Functional co-expression of two insect nicotinic receptor subunits (Nlα3 and Nlα8) reveals the effects of a resistance-associated mutation (Nlα3Y151S) on neonicotinoid insecticides

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively to control a variety of insect pest species. Previously, we have identified a nAChR point mutation (Y151S) associated with insecticide resistance in the brown planthopper Nilaparvata lugens . Although this mutation has been identified in two different N. lugens nAChR subunits (Nlα1 and Nlα3) because of difficulties in heterologous expression of Nlα3; its influence on agonist potency has been examined only in Nlα1-containing nAChRs. Here we describe the cloning of a novel nAChR subunit from N. lugens (Nlα8), together with evidence for its co-assembly with Nlα3 in native and recombinant nAChRs. This has, for the first time, enabled the functional effects of the Nlα3^Y151S mutation to be examined. The Nlα3^Y151S mutation has little effect on agonist potency of acetylcholine but has a dramatic effect on neonicotinoid insecticides (reducing I _max values and increasing EC₅₀ values). The apparent affinity of neonicotinoids was higher and the effect of the Y151S mutation on neonicotinoid agonist potency was more profound in Nlα3-containing, rather than Nlα1-containing nAChR. We conclude that Nlα3- and Nlα1-containing nAChRs may be representative of two distinct insect nAChR populations.     

Identification of two Lynx proteins in Nilaparvata lugens and the modulation on insect nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission in the insect brain and are targets for neonicotinoid insecticides. Some proteins, other than nAChRs themselves, might play important roles in insect nAChRs function in vivo and in vitro , such as the chaperone, regulator and modulator. Here we report the identification of two nAChR modulators (Nl-lynx1 and Nl-lynx2) in the brown planthopper, Nilaparvata lugens . Analysis of amino acid sequences of Nl-lynx1 and Nl-lynx2 reveals that they are two members of the Ly-6/neurotoxin superfamily, with a cysteine-rich consensus signature motif. Nl-lynx1 and Nl-lynx2 only increased agonist-evoked macroscopic currents of hybrid receptors Nlα1/β2 expressed in Xenopus oocytes, but not change the agonist sensitivity and desensitization properties. For example, Nl-lynx1 increased I _max of acetylcholine and imidacloprid to 3.56-fold and 1.72-fold of that of Nlα1/β2 alone, and these folds for Nl-lynx2 were 3.25 and 1.51. When the previously identified Nlα1^Y151S mutation was included (Nlα1^Y151S/β2), the effects of Nl-lynx1 and Nl-lynx2 on imidacloprid responses, but not acetylcholine response, were different from that in Nlα1/β2. The increased folds in imidacloprid responses by Nl-lynx1 and Nl-lynx2 were much higher in Nlα1^Y151S/β2 (3.25-fold and 2.86-fold) than in Nlα1/β2 (1.72-fold and 1.51-fold), which indicated Nl-lynx1 and Nl-lynx2 might also serve as an influencing factor in target-site insensitivity in N. lugens . These findings indicate that nAChRs chaperone, regulator and modulator may be of importance in assessing the likely impact of the target-site mutations such as Y151S upon neonicotinoid insecticide resistance.     

Heteromeric co-assembly of two insect nicotinic acetylcholine receptor α subunits: influence on sensitivity to neonicotinoid insecticides

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively in areas of crop protection and animal health to control a variety of insect pest species. Here, we describe studies performed with nAChR subunits Nlα1 and Nlα2 cloned from the brown planthopper Nilaparvata  lugens , a major insect pest of rice crops in many parts of Asia. The influence of Nlα1 and Nlα2 subunits upon the functional properties of recombinant nAChRs has been examined by expression in Xenopus oocytes. In addition, the influence of a Nlα1 mutation (Y151S), which has been linked to neonicotinoid lab generated resistance in N. lugens , has been examined. As in previous studies of insect α subunits, functional expression has been achieved by co-expression with the mammalian β2 subunit. This approach has revealed a significantly higher apparent affinity of imidacloprid for Nlα1/β2 than for Nlα2/β2 nAChRs. In addition, evidence has been obtained for the co-assembly of Nlα1 and Nlα2 subunits into 'triplet' nAChRs of subunit composition Nlα1/Nlα2/β2. Evidence has also been obtained which demonstrates that the resistance-associated Y151S mutation has a significantly reduced effect on neonicotinoid agonist activity when Nlα1 is co-assembled with Nlα2 than when expressed as the sole α subunit in a heteromeric nAChR. These findings may be of importance in assessing the likely impact of the target-site mutations such as Y151S upon neonicotinoid insecticide resistance in insect field populations.     

