogeneous time-resolved fluorescence (HTRF) as the detection readout. Using the 5-HT3 receptor as system model, we showed that the pharmacology of the HALO-5HT3 receptor was identical to that of the native receptor. After validation of the assay by using 5-HT3 agonists and antagonists of reference, a pilot screen enabled us to identify azelastine, a well-known histamine H1 antagonist, as a potent 5-HT3 antagonist. This interesting result was confirmed with electrophysiological experiments. The method described here is easy to implement and could be applicable for other LGICs, opening new ways for the screening of chemical libraries.     

Differential activation of individual subunits in heteromeric kainate receptors

Neuronal kainate receptors are assembled from subunits with dissimilar specificities for agonists and antagonists. The composite biophysical behavior of heteromeric kainate receptors is determined by intersubunit interactions whose nature is unclear. Here we use dysiherbaine, a selective kainate receptor agonist, to show that GluR5 subunits assembled in heteromeric GluR5/KA-2 kainate receptor complexes can gate current without concomitant activation of their partner KA-2 subunits. A long-lasting interaction between dysiherbaine and GluR5 subunits elicits a tonic current from GluR5/KA-2 receptors; subsequent cooperative gating of KA-2 subunits can be elicited by both agonists, such as glutamate, and some classically defined antagonists, such as CNQX. This study demonstrates that each type of subunit within a heteromeric kainate receptor contributes a distinct conductance upon activation by agonist binding, and therefore provides insight into the biophysical function of ionotropic glutamate receptors.     

Identification of Major Phylogenetic Branches of Inhibitory Ligand-Gated Channel Receptors

The gene superfamily of ligand-gated ion channel (LGIC) receptors is composed of members of excitatory LGIC receptors (ELGIC) and inhibitory LGIC receptors (ILGIC), all using amino acids as ligands. The ILGICs, including GABA_A, Gly, and GluCl receptors, conduct Cl⁻ when the ligand is bound. To evaluate the phylogenetic relationships among ILGIC members, 90 protein sequences were analyzed by both maximum-parsimony and distance matrix-based methods. The strength of the resulting phylogenetic trees was evaluated by means of bootstrap. Four major phylogenetic branches are recognized. Branch I, called BZ, for the majority of the members are known to be related to benzodiazepine binding, is subdivided into IA, composed of all GABA_A receptor α subunits, and IB, composed of the γ and ε subunits, which are shown to be tightly linked. Branch II, named NB for non–benzodiazepine binding, and consisting of GABA_A receptor β, δ, π, and ρ subunits, is further subdivided into IIA, containing β subunits; IIB, containing δ, and π subunits; and IIC, containing ρ subunits. Branch IIIA, composed of vertebrate Gly receptors, is loosely clustered with Branch IIIB, composed of invertebrate GluCl receptors, to form Branch III, which is designated NA for being non–GABA responsive. Branch IV is called UD for being undefined in specificity. The existence of primitive forms of GABA_A receptor non-β subunits in invertebrates is first suggested by the present analysis, and the identities of sequences p25123 from Drosophila melanogaster, s34469 from Lymnaea stagnalis, and u14635 and p41849 from C. aenorhabditis elegans are determined to be different from their previously given annotations. The proposed branching classification of ILGICs provides a phylogenetic map, based on protein sequences, for tracing the evolutionary pathways of ILGIC receptor subunits and determining the identities of newly discovered subunits on the basis of their protein sequences. Received: 15 April 1997 / Accepted: 11 March 1998     

