Diversity of A-conotoxins of three worm-hunting cone snails (Conus brunneus,Conus nux,and Conus princeps) from the Mexican Pacific coast

Conus marine snails (∼500 species) are tropical predators that use venoms mainly to capture prey and defend themselves from predators. The principal components of these venoms are peptides that are known as “conotoxins” and generally comprise 7–40 amino acid residues, including 0–5 disulfide bridges and distinct posttranslational modifications. The most common molecular targets of conotoxins are voltage- and ligand-gated ion channels, G protein-coupled receptors, and neurotransmitter transporters, to which they bind, typically, with high affinity and specificity. Due to these properties, several conotoxins have become molecular probes, medicines, and leads for drug design. Conotoxins have been classified into genetic superfamilies based on the signal sequence of their precursors, and into pharmacological families according to their molecular targets. The objective of this work was to identify and analyze partial cDNAs encoding conotoxin precursors belonging to the A superfamily from Conus brunneus, Conus nux, and Conus princeps. These are vermivorous species of the Mexican Pacific coast from which only one A-conotoxin, and few O- and I₂-conotoxins have been reported. Employing RT-PCR, we identified 30 distinct precursors that contain 13 different predicted mature toxins. With the exception of two groups of four highly similar peptides, these toxins are diverse at both the sequence and the physicochemical levels, and they belong to the 4/3, 4/4, 4/5, 4/6, and 4/7 structural subfamilies. These toxins are predicted to target diverse nicotinic acetylcholine receptor (nAChR) subtypes: nx1d, muscle; pi1a–pi1d, α₃β₂, α₇, and/or α₉α₁₀; br1a, muscle, α₃β₄, and/or α₄β₂; and nx1a–nx1c/pi1g and pi1h, α₃β₂, α₃β₄, α₉β₁₀, and/or α₇.     

Carbamoylcholine analogs as nicotinic acetylcholine receptor agonists—Structural modifications of 3-(dimethylamino)butyl dimethylcarbamate (DMABC)

Compounds based on the 3-(dimethylamino)butyl dimethylcarbamate (DMABC) scaffold were synthesized and pharmacologically characterized at the α₄β₂, α₃β_4, α₄β₄ and α₇ neuronal nicotinic acetylcholine receptors (nAChRs). The carbamate functionality and a small hydrophobic substituent in the C-3 position were found to be vital for the binding affinity to the nAChRs, whereas the carbamate nitrogen substituents were important for nAChR subtype selectivity. Finally, the compounds were found to be agonists at the α₃β₄ nAChR.     

Discovery of benzamide analogs as negative allosteric modulators of human neuronal nicotinic receptors: Pharmacophore modeling and structure–activity relationship studies

The present study describes our ongoing efforts toward the discovery of drugs that selectively target nAChR subtypes. We exploited knowledge on nAChR ligands and their binding site that were previously identified by our laboratory through virtual screenings and identified benzamide analogs as a novel chemical class of neuronal nicotinic receptor (nAChR) ligands. The lead molecule, compound 1 (4-(allyloxy)-N-(6-methylpyridin-2-yl)benzamide) inhibits nAChR activity with an IC₅₀ value of 6.0 (3.4–10.6) μM on human α4β2 nAChRs with a ～5-fold preference against human α3β4 nAChRs. Twenty-six analogs of compound 1 were also either synthesized or purchased for structure–activity relationship (SAR) studies and provided information relating the chemical/structural properties of the molecules to their ability to inhibit nAChR activity. The discovery of subtype-selective ligands of nAChRs described here should contribute significantly to our understanding of the involvement of specific nAChR subtypes in normal and pathophysiological states.     

Conotoxins: From the biodiversity of gastropods to new drugs

The review describes general trends in research of conotoxins, the peptide toxins isolated from sea gastropods of the genus Conus. It covers publications from conotoxin discovery in 1970th up to contemporary research and considers classification of conotoxins, their structural diversity and different ways of their action on molecular targets, particularly, ion channels. Special attention is paid to the applied aspect of conotoxin research especially to perspectives of the development of conotoxin based drugs. The first example of such conotoxin based drug is ziconotide an analgesic of a new generation.     