Amino acids outside of the loops that define the agonist binding site are important for ligand binding to insect nicotinic acetylcholine receptors

Nicotinic acetylcholine (ACh) receptors (nAChRs) are the targets of several kinds of insecticides. Based on the mutagenesis studies of Torpedo californica nAChRs and solved structure of a molluscan, glial-derived soluble ACh-binding protein, a model of the agonist site was constructed with contributing amino acids from three distinct loops (A, B, and C) of the α subunits and another three loops (D, E, and F) of the non-α subunits. According to this model, most insect nAChR subunits can form the functional heteromeric or homomeric receptors. Actually, insect subunits themselves did not form any functional receptor at various combinations as yet, and only part of them can form the functional receptors with vertebrate non-α subunits. These findings suggested that the agonist binding for insect nAChRs was not only contributed by those key amino acids in six loops, but also some unidentified amino acids from other regions. In our previous studies on nAChRs for Nilaparvata lugens , a target-site mutation (Y151S) was found within two α subunits (Nlα1 and Nlα3). In Drosophila S2 cells and Xenopus oocytes, Nlα1 can form functional receptors with rat β2 subunit. However, the same thing was not observed in Nlα3. In the present paper, by exchanging the corresponding regions between Nlα1 and Nlα3 to generate different chimeras, amino acid residues or residue clusters in the regions outside the six loops were found to play essential roles in agonist binding, especially for the amino acid clusters between loop B and C. This result indicated that the residues in the six loops could be necessary, but not enough for the activity of agonist binding.     

Regulation of Nicotinic Receptor Subtypes Following Chronic Nicotinic Agonist Exposure in M10 and SH-SY5Y Neuroblastoma Cells

Abstract: The present study further investigated whether nicotinic acetylcholine receptor (nAChR) subtypes differ in their ability to up-regulate following chronic exposure to nicotinic agonists. Seven nicotinic agonists were studied for their ability to influence the number of chick α4β2 nAChR binding sites stably transfected in fibroblasts (M10 cells) following 3 days of exposure. The result showed a positive correlation between the K _i values for binding inhibition and EC₅₀ values for agonist-induced α4β2 nAChR up-regulation. The effects of epibatidine and nicotine were further investigated in human neuroblastoma SH-SY5Y cells (expressing α3, α5, β2, and β4 nAChR subunits). Nicotine exhibited a 14 times lower affinity for the nAChRs in SH-SY5Y cells as compared with M10 cells, whereas epibatidine showed similar affinities for the nAChRs expressed in the two cell lines. The nicotine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was shifted to the right by two orders of magnitude as compared with that in M10 cells. The epibatidine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was one-fourth that in M10 cells. The levels of mRNA of the various nAChR subunits were measured following the nicotinic agonist exposure. In summary, the various nAChR subtypes show different properties in their response to chronic stimulation.     

Novel Neonicotinoid-Agarose Affinity Column for Drosophila and Musca Nicotinic Acetylcholine Receptors

Abstract: Neonicotinoids such as the insecticide imidacloprid (IMI) act as agonists at the insect nicotinic acetylcholine receptor (nAChR). Head membranes of Drosophila melanogaster and Musca domestica have a single high-affinity binding site for [³H]IMI with K _D values of 1–2 n M and B _max values of 560–850 fmol/mg of protein. Locusta and Periplaneta nAChRs isolated with an α-bungarotoxin (α-BGT)-agarose affinity column are known to be α-subunit homooligomers. This study uses 1 - [ N - (6 - chloro - 3 - pyridylmethyl) - N - ethyl]amino - 1 - amino-2-nitroethene (which inhibits [³H]IMI binding to Drosophila and Musca head membranes at 2–3 n M ) to develop a neonicotinoid-agarose affinity column. The procedure—introduction of Triton-solubilized Drosophila or Musca head membranes into this neonicotinoid-based column, elution with IMI, and analysis by lithium dodecyl sulfate-polyacrylamide gel electrophoresis—gives only three proteins (69, 66, and 61 kDa) tentatively assigned as putative subunits of the nAChR; the same three proteins are obtained with Musca using the α-BGT-agarose affinity column. Photoaffinity labeling of the Drosophila and Musca putative subunits from the neonicotinoid column with ¹²⁵I-α-BGT-4-azidosalicylic acid gives a labeled derivative of 66–69 kDa. The yield is 2–5 µg of receptor protein from 1 g of Drosophila or Musca heads. Neonicotinoid affinity chromatography to isolate native Drosophila and Musca receptors will facilitate studies on the structure and function of insect nAChRs.     

