Acetylcholine-binding proteins (AChBPs) from mollusks are suitable structural and functional surrogates of the nicotinic acetylcholine receptors when combined with transmembrane spans receptor. These assemble as a pentamer identical ACh binding sites at subunit interfaces show ligand specificities resembling those receptor for agonists antagonists. A subset ligands, termed neonicotinoids, exhibit specificity insect selective toxicity insecticides. AChBPs neither mammalian nor origin...

Spirolide and gymnodimine macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms shellfish toxicity. Analysis 13-desmethyl spirolide C A by binding voltage-clamp recordings on muscle-type α1 2 βγδ neuronal α3β2 α4β2 nicotinic acetylcholine receptors reveals subnanomolar affinities, potent antagonism, limited subtype selectivity. Their acetylcholine-binding proteins (AChBP), as soluble receptor surrogates, exhibits picomolar affinities...

We generated an acetylcholine-binding protein from Aplysia californica by synthesis of a cDNA found in existing data bases and its expression mammalian cell culture. Its subunit assembly ligand recognition behavior were compared with the binding previously derived Lymnaea stagnalis. The secreted proteins purified elution columns attached antibodies directed to FLAG epitope encoded construct. Although sequences two marine fresh water mollusks exhibit characteristic features extracellular...

Nicotinic acetylcholine receptors (nAChRs), which are responsible for mediating key physiological functions, ubiquitous in the central and peripheral nervous systems. As members of Cys loop ligand-gated ion channel family, neuronal nAChRs pentameric, composed various permutations α (α2 to α10) β (β2 β4) subunits forming functional heteromeric or homomeric receptors. Diversity nAChR subunit composition complicates development selective ligands specific subtypes, since five binding sites...

The recent characterization of an acetylcholine binding protein (AChBP) from the fresh water snail, <i>Lymnaea stagnalis</i>, shows it to be a structural homolog extracellular domain nicotinic receptor (nAChR). To ascertain whether AChBP exhibits recognition properties and functional states nAChR, we have expressed in milligram quantities synthetic cDNA transfected into human embryonic kidney (HEK) cells. secreted medium pentameric rosette structure with ligand stoichiometry approximating...

The nicotinic acetylcholine (ACh) receptor (nAChR) plays a crucial role in excitatory neurotransmission and is an important target for drugs insecticides. Diverse nAChR subtypes with various subunit combinations confer differential selectivity drugs. We investigated the subtype of agonists by comparing two ACh-binding proteins (AChBPs) as structural surrogates distinct pharmacological profiles [i.e., Lymnaea stagnalis ( Ls ) AChBP low neonicotinoid high nicotinoid sensitivities Aplysia...

Two types of structurally similar nicotinic agonists have very different biological and physicochemical properties. Neonicotinoids, important insecticides including imidacloprid thiacloprid, are nonprotonated selective for insects their receptors, whereas nicotinoids such as nicotine epibatidine cationic mammalian systems. We discovered that a mollusk acetylcholine binding protein (AChBP), structural surrogate the extracellular ligand-binding domain receptor, is similarly sensitive to...

We undertook cysteine substitution mutagenesis and fluorophore conjugation at selected residue positions to map sites of ligand binding changes in solvent exposure the acetylcholine-binding protein from Lymnaea stagnalis, a nicotinic receptor surrogate. Acrylodan fluorescence emission is highly sensitive its local environment, when bound protein, exhibits both intensity wavelength that are reflected degree exclusion effective dielectric constant environment fluorophore. Hence, mutants were...

The molluskan acetylcholine-binding protein (AChBP) is a homolog of the extracellular binding domain pentameric ligand-gated ion channel family. AChBP most closely resembles α-subunit nicotinic acetylcholine receptors and in particular homomeric α7 receptor. We report isolation characterization an α-conotoxin that has highest known affinity for Lymnaea also potently blocks nAChR subtype when expressed Xenopus oocytes. Remarkably, peptide high α3β2 indicating OmIA combination with may serve...