Autophagy protects neuron from Aβ-induced cytotoxicity

Autophagy is a degradation pathway for the turnover of dysfunctional organelles or aggregated proteins in cells. Extracellular accumulation of β-amyloid peptide has been reported to be a major cause of Alzheimer's disease (AD) and large numbers of autophagic vacuoles accumulate in the brain of AD patient. However, how autophagic process is involved in Aβ-induced neurotoxicity and how Aβ peptide is transported into neuron and metabolized is still unknown. In order to study the role of autophagic process in Aβ-induced neurotoxicity, EGFP-LC3 was over-expressed in SH-SY5Y cells (SH-SY5Y/pEGFP-LC3). It was found that treatment with Aβ_25-35, Aβ_1-42 or serum-starvation induced strong autophagy response in SH-SY5Y/pEGFP-LC3. Confocal double-staining image showed that exogenous application of Aβ1-42 in medium caused the co-localization of Aβ_1-42 with LC3 in neuronal cells. Concomitant treatment of Aβ with a selective α7nAChR antagonist, α-bungarotoxin (α-BTX), enhanced Aβ-induced neurotoxicity in SH-SY5Y cells. On the other hand, nicotine (nAChR agonist) enhanced the autophagic process and also inhibited cell death following Aβ application. In addition, nicotine but not α-BTX increased primary hippocampal neuronal survival following Aβ treatment. Furthermore, using Atg7 siRNA to inhibit autophagosome formation in an early step or α7nAChR siRNA to knockdown α7nAChR significantly enhanced Aβ-induced neurotoxicity. Confocal double-staining image shows that nicotine treatment in the presence of Aβ enhanced the co-localization of α7nAChR with autophagosomes. These results suggest that α7nAChR may act as a carrier to bind with eAβ and internalize into cytoplasm and further inhibit Aβ-induced neurotoxicity via autophagic degradation pathway. Our results suggest that autophagy process plays a neuroprotective role against Aβ-induced neurotoxicity. Defect in autophagic regulation or Aβ-α7nAChR transport system may impair the clearance of Aβ and enhance the neuronal death.     

3D-QSAR and QSSR studies of 3,8-diazabicyclo[4.2.0]octane derivatives as neuronal nicotinic acetylcholine receptors by comparative molecular field analysis (CoMFA)

High subtype selectivity (α4β2 over α2β3) of neuronal nicotinic acetylcholine receptor (nAChR) agonists is critical for the rational design of less toxic drugs used for the treatment of neurodegenerative and psychiatric diseases. Here, three CoMFA models of pEC₅₀(α4β2), pEC₅₀(α2β3) and p[EC₅₀(α4β2)/EC₅₀(α2β3)] (pEC₅₀(α4β2)pEC₅₀(α2β3)) were developed to study the quantitative structure–activity relationship (QSAR) and quantitative structure–selectivity relationship (QSSR) of the 3,8-diazabicyclo[4.2.0]octane derivatives as nAChRs agonists. The parameters of the three models were 0.584, 0.792, and 0.599 for cross-validated r² (r²_CV), 0.924, 0.935 and 0.875 for conventional r². Analyses indicated that both the steric and electrostatic factors should be considered in the rational design of more active and selective nAChR agonists.     

The 3,7-diazabicyclo[3.3.1]nonane scaffold for subtype selective nicotinic acetylcholine receptor ligands. Part 2: Carboxamide derivatives with different spacer motifs

3,7-Diazabicyclo[3.3.1]nonane (bispidine) based nicotinic acetylcholine receptor (nAChR) ligands have been synthesized and evaluated for nAChRs interaction. Diverse spacer motifs were incorporated between the hydrogen bond acceptor (HBA) part and a variety of substituted (hetero)aryl moieties. Bispidine carboxamides bearing spacer motifs often showed high affinity in the low nanomolar range and selectivity for the α4β2¹ nAChR. Compounds 15, 25, and 47 with K_i values of about 1 nM displayed the highest affinities for α4β2¹ nAChR. All evaluated compounds are partial agonists or antagonists at α4β2¹, with reduced or no effects on α3β4¹ with the exception of compound 15 (agonist), and reduced or no effect at α7 and muscle subtypes.     