Interaction of α-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor

α-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α-Conotoxins blocking muscle-type or α7 nAChRs compete with α-bungarotoxin. However, α-conotoxin ImII, a close homolog of the α7 nAChR-targeting α-conotoxin ImI, blocked α7 and muscle nAChRs without displacing α-bungarotoxin ( Ellison et al. 2003, 2004 ), suggesting binding at a different site. We synthesized α-conotoxin ImII, its ribbon isomer (ImII iso ), 'mutant' ImII(W10Y) and found similar potencies in blocking human α7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [¹²⁵I]-α-bungarotoxin from human α7 nAChRs in the cell line GH₄C₁ (IC₅₀ 17 and 23 μM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC₅₀ 2.0–9.0 μM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized α-bungarotoxin ( K _d and IC₅₀ 2.5–8.2 μM). On Torpedo nAChR, α-conotoxin [¹²⁵I]-ImII(W10Y) revealed specific binding ( K _d 1.5–6.1 μM) and could be displaced by α-conotoxin ImII, ImII iso and ImII(W10Y) with IC₅₀ 2.7, 2.2 and 3.1 μM, respectively. As α-cobratoxin and α-conotoxin ImI displaced [¹²⁵I]-ImII(W10Y) only at higher concentrations (IC₅₀≥ 90 μM), our results indicate that α-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.     

Role of the N-terminal α-helix in biogenesis of α7 nicotinic receptors

We studied the role of the α-helix present at the N-terminus of nicotinic acetylcholine receptor (nAChR) subunits in the expression of functional channels. Deletion of this motif in α7 subunits abolished expression of nAChRs at the membrane of Xenopus oocytes. The same effect was observed upon substitution by homologous motifs of other ligand-gated receptors. When residues from Gln4 to Tyr15 were individually mutated to proline, receptor expression strongly decreased or was totally abolished. Equivalent substitutions to alanine were less harmful, suggesting that proline-induced break of the α-helix is responsible for the low expression. Steady-state levels of wild-type and mutant subunits were similar but the formation of pentameric receptors was impaired in the latter. In addition, those mutants that reached the membrane showed a slightly increased internalization rate. Expression of α7 nAChRs in neuroblastoma cells confirmed that mutant subunits, although stable, were unable to reach the cell membrane. Analogous mutations in heteromeric nAChRs (α3β4 and α4β2) and 5-HT_3A receptors also abolished their expression at the membrane. We conclude that the N-terminal α-helix of nAChRs is an important requirement for receptor assembly and, therefore, for membrane expression.     

NIDA522131, a new radioligand for imaging extrathalamic nicotinic acetylcholine receptors: in vitro and in vivo evaluation

A novel radioligand, 6-chloro-3-((2-( S )-azetidinyl)methoxy)-5-(2-fluoropyridin-4-yl)pyridine (NIDA522131), for imaging extrathalamic nicotinic acetylcholine receptors (nAChRs) was characterized in vitro and in vivo using positron emission tomography. The K_d and T_1/2 of dissociation of NIDA522131 binding measured at 37°C in vitro were 4.9 ± 0.4 pmol/L and 81 ± 5 min, respectively. The patterns of radioactivity distribution in monkey brain in vivo was similar to that of 2-[¹⁸F]fluoro-3-(2( S )-azetidinylmethoxy)pyridine (2FA), a radioligand that has been successfully used in humans, and matched the α₄β₂* nAChRs distribution. Comparison between [¹⁸F]NIDA522131 and 2FA demonstrated better in vivo binding properties of the new radioligand and substantially greater radioactivity accumulation in brain. Consistent with [¹⁸F]NIDA522131 elevated affinity for nAChRs and its increased lipophilicity, both, the total and non-displaceable distribution volumes were substantially higher than those of 2FA. Estimated binding potential values in different brain regions, characterizing the specificity of receptor binding, were 3–4 fold higher for [¹⁸F]NIDA522131 than those of 2FA. Pharmacological evaluation in mice demonstrated a toxicity that was comparable to 2FA and is in agreement with a 2300 fold higher affinity at α₄β₂* versus α₃β₄* nAChRs. These results suggest that [¹⁸F]NIDA522131 is a promising positron emission tomography radioligand for studying extrathalamic nAChR in humans.     