Nicotinic acetylcholine (ACh) receptor (nAChR) agonists are potential therapeutic agents for neurological dysfunction. In the present study, homopentameric mollusk ACh binding protein (AChBP), used as a surrogate extracellular ligand-binding domain of nAChR, was specifically derivatized by highly potent agonist azidoepibatidine (AzEPI) prepared photoaffinity probe and radioligand. One EPI-nitrene photoactivated molecule incorporated in each subunit interface site based on analysis intact...

We present a cell based system and experimental approach to characterize agonist antagonist selectivity for ligand-gated ion channels (LGIC) by developing sensor cells stably expressing Ca2+ permeable LGIC genetically encoded Förster (or fluorescence) resonance energy transfer (FRET)-based calcium sensor. In particular, we describe separate lines with human α7 α4β2 nicotinic acetylcholine receptors, mouse 5-HT3A serotonin receptors chimera of α7/mouse receptors. Complete...

Biologically active macrocycles containing a cyclic imine were isolated for the first time from aquaculture sites in Nova Scotia, Canada, during 1990s. These compounds display "fast-acting" toxicity traditional mouse bioassay lipophilic marine toxins. Our work aimed at developing receptor-based detection method spirolides using microsphere/flow cytometry Luminex system. For assay, two alternatives considered as binding proteins, Torpedo marmorata nicotinic acetylcholine receptor (nAChR) and...

The discovery of the acetylcholine binding proteins (AChBPs) has provided critical soluble surrogates for examining structure and ligand interactions with nicotinic receptors related pentameric ligand-gated ion channels. multiple marine freshwater sources AChBP constitute a protein family substantial sequence divergence selectivity in recognition analyzing structure−activity relationships. purification quantities absence detergent enables one to conduct spectroscopic studies ligand−AChBP...

Alpha-conotoxins isolated from Conus venoms contain 11-19 residues and preferentially fold into the globular conformation that possesses a specific disulfide pairing pattern (C1-3, C2-4). We others new family of chi-conotoxins (also called lambda conotoxins) with conserved cysteine framework alpha-conotoxins but alternative (C1-4, C2-3) resulting in ribbon conformation. In both families, hence folding are important for their biological potency. By comparing structural differences, we...

Acetylcholine-binding protein (AChBP) recently emerged as a prototype for relating structure to function of the ligand binding domain nicotinic acetylcholine receptors (AChRs). To understand interactions competitive antagonists at atomic structural level, we studied curare derivatives d-tubocurarine (d-TC) and metocurine AChBP using computational methods, mutagenesis, measurements. account flexibility, used 2-ns molecular dynamics simulation generate multiple snapshots equilibrated dynamic...

Nicotinic acetylcholine receptors (nAChRs) containing α6 and β4 subunits are expressed by dorsal root ganglion neurons have been implicated in neuropathic pain. Rodent models often used to evaluate the efficacy of analgesic compounds, but species differences may affect activity some nAChR ligands. A previous candidate α-conotoxin-based therapeutic yielded promising results rodent models, failed human clinical trials, emphasizing importance understanding ligand activity. Here, we show that...

alpha-Cobratoxin (Cbtx), the neurotoxin isolated from venom of Thai cobra Naja kaouthia , causes paralysis by preventing acetylcholine (ACh) binding to nicotinic receptors (nAChRs). In current study, region Cbtx molecule that is directly involved in nAChRs used as target for anticobratoxin drug design. The crystal structure (1YI5) complex with protein (AChBP), a soluble homolog extracellular domain nAChRs, was selected prepare an alpha-cobratoxin active site docking. amino acid residues...

Previous work demonstrated kinetically that inhibition of mammalian acetylcholinesterase (AChE) by (1S)-isomalathions may proceed loss thiomethyl instead the expected diethyl thiosuccinate as primary leaving group followed one four possible modes rapid aging. This study sought to identify adduct renders AChE refractory toward reactivation after with (1S,3S)-stereoisomer. Electric eel (EEAChE) was inhibited stereoisomers isomalathion, and rate constants for spontaneous oxime-mediated (k3)...