Alpha S-conotoxin RVIIIA: a structurally unique conotoxin that broadly targets nicotinic acetylcholine receptors

We report the purification and characterization of a new conotoxin from the venom of Conus radiatus. The peptide, alphaS-conotoxin RVIIIA (alphaS-RVIIIA), is biochemically unique with respect to its amino acid sequence, post-translational modification, and molecular targets. In comparison to other nicotinic antagonists from Conus venoms, alphaS-RVIIIA exhibits an unusually broad targeting specificity for nicotinic acetylcholine receptor (nAChR) subtypes, as assayed by electrophysiology. The toxin is paralytic to mice and fish, consistent with its nearly irreversible block of the neuromuscular nAChR. Similar to other antagonists of certain neuronal nAChRs, the toxin also elicits seizures in mice upon intracranial injection. The only previously characterized conotoxin from the S superfamily, sigma-conotoxin GVIIIA, is a specific competitive antagonist of the 5-HT3 receptor; thus, alphaS-RVIIIA defines a novel family of nicotinic antagonists within the S superfamily. All previously characterized competitive conotoxin nAChR antagonists have been members of the A superfamily of conotoxins. Our working hypothesis is that the particular group of fish-hunting Conus species that includes Conus radiatus uses the alphaS-conotoxin family to target the muscle nAChR and paralyze prey.     

Exploring the Nicotinic Acetylcholine Receptor-associated Proteome with iTRAQ and Transgenic Mice

Neuronal nicotinic acetylcholine receptors (nAChRs) containing α4 and β2 subunits are the principal receptors in the mammalian central nervous system that bind nicotine with high affinity. These nAChRs are involved in nicotine dependence, mood disorders, neurodegeneration and neuroprotection. However, our understanding of the interactions between α4β2-containing (α4β2¹) nAChRs and other proteins remains limited. In this study, we identified proteins that interact with α4β2¹ nAChRs in a genedose dependent pattern by immunopurifying β2¹ nAChRs from mice that differ in α4 and β2 subunit expression and performing proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ). Reduced expression of either the α4 or the β2 subunit results in a correlated decline in the expression of a number of putative interacting proteins. We identified 208 proteins co-immunoprecipitated with these nAChRs. Furthermore, stratified linear regression analysis indicated that levels of 17 proteins was correlated significantly with expression of α4β2 nAChRs, including proteins involved in cytoskeletal rearrangement and calcium signaling. These findings represent the first application of quantitative proteomics to produce a β2¹ nAChR interactome and describe a novel technique used to discover potential targets for pharmacological manipulation of α4β2 nAChRs and their downstream signaling mechanisms.     

Structure and activity of alpha-conotoxin PeIA at nicotinic acetylcholine receptor subtypes and GABA(B) receptor-coupled N-type calcium channels

α-Conotoxins are peptides from cone snails that target the nicotinic acetylcholine receptor (nAChR). RgIA and Vc1.1 have analgesic activity in animal pain models. Both peptides target the α9α10 nAChR and inhibit N-type calcium channels via GABA(B) receptor activation, but the mechanism of action of analgesic activity is unknown. PeIA has previously been shown to inhibit the α9α10 and α3β2 nAChRs. In this study, we have determined the structure of PeIA and shown that it is also a potent inhibitor of N-type calcium channels via GABA(B) receptor activation. The characteristic α-conotoxin fold is present in PeIA, but it has a different distribution of surface-exposed hydrophobic and charged residues compared with Vc1.1. Thus, the surface residue distribution, rather than the overall fold, appears to be responsible for the 50-fold increase in selectivity at the α3β2 nAChR by PeIA relative to Vc1.1. In contrast to their difference in potency at the nAChR, the equipotent activity of PeIA and Vc1.1 at the GABA(B) receptor suggests that the GABA(B) receptor is more tolerant to changes in surface residues than is the nAChR. The conserved Asp-Pro-Arg motif of Vc1.1 and RgIA, which is crucial for potency at the α9α10 nAChR, is not required for activity at GABA(B) receptor/N-type calcium channels because PeIA has a His-Pro-Ala motif in the equivalent position. This study shows that different structure-activity relationships are associated with the targeting of the GABA(B) receptor versus nAChRs. Furthermore, there is probably a much more diverse range of conotoxins that target the GABA(B) receptor than currently realized.     

Anesthetic effects on the structure and dynamics of the second transmembrane domains of nAChR α4β2