Amino acids within loops D, E and F of insect nicotinic acetylcholine receptor beta subunits influence neonicotinoid selectivity

Nicotinic acetylcholine (ACh) receptors (nAChRs) are ligand-gated ion channels which mediate fast cholinergic synaptic transmission in insect and vertebrate nervous systems. The nAChR agonist-binding site is present at the interface of adjacent subunits and is formed by loops A-C present in alpha subunits together with loops D-F present in either non-alpha subunits or homomer-forming alpha subunits. To investigate the mechanism of neonicotinoid selectivity, we have examined the effects of altering insect-specific loops D, E and F in hybrid nAChRs containing insect and mammalian subunits (Nlalpha1 from the brown planthopper Nilaparvata lugens and beta2 from rat). Introduction of the insect-specific loops D, E and F, singly or together, into rat beta2 subunit resulted in a leftward shift of the imidacloprid dose-response curves for nAChRs Nlalpha1-beta2 chimeras, reflecting decreases in EC(50), compared to wildtype nAChRs Nlalpha1-beta2. By contrast, the influences on ACh potency were minimal or negligible. The effects of loop D could be interpreted by the earlier findings of Shimomura et al. [2006. Role in the selectivity of neonicotinoids of insect-specific basic residues in loop D of the nicotinic acetylcholine receptor agonist-binding site. Mol. Pharmacol. 70, 1255-1263.], in which T77R and E79V were shown to be responsible for neonicotinoid selectivity. In the present study, S131Y(R) and D133N in loop E and T191W and P192K in loop F were found to contribute to the neonicotinoid selectivity of insect-specific loops E and F. These results indicated the insect-specific loops D, E and F each play important roles in neonicotinoids selectivity. This study contributes to our understanding of the molecular mechanism underlying selectivity of neonicotinoids against insects over vertebrates.     

Molecular Characterization and Imidacloprid Selectivity of Nicotinic Acetylcholine Receptor Subunits from the Peach-Potato Aphid Myzus persicae

The recent introduction of the chloronicotinyl insecticide imidacloprid, targeting insect nicotinic acetylcholine receptors (nAChRs), emphasises the importance of a detailed molecular characterisation of these receptors. We are investigating the molecular diversity of insect nAChR subunit genes in an important agricultural pest, the peach-potato aphid Myzus persicae. Two M. persicae alpha-subunit cDNAs, Mp alpha1 and Mp alpha2, have been cloned previously. Here we report the isolation of three novel alpha-subunit genes (Mp alpha3-5) with overall amino acid sequence identities between 43 and 76% to characterised insect nAChR subunits. Alignment of their amino acid sequences with other invertebrate and vertebrate nAChR subunits suggests that the insect alpha subunits evolved in parallel to the vertebrate neuronal nAChRs and that the insect non-alpha subunits are clearly different from vertebrate neuronal beta and muscle non-alpha subunits. The discovery of novel subtypes in M. persicae is a further indicator of the complexity of the insect nAChR gene family. Heterologous co-expression of M. persicae nAChR alpha-subunit cDNAs with the rat beta2 in Drosophila S2 cells resulted in high-affinity binding of nicotinic radioligands. The affinity of recombinant nAChRs for [3H]imidacloprid was influenced strongly by the alpha subtype. This is the first demonstration that imidacloprid selectively acts on Mp alpha2 and Mp alpha3 subunits, but not Mp alpha1, in M. persicae.     