Structural organization of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptor of the electric ray Torpedo is the most comprehensively characterized neurotransmitter receptor. It consists of five subunits (alpha2beta gammadelta) amino acid sequences of which were determined by cDNA cloning and sequencing. The shape and size of the receptor were determined by electron cryomicroscopy. It has two agonist/competitive antagonist binding sites which are located between subunits near the membrane surface. The receptor ion channel is formed by five transmembrane helices (M2) of all five subunits. The position of the binding site for noncompetitive ion channel blockers was found by photoaffinity labelling and site-directed mutagenesis. The intrinsic feature of the receptor structure is the position of the agonist/competitive antagonist binding sites in close vicinity to the ion channel spanning the bilayer membrane. This peculiarity may substantially enhance allosteric transitions transforming the ligand binding into the channel opening and physiological response. Muscle nicotinic acetylcholine receptors from birds and mammals are also pentaoligomers consisting of four different subunits (alpha2beta gammadelta or alpha2beta epsilondelta) with high homology to the Torpedo receptor. Apparently, the pentaoligomeric structure is the main feature of all nicotinic, both muscle and neuronal, receptors. However, the neuronal receptors are formed only by two subunit types (alpha and beta) or are even pentahomomers (alpha7 neuronal receptors). All nicotinic receptors are ligand-gated ion channel, the properties of the channels being essentially determined by amino acid residues forming M2 transmembrane fragments.     

Modeling of agonist binding to the ligand-gated ion channel superfamily of receptors

A generalized model is presented of agonist binding to ligand-gated ion channels (LGICs). Broad similarity in the structure of agonists suggests that the binding sites of LGICs may have evolved from a protobinding site. Aligned sequence data identified as a candidate for such a site a highly conserved 15 residue stretch of primary structure in the N-terminal extracellular region of all known LGIC subunits. We modeled this subregion, termed the cys-loop, as a rigid, amphiphilic beta-hairpin and propose that it may form a major determinant of a conserved structural binding cleft. In the model of the binding complex (1) an invariant aspartate residue at position 11 of the cys-loop is the anionic site interacting with the positively charged amine group of agonists, (2) a local dipole within the pi-electron system of agonists is favorably oriented in the electrostatic field of the invariant aspartate, (3) the epsilon ring-proton of a conserved aromatic residue at the turn of the cys-loop interacts orthogonally with the agonist pi-electron density at its electronegative center, and (4) selective recognition is partly a result of the type of amino acid residue at position 6 of the cys-loop. Additionally, formation of a hydrogen bond between the electronegative atom of the pi-electron system of agonist and a complementary group in the receptor may be important in the high-affinity binding of agonists.     

Identification of an Ascaris G protein-coupled acetylcholine receptor with atypical muscarinic pharmacology

Acetylcholine (ACh) is a neurotransmitter/neuromodulator in the nematode nervous system and induces its effects through interaction with both ligand-gated ion channels (LGICs) and G protein-coupled receptors (GPCRs). The structure, pharmacology and physiological importance of LGICs have been appreciably elucidated in model nematodes, including parasitic species where they are targets for anthelmintic drugs. Significantly less, however, is understood about nematode ACh GPCRs, termed GARs (G protein-linked ACh receptors). What is known comes from the free-living Caenorhabditis elegans as no GARs have been characterized from parasitic species. Here we clone a putative GAR from the pig gastrointestinal nematode Ascaris suum with high structural homology to the C. elegans receptor GAR-1. Our GPCR, dubbed AsGAR-1, is alternatively spliced and expressed in the head and tail of adult worms but not in dorsal or ventral body wall muscle, or the ovijector. ACh activated AsGAR-1 in a concentration-dependent manner but the receptor was not activated by other small neurotransmitters. The classical muscarinic agonists carbachol, arecoline, oxotremorine M and bethanechol were also AsGAR-1 agonists but pilocarpine was ineffective. AsGAR-1 activation by ACh was partially antagonized by the muscarinic blocker atropine but pirenzepine and scopolamine were largely ineffective. Certain biogenic amine GPCR antagonists were also found to block AsGAR-1. Our conclusion is that Ascaris possesses G protein-coupled ACh receptors that are homologous in structure to those present in C. elegans, and that although they have some sequence homology to vertebrate muscarinic receptors, their pharmacology is atypically muscarinic.