Channel functions of the neuronal α4β2 nicotinic acetylcholine receptor (nAChR), one of the most widely expressed subtypes in the brain, can be inhibited by volatile anesthetics. Our Na⁺ flux experiments confirmed that the second transmembrane domains (TM2) of α4 and β2 in 2:3 stoichiometry, (α4)₂(β2)₃, could form pentameric channels, whereas the α4 TM2 alone could not. The structure, topology, and dynamics of the α4 TM2 and (α4)₂(β2)₃ TM2 in magnetically aligned phospholipid bicelles were investigated using solid-state NMR spectroscopy in the absence and presence of halothane and isoflurane, two clinically used volatile anesthetics. ²H NMR demonstrated that anesthetics increased lipid conformational heterogeneity. Such anesthetic effects on lipids became more profound in the presence of transmembrane proteins. PISEMA experiments on the selectively ¹⁵N-labeled α4 TM2 showed that the TM2 formed transmembrane helices with tilt angles of 12° ± 1° and 16° ± 1° relative to the bicelle normal for the α4 and (α4)₂(β2)₃ samples, respectively. Anesthetics changed the tilt angle of the α4 TM2 from 12° ± 1° to 14° ± 1°, but had only a subtle effect on the tilt angle of the (α4)₂(β2)₃ TM2. A small degree of wobbling motion of the helix axis occurred in the (α4)₂(β2)₃ TM2. In addition, a subset of the (α4)₂(β2)₃ TM2 exhibited counterclockwise rotational motion around the helix axis on a time scale slower than 10^- 4 s in the presence of anesthetics. Both helical tilting and rotational motions have been identified computationally as critical elements for ion channel functions. This study suggested that anesthetics could alter these motions to modulate channel functions.     

Synthesis and evaluation of N(1)-alkylindole-3-ylalkylammonium compounds as nicotinic acetylcholine receptor ligands

In this study thirty-three novel indole derivatives were designed and synthesized based on the structure of deformylflustrabromine B (1), a metabolite isolated from the marine bryozoan Flustra foliacea L. The syntheses were carried out using standard methodologies and in good yields. The molecules were tested for their affinities for the α4β2¹, α3β4¹, α7¹ and (α1)₂β1γδ nicotinic acetylcholine receptor (nAChR) subtypes. Binding assays showed that, among these ligands, compound 7c exhibited the highest affinity with K_i = 136.1, 93.9 and 862.4 nM for the α4β2¹, α3β4¹, and α7¹ nAChRs subtypes, respectively. These results indicated that the indole core might be a useful scaffold for the development of new potent and selective nAChR ligands.     

Differential physiologic responses of α7 nicotinic acetylcholine receptors to β‐amyloid1–40 and β‐amyloid1–42

The β‐amyloid peptides (Aβ), Aβ_1–40 and Aβ_1–42, have been implicated in Alzheimer's disease (AD) pathology. Although Aβ_1–42 is generally considered to be the pathological peptide in AD, both Aβ_1–40 and Aβ_1–42 have been used in a variety of experimental models without discrimination. Here we show that monomeric or oligomeric forms of the two Aβ peptides, when interact with the neuronal cation channel, α7 nicotinic acetylcholine receptors (α7nAChR), would result in distinct physiologic responses as measured by acetylcholine release and calcium influx experiments. While Aβ_1–42 effectively attenuated these α7nAChR‐dependent physiology to an extent that was apparently irreversible, Aβ_1–40 showed a lower inhibitory activity that could be restored upon washings with physiologic buffers or treatment with α7nAChR antagonists. Our data suggest a clear pharmacological distinction between Aβ_1–40 and Aβ_1–42. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 25–30, 2003     

Metabotropic signaling cascade involved in α4β2 nicotinic acetylcholine receptor-mediated PKCβII activation

Nicotinic acetylcholine receptors (nAChRs) belong to the ionophore receptor family, which regulates plasma membrane conductance to Na⁺, K⁺, and Ca²⁺ ions. Some studies, however, have shown that nAChRs also employ second messengers for intracellular signaling. We previously showed that α4β2 nAChR mediates the translocation of protein kinase CβII (PKCβII) from the cytoplasm to the plasma membrane, which is a typical activation marker for PKCβII. In this study, we investigated the molecular mechanisms underlying PKCβII activation through α4β2 nAChR. α4β2 nAChR is the most abundant nAChR subtype and is implicated in various brain functions and diseases. Putative α4β2 nAChR signaling components were identified by knockdown or chemical inhibition of candidate proteins, and the signaling cascade was deduced by protein interactions in predicted cellular components. α4β2 nAChR-mediated PKCβII translocation was found to occur in an ionophore activity-independent manner. Nicotinic stimulation of α4β2 nAChR activated Src in a β-arrestin1 and 14–3-3η-dependent manner. Activated Src phosphorylated the tyrosine residue(s) on Syk molecules, which in turn interacted with phospholipase C γ1 to trigger the translocation of PKCβII to the cell membrane by elevating cellular diacylglycerol levels. The activated PKCβII in turn exerted a positive feedback effect on Src activation, suggesting that α4β2 nAChR signaling is amplified by a positive feedback loop. These findings provide novel information for unveiling the previously unclear metabotropic second messenger-based signal transduction pathway of nAChRs.     

Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [I]epibatidine and 5[I]A-85380

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [¹²⁵I]epibatidine and 5[¹²⁵I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [¹²⁵I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [¹²⁵I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [¹²⁵I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.     

NMR structures of the transmembrane domains of the α4β2 nAChR

The α4β2 nicotinic acetylcholine receptor (nAChR) is the predominant heteromeric subtype of nAChRs in the brain, which has been implicated in numerous neurological conditions. The structural information specifically for the α4β2 and other neuronal nAChRs is presently limited. In this study, we determined structures of the transmembrane (TM) domains of the α4 and β2 subunits in lauryldimethylamine-oxide (LDAO) micelles using solution NMR spectroscopy. NMR experiments and size exclusion chromatography-multi-angle light scattering (SEC-MALS) analysis demonstrated that the TM domains of α4 and β2 interacted with each other and spontaneously formed pentameric assemblies in the LDAO micelles. The Na(+) flux assay revealed that α4β2 formed Na(+) permeable channels in lipid vesicles. Efflux of Na(+) through the α4β2 channels reduced intra-vesicle Sodium Green? fluorescence in a time-dependent manner that was not observed in vesicles without incorporating α4β2. The study provides structural insight into the TM domains of the α4β2 nAChR. It offers a valuable structural framework for rationalizing extensive biochemical data collected previously on the α4β2 nAChR and for designing new therapeutic modulators.     

Structural basis for α-conotoxin potency and selectivity

Parkinson’s disease is a debilitating movement disorder characterized by altered levels of α₆β₂1 (1 indicates the possible presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) localized on presynaptic striatal catecholaminergic neurons. α-Conotoxin MII (α-CTx MII) is a highly useful ligand to probe α₆β₂ nAChRs structure and function, but it does not discriminate among closely related α₆1 nAChR subtypes. Modification of the α-CTx MII primary sequence led to the identification of α-CTx MII[E11A], an analog with 500–5300-fold discrimination between α₆1 subtypes found in both human and non-human primates. α-CTx MII[E11A] binds most strongly (femtomolar dissociation constant) to the high affinity α₆ nAChR, a subtype that is selectively lost in Parkinson’s disease. Here, we present the three-dimensional solution structure for α-CTx MII[E11A] as determined by two-dimensional ¹H NMR spectroscopy to 0.13 ± 0.09 ? backbone and 0.45 ± 0.08 ? heavy atom root-mean-square deviation from mean structure. Structural comparisons suggest that the increased hydrophobic area of α-CTx MII[E11A] relative to other members of the α-CTx family may be responsible for its exceptionally high affinity for α6α4β21 nAChR as well as discrimination between α₆β₂ and α₃β₂ containing nAChRs. This finding may enable the rational design of novel peptide analogs that demonstrate enhanced specificity for α₆1 nAChR subunit interfaces and provide a means to better understand nAChR structural determinants that modulate brain dopamine levels and the pathophysiology of Parkinson’s disease.     

Mouse models to unravel the role of inhaled pollutants on allergic sensitization and airway inflammation

Background

Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study, we determine the nAChR inventory of rat alveolar macrophages (AM), and investigate the cellular events evoked by stimulation with nicotine.

Methods

Rat AM were isolated freshly by bronchoalveolar lavage. The expression of nAChR subunits was analyzed by RT-PCR, immunohistochemistry, and Western blotting. To evaluate function of nAChR subunits, electrophysiological recordings and measurements of intracellular calcium concentration ([Ca²⁺]_i) were conducted.

Results

Positive RT-PCR results were obtained for nAChR subunits α3, α5, α9, α10, β1, and β2, with most stable expression being noted for subunits α9, α10, β1, and β2. Notably, mRNA coding for subunit α7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. RT-PCR data were supported by immunohistochemistry on AM isolated by lavage, as well as in lung tissue sections and by Western blotting. Neither whole-cell patch clamp recordings nor measurements of [Ca²⁺]_i revealed changes in membrane current in response to ACh and in [Ca²⁺]_i in response to nicotine, respectively. However, nicotine (100 μM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in [Ca²⁺]_i by 30%. This effect was blocked by α-bungarotoxin and did not depend on the presence of extracellular calcium.

Conclusions

Rat AM are equipped with modulatory nAChR with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system. Their stimulation with nicotine dampens ATP-induced Ca²⁺-release from intracellular stores. Thus, the present study identifies the first acute receptor-mediated nicotinic effect on AM with anti-inflammatory potential.