A nicotinic acetylcholine receptor mutation (Y151S) causes reduced agonist potency to a range of neonicotinoid insecticides

Neonicotinoid insecticides are potent selective agonists of insect nicotinic acetylcholine receptors (nAChRs). Since their introduction in 1991, resistance to neonicotinoids has been slow to develop, but it is now established in some insect field populations such as the planthopper, Nilaparvata lugens, a major rice pest in many parts of Asia. We have reported recently the identification of a target-site mutation (Y151S) within two nAChR subunits (Nlalpha1 and Nlalpha3) from a laboratory-selected field population of N. lugens. In the present study, we have examined the influence of this mutation upon the functional properties of recombinant nAChRs expressed in Xenopus oocytes (as hybrid nAChRs, co-expressed with a rat beta2 subunit). The agonist potency of several nicotinic agonists has been examined, including all of the neonicotinoid insecticides that are currently licensed for either crop protection or animal health applications (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam). The Y151S mutation was found to have no significant effect on the maximal current (I(max)) observed with the endogenous agonist, acetylcholine. In contrast, a significant reduction in I(max) was observed for all neonicotinoids (the I(max) for mutant nAChRs ranged from 13 to 81% of that observed on wild-type receptors). In addition, nAChRs containing the Y151S mutation caused a significant rightward shift in agonist dose-response curves for all neonicotinoids, but of varying magnitude (shifts in EC(50) values ranged from 1.3 to 3.6-fold). The relationship between neonicotinoid structure and their potency on nAChRs containing the Y151S target-site mutation is discussed.     

Temperature-Sensitive Expression of Drosophila Neuronal Nicotinic Acetylcholine Receptors

Abstract: Heterologous expression of cloned Drosophila nicotinic acetylcholine receptor (nAChR) subunits indicates that these proteins misfold when expressed in mammalian cell lines at 37°C. This misfolding can, however, be overcome either by growing transfected mammalian cells at lower temperatures or by the expression of Drosophila nAChR subunits in a Drosophila cell line. Whereas the Drosophila nAChR β subunit (SBD) cDNA, reported previously, lacked part of the SBD coding sequence, here we report the construction and expression of a full-length SBD cDNA. We have examined whether problems in expressing functional Drosophila nAChRs in either Xenopus oocytes or mammalian cell lines can be attributed to an inability of these expression systems to assemble correctly Drosophila nAChRs. Despite expression in what might be considered a more native cellular environment, we have been unable to detect functional nAChRs in a Drosophila cell line unless Drosophila nAChR subunit cDNAs are coexpressed with vertebrate nAChR subunits. Our results indicate that the folding of Drosophila nAChR subunits is temperature-sensitive and strongly suggest that the inability of these Drosophila nAChR subunits to generate functional channels in the absence of vertebrate subunits is due to a requirement for coassembly with as yet unidentified Drosophila nAChR subunits.     

Functional interactions between the SK2 channel and the nicotinic acetylcholine receptor in enteric neurons of the guinea pig ileum

The neurotransmitter acetylcholine (ACh) plays a critical role in gastrointestinal function. The role of the small conductance Ca²⁺-activated K⁺ (SK) channel in ACh release was examined using myenteric plexus preparations of guinea pig ileum. Apamin, an inhibitor of the SK channel, significantly enhanced nicotine-induced ACh release, but neither electrical field stimulation- nor 5-hydroxytryptamine-induced ACh release, suggesting that SK channels might be selectively involved in the regulation of nicotine-induced ACh release. Therefore, we investigated the distribution of SK2 and SK3 subunits and the interaction between SK2 channels and nicotinic ACh receptors (nAChRs) in the guinea pig ileum. The immunoreactivity of SK2 subunits was located in enteric neuronal cells. Furthermore, SK2-immunoreactive cells stained with an antibody for choline acetyltransferase, a marker for cholinergic neurons, and with an antibody for the α3/5 subunits of nAChR. In contrast, immunoreactivity of SK3 subunits was not found in enteric neurons. A co-immunoprecipitation assay with Triton X-100-soluble membrane fractions prepared from the ileum revealed an association of the SK2 subunit with the α3/5 subunits of nAChR. These results suggest that SK2 channels negatively regulate the excitation of enteric neurons via functional interactions with nAChRs.     

Effects of Steroid Exposure on Ligand Binding and Functional Activities of Diverse Nicotinic Acetylcholine Receptor Subtypes

Abstract: Nicotinic acetylcholine receptors (nAChR) are diverse members of the ligand-gated ion channel superfamily of neurotransmitter receptors and play critical roles in chemical signaling throughout the nervous system. The present study tests whether nAChR are potential targets for steroids. Acute or short-term (5 min) preexposure to steroids such as progesterone (which acts most potently), estradiol, corticosterone, or dexamethasone inhibits function of human muscle-type (α1β1γδ) or ganglionic (α3β4) nAChR measured using ⁸⁶Rb⁺ efflux assays in TE671/RD clonal or SH-SY5Y neuroblastoma cells. Absolute (high nanomolar to intermediate micromolar range) and rank-order potencies for steroid-mediated functional inhibition are similar across nAChR subtypes but differ for some steroid derivatives. At concentrations that produce blockade of nAChR function, steroids do not affect binding of radioligands such as ¹²⁵I-labeled α-bungarotoxin or [³H]acetylcholine to muscle-type or ganglionic nAChR or to neuronal toxin-binding nAChR that contain α7 subunits (α7-nAChR). Steroid-mediated blockade of nAChR function is insurmountable by increasing agonist concentrations, and cell-impermeant progesterone:bovine serum albumin conjugates have full potency as inhibitors of ganglionic or muscle-type nAChR function. Chronic (48 h) exposure to progesterone or estradiol, but not the other steroids, also produces blockade of nAChR function, without significant effects on numbers of nAChR radioligand-binding sites. Collectively, these results suggest that steroids act noncompetitively at extracellular sites to inhibit nAChR function with unique potencies for different steroid-nAChR subtype combinations. Thus, nAChR could be among the targets mediating physiologically relevant effects of steroid action in the nervous system.     

Role of GSK-3beta activation and alpha7 nAChRs in Abeta(1-42)-induced tau phosphorylation in PC12 cells

β-amyloid peptide 1–42 (Aβ_1–42) and hyperphosphorylated tau are associated with neurodegeneration in Alzheimer's disease. Emerging evidence indicates that Aβ_1–42 can potentiate hyperphosphorylation of tau in cell lines and in transgenic mice, but the underlying mechanism(s) remains unclear. In this study, Aβ_1–42-induced tau phosphorylation was investigated in differentiated PC12 cells. Treatment of cells with Aβ_1–42 increased phosphorylation of tau at serine-202 as detected by AT8 antibody. This Aβ_1–42-induced tau phosphorylation paralleled phosphorylation of glycogen synthase kinase-3β (GSK-3β) at tyrosine-216 (GSK-3β-pY216), which was partially inhibited by the GSK-3β inhibitor, CHIR98023. Aβ_1–42-induced tau phosphorylation and increase in GSK-3β-pY216 phosphorylation were also partially attenuated by α7 nicotinic acetylcholine receptor (α7 nAChR) selective ligands including agonist A-582941 and antagonists methyllycaconitine and α-bungarotoxin. The α7 nAChR agonist and the GSK-3β inhibitor had no additive effect. These observations suggest that α7 nAChR modulation can influence Aβ_1–42-induced tau phosphorylation, possibly involving GSK-3β. This study provides evidence of nAChR mechanisms underlying Aβ_1–42 toxicity and tau phosphorylation, which, if translated in vivo , could provide additional basis for the utility of α7 nAChR ligands in the treatment of Alzheimer's disease.     

Host Cell-Specific Folding and Assembly of the Neuronal Nicotinic Acetylcholine Receptor α7 Subunit

Abstract: Expression of the cloned neuronal nicotinic acetylcholine receptor (nAChR) α7 subunit in several cultured mammalian cell lines has revealed that the folding, assembly, and subcellular localization of this protein are critically dependent upon the nature of the host cell. In all cell lines that were examined, high levels of α7 protein were detected by metabolic labelling and immunoprecipitation after transfection with the cloned α7 cDNA. In contrast, elevated levels of α-bungarotoxin binding could be detected in only two of the nine cell lines. Both of these "α7-permissive" cell lines [rat phaeochromocytoma (PC12) and human neuroblastoma (SH-SY5Y)] express an endogenous α7 subunit. However, by expression of an epitope-tagged α7 subunit, it has been possible to show that the elevation in surface α-bungarotoxin binding in these two cell lines is due to expression of cDNA-encoded α7. The cell-specific misfolding of the neuronal nAChR α7 subunit is a phenomenon that is not shared by either the hetero-oligomeric muscle nAChR or the homo-oligomeric serotonin receptor 5-HT₃ subunit. Our data also indicate that the cell-specific misfolding cannot be explained by a requirement for the coassembly with other known nAChR subunits and cannot be alleviated by treatments that have been reported to affect the assembly efficiency of other neurotransmitter-gated ion channels.     

褐飞虱对吡虫啉的抗性机理和靶标分子毒理学

褐飞虱Nilaparvata lugens是水稻最重要的害虫之一,长期依赖化学防治导致了该害虫对不同类型杀虫剂抗性的产生,对新烟碱类杀虫剂吡虫啉高水平抗性的产生更是造成了巨大的粮食生产损失。近年来在褐飞虱对吡虫啉抗性机理,以及在抗药性机理研究推动下吡虫啉作用靶标褐飞虱神经系统烟碱型乙酰胆碱受体（nicotinic acetylcholine receptors, nAChRs）毒理学等方面取得了许多研究进展。nAChRs是昆虫神经系统中最重要的神经递质受体,是几类重要杀虫剂的作用靶标,其中以新烟碱类杀虫剂为代表。通过对比敏感品系和室内连续筛选获得的高抗吡虫啉品系,在褐飞虱两个nAChRs亚基Nlα1和Nlα3中均发现了抗性相关点突变Y151S,该突变导致了受体与吡虫啉结合亲和力的显著下降,而对内源神经递质乙酰胆碱的亲和力影响很小。Nlα1与褐飞虱另外两个亚基Nlα2和Nlβ1共聚成一个受体,构成吡虫啉低亲和力结合位点;Nlα3与褐飞虱另外两个亚基Nlα8和Nlβ1共聚成一个受体,构成吡虫啉高亲和力结合位点。不仅褐飞虱nAChRs与吡虫啉抗性相关,某些nAChRs附属蛋白也直接影响褐飞虱对吡虫啉的抗性,如Lynx蛋白。关于褐飞虱nAChRs组成、抗药性相关变异、受体附属蛋白对抗药性的影响等方面的研究,均为国内外前沿报道,不仅有助于对新烟碱类杀虫剂抗性机理的理解,对昆虫nAChRs毒理学同样具有很大的推动作用。     

The potential subunits involved in two subtypes of α-Bgt-resistant nAChRs in cockroach dorsal unpaired median (DUM) neurons

The american cockroach (Periplaneta americana) dorsal unpaired median (DUM) neurons provide an native tool to analyze the functional and pharmacological properties of ion channels and membrane receptors, such as nicotine acetylcholine receptors (nAChRs). Here the imidacloprid-activated nAChR subtypes were examined in DUM neurons by the patch-clamp technique and the potential subunits involved in important subtypes were analyzed by combining with RNA interference (RNAi) technique. Imidacloprid exerted agonist activities on one subtype in α-Bgt-sensitive nAChRs and another subtype in α-Bgt-resistant nAChRs, in which the α-Bgt-resistant subtype showed much higher sensitivity to imidacloprid than the α-Bgt-sensitive subtype, with the difference close to 200-fold. In α-Bgt-resistant nAChRs, nicotine exerted the agonist activity on two subtypes (nAChR1 and nAChR2), although imidacloprid only activated nAChR1. RNAi against Paα3, Paα8 and Paβ1 significantly reduced both imidacloprid- and nicotine-activated currents on nAChR1. In contrast, RNAi against Paα1, Paα2 and Paβ1 decreased nicotine-activated currents on nAChR2. The results indicated that, in α-Bgt-resistant nAChRs, Paα3, Paα8 and Paβ1 might be involved in the subunit composition of nAChR1, and Paα1, Paα2 and Paβ1 in nAChR2. In summary, from the present study and previous reports, we deduced that there are at least three nAChR subtypes that are sensitive to imidacloprid in the cockroach DUM neurons.